Biochemistry of the Fed State (Absorptive/Postprandial

Phase)
1. Definition & Timing:

• Fed State: The metabolic state occurring 2-4 hours after consuming food.
• Also known as: Absorptive Phase, Postprandial State.
• Characterized by: Digestion and absorption of nutrients (Carbohydrates, Proteins, Lipids).
• Resulting Plasma Changes:
◦ Increased: Glucose, Amino Acids, Fatty Acids, Triacylglycerols (TAGs).
◦ Increased: Chylomicrons (transport dietary TAGs).
2. Hormonal Control: Insulin

• Primary Hormone: Insulin.

• Secretion Trigger: Blood glucose levels rise above 3.9 mmol/L (70 mg/dL).
• Secreting Cells: Beta cells of the pancreas.
3. Insulin Secretion Mechanism (Pancreatic Beta Cells):

• Glucose Entry: Via GLUT2 transporter (Low affinity - transports glucose effectively only when blood
glucose is high).
• Glucose Metabolism:
◦ Glucose → Glucose-6-Phosphate (by Glucokinase).
◦ Glucose-6-Phosphate → Pyruvate (Glycolysis).
◦ ATP production increases.
• ATP/ADP Ratio: Rises significantly.
• K+ Channel Closure: Increased ATP closes ATP-sensitive K+ channels.
• Depolarization: K+ cannot exit the cell, leading to membrane depolarization.
• Ca2+ Channel Opening: Depolarization opens voltage-gated Ca2+ channels.
• Ca2+ Influx: Calcium ions (Ca2+) enter the beta cell.
• Insulin Exocytosis: Increased intracellular Ca2+ stimulates secretory vesicles containing insulin to fuse
with the cell membrane and release insulin.
• Co-Secretion: C-peptide is released along with insulin in equimolar amounts (Marker for endogenous
insulin production).
4. Role of Incretins:

• Definition: Hormones that enhance glucose-stimulated insulin secretion.

• Examples:
◦ GIP (Glucose-dependent insulinotropic peptide)
◦ GLP-1 (Glucagon-like peptide-1)
• Source: Neuroendocrine cells (L-cells) of the GIT; possibly intrapancreatic alpha cells.
• Mechanism:
◦ Bind to incretin receptors on beta cells.
◦ Increase intracellular cyclic AMP (cAMP) levels.
 ◦ cAMP enhances the exocytosis of insulin-containing vesicles.
• Clinical Note: Incretin analogues are used to treat diabetes mellitus.
5. Insulin Receptor & Mechanism of Action:

• Receptor Structure: Insulin Receptor is a heterotetramer (2 alpha, 2 beta subunits).

◦ Alpha subunits: Extracellular, bind insulin.
◦ Beta subunits: Transmembrane, possess intracellular intrinsic tyrosine kinase activity.
• Activation Pathway:
1. Insulin binds to alpha subunits.
2. Conformational change activates tyrosine kinase activity in beta subunits.
3. Autophosphorylation: Beta subunits phosphorylate tyrosine residues on each other.
4. Signal Cascade Initiation: Phosphorylated receptor recruits and phosphorylates Insulin Receptor
Substrates (IRS), specifically IRS 1-4.
• Downstream Effects via IRS:
◦ IRS binds PI3 Kinase (Phosphoinositide 3-kinase) via SH2 domain:
▪ Protein Translocation: Increases movement of GLUT4 transporters and Insulin Receptors to
the cell membrane.
▪ Gene Transcription: Increases synthesis of enzymes like Glucokinase.
▪ Enzyme Activity Modulation: Increases activity of Phosphodiesterase (PDE) (breaks down
cAMP) and Phosphatases (dephosphorylate target proteins, often activating anabolic pathways).
◦ IRS binds GRB2 via SH2 domain:
▪ Promotes cell growth.
• Mnemonic (IRS Actions via PI3K): IRS -> PI3K -> Protein moves (GLUT4), Genes transcribed
(Glucokinase), Enzymes activated (PDE/Phosphatase) -> PGE.
6. Organ-Specific Metabolism in the Fed State (Anabolism Favored):

• A. Liver:
◦ Glucose Uptake: Via GLUT2 (low affinity, high capacity).
◦ Carbohydrate Metabolism:
▪ Glycolysis: Glucose → G6P (Glucokinase) → Pyruvate → Acetyl-CoA (Pyruvate Dehydrogenase
- PDH).
▪ Glycogen Synthesis: Glucose stored as glycogen.
▪ HMP Shunt (Pentose Phosphate Pathway): Produces NADPH for biosynthesis.
◦ Lipid Metabolism:
▪ Fatty Acid Synthesis: Excess Acetyl-CoA → Fatty Acids (requires NADPH).
▪ Lipogenesis: Fatty Acids + Glycerol → Triacylglycerol (TAG).
▪ VLDL Synthesis & Secretion: Endogenously synthesized TAG packaged into VLDL and
released into blood.
◦ Amino Acid Metabolism:
▪ Protein Synthesis: Increased.
▪ Deamination: Amino acids → Carbon skeletons (keto acids like pyruvate, oxaloacetate) + Amino
group.
▪ Carbon Skeletons: Used for anabolic purposes (e.g., enter TCA cycle).
 • B. Adipose Tissue:
◦ Glucose Uptake: Via GLUT4 (insulin-dependent).
◦ Carbohydrate Metabolism:
▪ Glycolysis: Glucose → G6P → Pyruvate → Acetyl-CoA (PDH). (Provides glycerol backbone for
TAG synthesis).
▪ HMP Shunt: Produces NADPH for fatty acid synthesis.
▪ (No significant glycogen synthesis).
◦ Lipid Metabolism:
▪ Fatty Acid Synthesis: Acetyl-CoA → Fatty Acids (requires NADPH).
▪ Lipogenesis: Fatty Acids + Glycerol (from glycolysis) → TAG storage.
▪ TAG Uptake: Fatty acids from Chylomicrons (dietary) and VLDL (liver-derived) are taken up
(via Lipoprotein Lipase - LPL, implied) and re-esterified into TAG for storage.
◦ Primary Function: Store TAG.
• C. Skeletal Muscle:
◦ Glucose Uptake: Via GLUT4 (insulin-dependent).
◦ Carbohydrate Metabolism:
▪ Glycolysis: Glucose → G6P → Pyruvate → Acetyl-CoA (PDH).
▪ Glycogen Synthesis: Glucose stored as glycogen (major storage site besides liver).
▪ TCA Cycle: Acetyl-CoA oxidation for ATP (energy for muscle function).
◦ Amino Acid Metabolism:
▪ Protein Synthesis: Increased uptake and synthesis of muscle protein.
◦ Lipid Metabolism: Less significant than in liver or adipose tissue.
• D. Brain:
◦ Glucose Uptake: Via GLUT1 (Blood-Brain Barrier) and GLUT3 (Neurons). (Both high affinity,
insulin-independent).
◦ Carbohydrate Metabolism:
▪ Glycolysis: Glucose → G6P → Pyruvate → Acetyl-CoA (PDH).
▪ TCA Cycle: Acetyl-CoA oxidation for ATP.
◦ Fuel Dependence: Highly dependent on glucose via aerobic/oxidative pathways.
7. Metabolic Fuel Preferences in the Fed State:

• Brain: Glucose ONLY.

• RBCs: Glucose ONLY (anaerobic glycolysis).
• Liver: Prefers Glucose (high availability via GLUT2).
• Skeletal Muscle: Prefers Glucose (insulin stimulates GLUT4 uptake).
• Adipose Tissue: Uses Glucose (via GLUT4) for energy and TAG synthesis backbone; takes up fatty
acids for storage.
• Heart: Prefers Free Fatty Acids (despite GLUT4 presence, due to low glycolytic capacity).
• Mnemonic (Fuel Preference): BRain/RBCs = Glucose Only (BRinG Only). Heart Favors Acids
(HFA). Most others prefer Glucose when abundant (Fed State).
8. Important Points to Remember:

• Fed state = Postprandial = Absorptive phase (2-4 hrs post-meal).

• Insulin is the key anabolic hormone.
• Insulin secretion depends on glucose uptake (GLUT2), metabolism (Glucokinase, ATP↑), K+
channel closure, and Ca2+ influx in beta cells.
• Incretins (GIP, GLP-1) enhance insulin release via cAMP.
• Insulin acts via a Receptor Tyrosine Kinase, activating IRS -> PI3K and GRB2 pathways.
• Major actions via PI3K: GLUT4 translocation, increased gene transcription (e.g., Glucokinase),
enzyme modulation (PDE, Phosphatases).
• Organ metabolic profiles shift towards synthesis and storage:
◦ Liver: Glycogen synthesis, Fatty Acid/TAG synthesis, VLDL export, Protein synthesis.
◦ Adipose: TAG synthesis and storage (from glucose and circulating lipids).
◦ Muscle: Glycogen synthesis, Protein synthesis.
• Fuel Use: Most tissues utilize abundant glucose (via GLUT2 or insulin-stimulated GLUT4), except
Brain/RBCs (always glucose) and Heart (prefers fatty acids).
• C-peptide is a marker of endogenous insulin secretion.

Biochemistry of Fasting Stages: Summary

I. Introduction & Importance

• Fasting stages commence after the well-fed state (2-4 hours post-meal).
• Understanding fasting biochemistry is crucial (illustrated by a case scenario).
• Case Scenario: Dinner at 8 PM, breakfast at 9 AM (13 hours fasting). Blood glucose = 100 mg/dL.
Question: What pathway maintains this glucose level? (Answered later).
II. Timeline of Feed-Fast Cycle & Fasting Stages

• Well-Fed State: 2-4 hours post-meal.

• Fasting Stages (Post-Absorptive Phases):
◦ 1. Early Fasting: 4 - 16 hours without food.
◦ 2. Fasting: 16 - 48 hours without food.
◦ 3. Starvation (Prolonged Fasting): 2 - 5 days without food (duration varies based on individual
adipocyte TAG stores).
◦ 4. Prolonged Starvation: >5 days without food (or when TAG stores are depleted).
III. Metabolic Changes During Fasting Stages

• 1. Early Fasting (4-16 hours):

◦ Primary Source of Blood Glucose: Hepatic Glycogenolysis (breakdown of liver glycogen).
◦ Muscle glycogenolysis does not contribute directly to blood glucose because muscle lacks the
enzyme Glucose-6-Phosphatase.
◦ Glycogen stores become depleted by 16-18 hours.
• 2. Fasting (16-48 hours):
◦ Glycogen depleted; body seeks alternative fuels.
 ◦ Primary Source of Blood Glucose: Gluconeogenesis (synthesis of glucose from non-carbohydrate
precursors like amino acids, lactate, glycerol).
◦ ATP Source for Gluconeogenesis: Fatty Acid Beta-Oxidation.
▪ Stored Triacylglycerols (TAG) in adipose tissue are broken down (lipolysis) -> Fatty Acids +
Glycerol.
▪ Fatty acids undergo beta-oxidation -> ATP.
▪ Glycerol serves as a substrate for gluconeogenesis.
• 3. Starvation / Prolonged Fasting (2-5 days):
◦ Gluconeogenesis decreases due to depletion of non-carbohydrate substrates (including
Oxaloacetate, an intermediate used in both TCA and gluconeogenesis).
◦ Primary Metabolic Activity: TAG breakdown continues -> Fatty Acids -> Beta-Oxidation ->
Acetyl-CoA.
◦ Fate of Acetyl-CoA: Enters Ketone Body Synthesis in the liver.
▪ Reason: TCA cycle slows down due to lack of Oxaloacetate to combine with Acetyl-CoA.
◦ Ketone bodies become a crucial fuel source for vital organs (brain, heart, muscle) as blood glucose
drops.
• 4. Prolonged Starvation (>5 days / TAG Depleted):
◦ TAG stores exhausted; Fatty acid availability drops.
◦ Ketone body synthesis decreases.
◦ Body resorts to Muscle Proteolysis (breakdown of structural muscle proteins) for amino acids
(potential gluconeogenic substrates, but primarily fuel).
▪ This is initially prevented as long as ketone bodies are available (protective mechanism).
◦ Leads to severe muscle wasting and Cachexia.
IV. Hormones of Fasting

• Key Hormones: Glucagon and Epinephrine.

• Glucagon Secretion: Triggered when blood glucose < 50 mg/dL.
• Mechanism of Action (Similar for both):
1. Hormone binds to G Protein-Coupled Receptor (GPCR) on plasma membrane.
2. Activates G protein.
3. Activates Adenylyl Cyclase.
4. Converts ATP -> Cyclic AMP (cAMP).
5. cAMP activates Protein Kinase A (PKA).
6. PKA phosphorylates target enzymes.
7. Result: Enzymes involved in fasting pathways (glycogenolysis, gluconeogenesis, lipolysis) are
generally activated by phosphorylation.
V. Organ-Specific Metabolism During Fasting

• A. Liver: (Mnemonic: Liver Gives Glucose & Ketones)

◦ Glucose Transporter: GLUT2 (low affinity; transports glucose out into blood when liver glucose
concentration is high due to glycogenolysis/gluconeogenesis).
◦ Pathways:
▪ Hepatic Glycogenolysis (Early Fasting).
 ▪ Gluconeogenesis (Fasting stage, using lactate, glycerol, amino acids). Requires ATP from fatty
acid oxidation.
▪ Fatty Acid Beta-Oxidation (Provides ATP).
▪ Ketone Body Synthesis (Starvation stage, from excess Acetyl-CoA). Exports ketone bodies.
◦ Key Role: Produces glucose and ketone bodies for other tissues. Does not use ketone bodies as fuel.
"Selfless organ".
• B. Adipose Tissue:
◦ Glucose Transporter: GLUT4 (Insulin-dependent; low activity during fasting -> less glucose
uptake).
◦ Primary Pathway: Lipolysis (breakdown of stored TAG).
◦ Key Enzyme: Hormone-Sensitive Lipase (HSL), activated by glucagon/epinephrine via PKA.
◦ Products:
▪ Fatty Acids: Released into blood, used by liver, muscle, heart. Used locally for ATP via beta-
oxidation.
▪ Glycerol: Released into blood, used by liver for gluconeogenesis.
• C. Resting Skeletal Muscle: (Mnemonic: Muscle Feeds on Fat & Ketones)
◦ Glucose Transporter: GLUT4 (Insulin-dependent; low activity during fasting -> less glucose
uptake).
◦ Protein Breakdown: Releases amino acids (especially Alanine) into blood -> used by liver for
gluconeogenesis.
◦ Glycogen Use: Muscle glycogen breakdown -> Glucose-6-Phosphate -> Pyruvate -> Alanine
(indirectly supports gluconeogenesis via Glucose-Alanine cycle). Cannot release free glucose.
◦ Primary Fuels:
▪ Fatty Acids (via beta-oxidation).
▪ Ketone Bodies (via ketolysis, during starvation).
• D. Brain: (Mnemonic: Brain Gets Glucose or Ketones)
◦ Glucose Transporters: GLUT1 (Blood-Brain Barrier) & GLUT3 (Neurons). Both are high affinity,
ensuring glucose uptake even at low blood glucose levels.
◦ Primary Fuel: Glucose (via oxidative metabolism: Glycolysis -> Pyruvate -> Acetyl-CoA -> TCA
cycle -> ATP).
◦ Alternate Fuel (Starvation): Ketone Bodies (via ketolysis).
▪ Can only supply a portion of energy needs (mentioned as 20%, leading to sluggish function).
Contextual Note: Often cited as covering up to 60-70% of brain energy needs in prolonged
starvation, but the lecture states 20%. We stick to the transcript.
◦ Cannot use Fatty Acids: Albumin-bound fatty acids cannot cross the Blood-Brain Barrier.
• E. Red Blood Cells (RBC):
◦ Sole Fuel: Glucose (lack mitochondria, rely on anaerobic glycolysis).
◦ Cannot use fatty acids or ketone bodies.
◦ Risk of hemolysis if glucose becomes unavailable.
VI. Summary of Metabolic Fuels
 Tissue Fed State Early Fasting/ Starvation Fuel Key Notes
Fuel Fasting Fuel

Liver Glucose > FFA > Glucose FFA, Amino Acids Produces Glucose & Ketones;
FFA Doesn't use Ketones

Heart FFA > FFA > Glucose FFA, Ketone Bodies Low glycolytic capacity
Glucose

Brain Glucose Glucose Glucose (if avail.), Cannot use FFA; High-affinity
Ketone Bodies GLUTs

Skeletal Glucose > FFA > Glucose FFA, Ketone Bodies Releases Alanine; Cannot release
Muscle FFA Glucose

RBC Glucose Glucose Glucose Glucose ONLY; Risk of hemolysis

Adipose Glucose > FFA > Glucose FFA, Ketone Bodies Releases FFA & Glycerol
Tissue FFA (Lipolysis)

• Major Fuel Shift: From Glucose (fed) to Free Fatty Acids (FFA) (fasting/starvation).
• Secondary Fuel (Starvation): Ketone Bodies (for Brain, Heart, Muscle, Adipose).
VII. Case Scenario Answer

• The person fasted for 13 hours (8 PM to 9 AM).

• This falls within the Early Fasting stage (4-16 hours).
• The primary pathway maintaining blood glucose during this time is Hepatic Glycogenolysis.
VIII. Important Points to Remember

• Timelines: Know the approximate hours/days defining Early Fasting, Fasting, Starvation, Prolonged
Starvation.
• Key Pathways by Stage:
◦ Early Fasting: Hepatic Glycogenolysis.
◦ Fasting: Gluconeogenesis (fueled by Fatty Acid Oxidation).
◦ Starvation: Ketone Body Synthesis (fueled by Fatty Acid Oxidation).
◦ Prolonged Starvation: Muscle Proteolysis.
• Hormonal Control: Glucagon/Epinephrine -> GPCR -> Adenylyl Cyclase -> cAMP -> PKA ->
Phosphorylation (activates fasting pathways like HSL, glycogen phosphorylase, gluconeogenic enzymes).
• Organ Roles:
◦ Liver: Central regulator; makes glucose & ketones for others; uses FFA.
◦ Adipose: Stores & releases FFA & glycerol (via HSL).
◦ Muscle: Uses FFA/Ketones; releases amino acids (Alanine); cannot release glucose.
◦ Brain: Prioritizes Glucose (GLUT1/3); uses Ketones in starvation; cannot use FFA.
◦ RBC: Glucose only.
• Enzyme Deficiencies: Muscle lacks Glucose-6-Phosphatase.
• Fuel Restrictions: Brain cannot use FFA; RBC uses only Glucose; Liver doesn't use Ketones.
• Interdependence: Gluconeogenesis needs ATP from fat breakdown; ketone synthesis occurs when TCA
slows (low OAA). Muscle proteolysis is spared by ketone availability.
Summary: Concepts of Enzyme Regulation
This summary covers the core concepts of hormonal and allosteric enzyme regulation as presented in the
lecture.
I. Introduction * Enzyme regulation is fundamental to understanding metabolic pathways. * Two primary
mechanisms discussed: 1. Hormonal Regulation 2. Allosteric Regulation
II. Hormonal Regulation

• Physiological States & Key Hormones:

◦ Well-Fed State (Absorptive/Postprandial):
▪ Hormone: Insulin
▪ Ratio: High Insulin/Glucagon ratio
◦ Fasting State (Post-absorptive):
▪ Hormone: Glucagon
▪ Ratio: Low Insulin/Glucagon ratio
• Mechanism of Glucagon Action:
1. Glucagon binds to G Protein-Coupled Receptor (GPCR) on the plasma membrane.
2. Activates G protein.
3. G protein activates Adenylyl Cyclase.
4. Adenylyl Cyclase converts ATP → cyclic AMP (cAMP).
5. cAMP activates cAMP-dependent Protein Kinase (Protein Kinase A - PKA).
6. PKA phosphorylates target enzymes (proteins) at hydroxyl groups of Serine, Threonine, or Tyrosine
residues, using ATP.
7. Result: Target enzyme exists in a Phosphorylated State.
• Mechanism of Insulin Action (Dual Role):
1. Reduces cAMP: Insulin activates Phosphodiesterase, which converts cAMP → 5' AMP. This
lowers intracellular cAMP levels, counteracting the glucagon signal cascade.
2. Promotes Dephosphorylation: Insulin leads to the removal of phosphate groups from target
enzymes (activating protein phosphatases - implied). Water is used, and inorganic phosphate (Pi) is
released.
3. Result: Target enzyme exists in a Dephosphorylated State.
• Core Concept & Application Strategy:
◦ Under Insulin (Well-fed): Enzymes are typically active in the DEPHOSPHORYLATED state.
◦ Under Glucagon (Fasting): Enzymes are typically active in the PHOSPHORYLATED state.
◦ Mnemonic: Insulin -> Inactivates Phosphate (Dephosphorylated); Glucagon -> Gets Phosphate
(Phosphorylated).
◦ Problem-Solving: For any pathway, ask:
1. Active in Well-fed or Fasting state?
2. Active under Insulin or Glucagon?
3. If Well-fed/Insulin: Active when Dephosphorylated.
4. If Fasting/Glucagon: Active when Phosphorylated.
 • Examples:
◦ Glycolysis: Well-fed, Insulin -> Active Dephosphorylated.
◦ Gluconeogenesis: Fasting, Glucagon -> Active Phosphorylated.
◦ Glycogen Synthesis: Well-fed, Insulin -> Active Dephosphorylated.
◦ Glycogen Degradation (Glycogenolysis): Fasting, Glucagon -> Active Phosphorylated.
◦ Pyruvate Dehydrogenase (PDH): Well-fed, Insulin (links Glycolysis to TCA) -> Active
Dephosphorylated.
◦ Fatty Acid Synthesis: Well-fed, Insulin -> Active Phosphorylated (as stated in the transcript).
◦ Cholesterol Synthesis: Well-fed, Insulin -> Active Dephosphorylated.
III. Allosteric Regulation

• Core Concept:
◦ Substrate: Favors the forward reaction -> Activator.
◦ Product: Inhibits the forward reaction -> Inhibitor.
◦ Mnemonic: Substrate -> Stimulates; Product -> Prohibits.
• Terminology:
◦ Feed-Forward Regulation: Substrate activates an enzyme downstream.
◦ Feedback Inhibition: End-product of a pathway inhibits an earlier enzyme (often the regulatory/rate-
limiting step).
• Examples:
◦ Phosphofructokinase-1 (PFK-1) in Glycolysis: (Converts Fructose-6-P → Fructose-1,6-bisP)
▪ Activators:
▪ Fructose-6-Phosphate (Substrate)
▪ AMP (Indicates low energy state, need for ATP synthesis; acts like substrate indicator)
▪ Inhibitors:
▪ ATP (Product of pathway; indicates high energy state)
▪ Low pH (Product of anaerobic glycolysis - Lactic Acid)
▪ Citrate (Product of TCA cycle; indicates downstream pathway is saturated)
◦ ALA Synthase (Heme Synthesis):
▪ Inhibitor: Heme (End-product) -> Feedback Inhibition.
◦ Acetyl-CoA Carboxylase (Fatty Acid Synthesis):
▪ Activator: Citrate (Source of Acetyl-CoA for FA synthesis in cytosol; acts as substrate indicator)
▪ Inhibitor: Acyl-CoA (Fatty acid product) -> Feedback Inhibition.
IV. Important Points to Remember

1. Hormonal State Dictates Phosphorylation: Insulin = Dephosphorylation; Glucagon = Phosphorylation.

Know the dominant hormone in well-fed vs. fasting states.
2. Two Questions for Hormonal Regulation: Ask if a pathway is active in (1) Well-fed/Fasting and (2)
under Insulin/Glucagon to determine its active phosphorylation state.
3. Allosteric Basics: Substrates generally activate, Products generally inhibit.
4. Key Allosteric Regulators: Understand the roles of ATP/AMP (energy state), Citrate (TCA cycle link),
and pathway end-products (feedback inhibition).
 5. Enzyme State Matters: The activity of many key metabolic enzymes depends critically on whether they
are phosphorylated or dephosphorylated, or bound by allosteric effectors.
6. (Transcript Specific Note): The lecture stated Fatty Acid Synthesis is active when phosphorylated under
insulin, which contradicts the general Insulin -> Dephosphorylated rule presented earlier in the same
lecture. Remember this specific point as stated.
Carbohydrates Lecture Summary
I. Introduction & Importance

• Carbohydrates are the major dietary source of calories globally.

• Focus is shifting towards clinical applications of biochemistry, not just deep chemistry.
• Understanding basic chemistry and metabolism is crucial for grasping applications.
II. Chemistry of Carbohydrates

• A. Definition:
◦ "Carbo-hydrate": Hydrates of Carbon.
◦ Chemically: Aldehyde or Keto derivatives of polyhydroxy alcohols.
◦ Parent alcohol example: Glycerol (a 3-carbon polyhydroxy alcohol).
• B. General Formula (Monosaccharides):
◦ CnH2On (or Cn(H2O)n), where 'n' is the number of carbon atoms.

• C. Simplest Carbohydrates (Trioses - 3C):

◦ Derived from Glycerol.
◦ Glyceraldehyde: Aldehyde derivative (Aldotriose). Functional group (CHO) typically on C1.
◦ Dihydroxyacetone: Keto derivative (Ketotriose). Functional group (C=O) typically on C2.
◦ These are the parent/simplest carbohydrates.
• D. Classification:
◦ Based on Sugar Units:
▪ Monosaccharides: 1 sugar unit.
▪ Disaccharides: 2 sugar units.
▪ Oligosaccharides: 3-10 sugar units.
▪ Polysaccharides: >10 sugar units.
◦ Monosaccharide Classification:
▪ By Carbon Number: Trioses (3C), Tetroses (4C), Pentoses (5C), Hexoses (6C).
▪ By Functional Group:
▪ Aldoses: Contain aldehyde group (end in -ose). Examples: Glyceraldehyde, Erythrose,
Ribose, Xylose, Glucose, Galactose, Mannose.
▪ Ketoses: Contain keto group (end in -ulose). Examples: Dihydroxyacetone, Erythrulose,
Ribulose, Xylulose, Fructose.
▪ Mnemonic: Ketose sounds like 'you-lose' (ulose ending), like fructose.
• E. Biologically Important Monosaccharides:
◦ Hexoses: Glucose and Fructose are most important.
• F. Ring Structures (Crucial for Image-Based Questions - IBQs):
◦ In solution (and body), monosaccharides exist predominantly in ring forms.
 ◦ Formed by reaction between the functional group (aldehyde/keto) and a hydroxyl group on another
carbon (typically C5 for hexoses).
◦ Glucose:
▪ Forms a 6-membered ring (5 Carbons + 1 Oxygen) called a Pyranose ring.
▪ Proper name: Glucopyranose.
▪ C6 is outside the ring.
◦ Fructose:
▪ Forms a 5-membered ring (4 Carbons + 1 Oxygen) called a Furanose ring.
▪ Proper name: Fructofuranose.
▪ C1 and C6 are outside the ring.
▪ Mnemonic: Fructose = Five-membered Furanose.
• G. Disaccharides:
◦ Reducing vs. Non-reducing:
▪ Depends on whether an anomeric carbon (the C1 of an aldose or C2 of a ketose) is free (not
involved in the glycosidic bond).
▪ If a free anomeric carbon exists, the sugar is reducing.
▪ All monosaccharides are reducing.
◦ Examples:
▪ Reducing:
▪ Maltose: Glucose + Glucose (α-1,4 glycosidic linkage).
▪ Isomaltose: Glucose + Glucose (α-1,6 glycosidic linkage).
▪ Lactose: Galactose + Glucose (β-1,4 glycosidic linkage).
▪ Lactulose: Galactose + Fructose (linkage not specified as important). Has '-ulose' ending due
to fructose (ketose).
▪ Non-reducing: (Anomeric carbons of both units are involved in the bond)
▪ Sucrose: Glucose + Fructose (α-1, β-2 glycosidic linkage).
▪ Trehalose: Glucose + Glucose (α-1, α-1 glycosidic linkage).
• H. Polysaccharides:
◦ Homopolysaccharides: Made of only one type of monomer unit.
◦ Heteropolysaccharides: Made of more than one type of monomer unit.
◦ Important Examples:
▪ Glycogen:
▪ Storage carbohydrate in animals.
▪ Monomer: α-D-Glucose.
▪ Branched structure: Linear chains (α-1,4 linkages) with branches (α-1,6 linkages).
▪ Has one reducing end (the start) and multiple non-reducing ends (where glucose is added/
removed).
▪ Starch:
▪ Storage carbohydrate in plants.
▪ Monomer: α-D-Glucose.
▪ Two components:
▪ Amylose: Linear (α-1,4 linkages only).
▪ Amylopectin: Branched (α-1,4 and α-1,6 linkages).
 ▪ Cellulose:
▪ Structural component in plants.
▪ Monomer: β-D-Glucose.
▪ Linear structure (β-1,4 linkages).
▪ Indigestible by humans (lack enzyme for β-1,4 bond). Major component of dietary fiber.
▪ Inulin:
▪ Homopolysaccharide of Fructose (a fructosan).
▪ Used in Inulin Clearance Test to estimate Glomerular Filtration Rate (GFR) - ideal marker
but exogenous.
▪ Chitin:
▪ Found in exoskeleton of crustaceans, insects, fungi cell walls.
▪ Monomer: N-acetyl-D-glucosamine.
▪ Pectin:
▪ Heteropolysaccharide (predominantly Galacturonic acid).
▪ Soluble dietary fiber.
▪ Dextrin:
▪ Hydrolytic product of starch (α-D-Glucose polymer).
▪ Component of dental plaque.
▪ Mentioned as potential plasma volume expander and used in size exclusion chromatography
(Note: Dextran is more commonly cited for these uses, but the text specifies Dextrin).
III. Dietary Fiber

• A. Definition: Remnant of edible plant parts & analogous carbohydrates resistant to digestion and
absorption in the human small intestine. Fermented completely or partially in the large intestine. Also
called Non-Starch Polysaccharides (NSP).
• B. Source: Exclusively from plants (not found in animal foods).
• C. Resistance: Humans lack enzymes (e.g., cannot break β-1,4 bonds of cellulose).
• D. Fermentation: Occurs in the large intestine, produces Short-Chain Fatty Acids (SCFAs) like acetic,
propionic, butyric acid.
• E. Classification:
◦ Insoluble (Crude Fiber): Cellulose, Hemicellulose, Lignin (Note: Text mentioned 'lectin', Lignin is
the correct fiber component contextually). Lignin is generally non-fermentable.
◦ Soluble: Pectin, Gums, Mucilage.
• F. Recommended Daily Allowance (RDA): 40 grams per 2000 kcal diet.
• G. Energy Value: Approx. 2 kcal/gram (from absorbed SCFAs).
• H. Benefits:
◦ Acts as a Prebiotic (promotes growth of beneficial gut bacteria/probiotics).
◦ Increases fecal bulk, softens stools, aids bowel regularity.
◦ Reduces cholesterol (binds bile acids, increasing cholesterol excretion).
◦ Promotes satiety (helps control appetite/obesity).
◦ Slows glucose absorption, reducing postprandial blood glucose (especially soluble fibers like gums
- e.g., fenugreek - and pectins). Important for diabetes management.
IV. Isomerism in Carbohydrates

• A. Definition: Compounds with the same molecular formula but different structural arrangements.
• B. Asymmetric (Chiral) Carbon Atom: A carbon atom attached to four different groups.
◦ Glucose: 4 asymmetric carbons (C2, C3, C4, C5) in straight chain; 5 in pyranose ring form (C1
becomes asymmetric upon ring formation).
◦ Dihydroxyacetone: Has no asymmetric carbon atoms.
◦ Ketoses generally have one less asymmetric carbon than their corresponding aldose.
• C. Number of Stereoisomers: Calculated by 2n (Le Bel-Van't Hoff rule), where 'n' = number of
asymmetric carbons.
• D. Types of Isomerism:
◦ 1. Structural / Stereoisomers:
▪ D and L Isomerism (Enantiomers):
▪ Configuration differs at the penultimate (reference) carbon atom (second to last, e.g., C5 in
glucose).
▪ They are mirror images of each other.
▪ Designated Capital D or Capital L.
▪ Most naturally occurring sugars are in the D-form. (Most amino acids are L-form).
▪ Example: D-Glyceraldehyde vs L-Glyceraldehyde; D-Glucose vs L-Glucose.
▪ Anomerism:
▪ Configuration differs only at the anomeric carbon (the functional carbonyl carbon involved
in ring formation - C1 for aldoses, C2 for ketoses).
▪ Exists only in ring structures.
▪ Designated alpha (α) or beta (β).
▪ Rule: In Haworth projection, if OH on anomeric carbon is below the ring plane = α; if above
= β. Mnemonic: Beta = OH points up like a Bird.
▪ Examples: α-D-Glucopyranose vs β-D-Glucopyranose.
▪ Most predominant free form of glucose: β-D-Glucopyranose.
▪ Epimerism:
▪ Configuration differs at only one single asymmetric carbon, other than the anomeric or
penultimate carbon.
▪ Examples (Epimers of Glucose):
▪ Mannose: C2 epimer of Glucose.
▪ Galactose: C4 epimer of Glucose.
▪ Allose: C3 epimer of Glucose (less common).
▪ Diastereoisomers:
▪ Stereoisomers that are not mirror images (i.e., not enantiomers).
▪ Differ at more than one asymmetric center, but not all.
▪ Epimers are a subset of diastereoisomers.
▪ Example: Mannose and Galactose are diastereoisomers of each other (differ at C2 and C4
relative to glucose).
◦ 2. Optical Isomerism:
▪ Ability of a compound (due to asymmetric carbons) to rotate the plane of polarized light.
 ▪ Dextrorotatory (d or +): Rotates light clockwise (to the right). Example: Glucose (also called
Dextrose).
▪ Levorotatory (l or -): Rotates light anti-clockwise (to the left). Example: Fructose (also called
Levulose).
▪ Note: D/L notation (capital) refers to structure relative to glyceraldehyde; d/l notation (lowercase)
refers to optical activity. They are not necessarily related.
▪ Invert Sugar: Sucrose is initially dextrorotatory (+). Upon hydrolysis, it yields glucose (+) and
fructose (-). Since fructose's levorotation is stronger than glucose's dextrorotation, the resulting
mixture becomes levorotatory. This change (inversion) of rotation gives sucrose the name 'invert
sugar'.
V. Important Points to Remember

• Glucose exists mainly as β-D-Glucopyranose in solution.

• Humans cannot digest β-1,4 linkages (like in Cellulose).
• Glycogen (animal storage) and Starch (plant storage) are polymers of α-D-Glucose.
• Dietary fiber is crucial for health (gut health, cholesterol, blood sugar control).
• Understand the difference between Reducing and Non-reducing sugars (based on free anomeric carbon).
• Know the definitions and examples of Isomers:
◦ D/L (Enantiomers): Penultimate Carbon, Mirror Images.
◦ α/β (Anomers): Anomeric Carbon (in rings).
◦ Epimers: Single other asymmetric carbon.
◦ d/l (Optical): Direction of light rotation (+/-).
• Clinical Relevance: Fiber intake, GFR estimation (Inulin), Blood sugar control, plasma expanders
(Dextrin/Dextran).
• Simplest aldose: Glyceraldehyde. Simplest ketose: Dihydroxyacetone.
• Carbohydrate without asymmetric carbon: Dihydroxyacetone.
(Concluding thought from lecture): Eat less sugar, you are sweet enough already.
Glycosaminoglycans (GAGs) / Mucopolysaccharides

• Definition: Long, unbranched heteropolysaccharides. Also known as mucopolysaccharides.

• Structure: Composed of repeating disaccharide units.
• Repeating Disaccharide Unit:
◦ Component 1: An Amino Sugar (usually N-acetylated)
▪ Glucosamine (N-acetylglucosamine)
▪ Galactosamine (N-acetylgalactosamine)
◦ Component 2: An Acidic Sugar
▪ Usually a Uronic Acid:
▪ Glucuronic acid
▪ Iduronic acid (C5 epimer of Glucuronic acid)
▪ Exception: Keratin sulfate has Galactose instead of a uronic acid.
• Key Chemical Feature: Highly negatively charged (polyanions) due to:
◦ Carboxyl groups (COO-) from uronic acids.
◦ Sulfate groups (SO3-) often added to amino sugars.
 ◦ (Sometimes acetyl groups, CH3COO-, are added, but sulfate and carboxyl groups dominate the
charge).
• Properties (due to negative charge):
◦ Bind large amounts of water via hydrogen bonds, forming a hydrated gel.
◦ Act as lubricants and shock absorbers (explains cartilage compressibility, joint mobility/resilience).
◦ Occupy large volumes due to charge repulsion.
◦ Form a molecular sieve, allowing selective passage of molecules.
Major GAGs: Composition, Location & Key Features

GAG Name Repeating Disaccharide Key Locations Key Features/Functions

Unit

Hyaluronic N-acetylglucosamine + Skin, synovial fluid, Unique: Not sulfated, not typically covalently
Acid Glucuronic acid bone, cartilage, loose bound to core protein. Involved in cell
CT migration (wound repair, embryogenesis,
metastasis).

Chondroitin N-acetylgalactosamine + Cartilage, bone, CNS Most abundant GAG. Responsible for
Sulfate Glucuronic acid cartilage compressibility.

Keratin N-acetylglucosamine + Cornea (KS I), Unique: Contains Galactose instead of uronic
Sulfate Galactose cartilage, loose CT acid. KS I crucial for corneal transparency.
(KS II) Most heterogeneous GAG.

Heparin Glucosamine + Iduronic Intracellular in Mast Unique: Primarily intracellular. Potent natural
acid (highly sulfated) cells, also lung, liver, anticoagulant (binds Antithrombin III).
skin Injection dislodges LPL.

Heparan Glucosamine + Glucuronic Skin, Kidney basement Crucial for charge selectivity of the glomerular
Sulfate acid (variable sulfation) membrane, synaptic basement membrane. Anchors Lipoprotein
vesicles Lipase (LPL). Acts as plasma membrane
receptor.

Dermatan N-acetylgalactosamine + Dermis of skin, blood Provides structure to sclera. Synthesized by

Sulfate Iduronic acid vessels, heart valves arterial smooth muscle cells, binds LDL,
considered atherogenic.

(Mnemonic Hint: Think Heparan sulfate for Held LPL and kHidney filter charge)
Proteoglycans

• Structure: Core protein + one or more covalently attached GAG chains.

◦ Linkage Region: GAGs attach to serine residues on the core protein via a trisaccharide linker
(Xylose-Galactose-Galactose).
◦ Analogy: "Bottle Brush" shape (Protein core = handle, GAGs = bristles). GAG negative charges
cause bristles to repel and extend.
• Proteoglycan Aggregates:
◦ Multiple proteoglycan monomers non-covalently associate with a long molecule of Hyaluronic Acid.
◦ Association is stabilized by Link Proteins.
◦ Forms massive complexes, important in extracellular matrix (e.g., cartilage).
GAG Synthesis & Degradation

• Synthesis: Occurs primarily in the Endoplasmic Reticulum (ER) and Golgi apparatus.
• Degradation: Occurs sequentially within Lysosomes by specific lysosomal acid hydrolases.
Mucopolysaccharidoses (MPS)

• Definition: A group of inherited Lysosomal Storage Disorders.

• Cause: Deficiency of specific lysosomal enzymes required for GAG degradation.
• Result: Accumulation of undegraded or partially degraded GAGs within lysosomes, leading to cellular
dysfunction and multi-systemic clinical features.
• Inheritance: Most are Autosomal Recessive (AR).
◦ Exception: MPS II (Hunter Syndrome) is X-Linked Recessive.
General Clinical Features of MPS:

• Coarse Facial Features: Frontal bossing, depressed nasal bridge, thick lips, large head.
• Corneal Clouding (absent in Hunter & Sanfilippo).
• Gingival hypertrophy.
• Macroglossia (large tongue) -> recurrent upper respiratory infections, hearing loss.
• Claw Hand deformity.
• Intellectual Disability / Developmental Delay (variable, notably absent in Scheie, Morquio, Maroteaux-
Lamy).
• Visceromegaly: Hepatosplenomegaly -> protuberant abdomen, umbilical/inguinal hernias (absent in
Morquio).
• Short Stature.
• Skeletal Abnormalities (Dysostosis Multiplex):
◦ Abnormalities in multiple bones.
◦ Beaking of Vertebrae.
◦ Bullet-shaped Phalanges (metacarpals).
• Leukocyte Inclusions: Alder-Reilly bodies may be seen in peripheral blood smears (cytoplasmic
granules).
Specific MPS Disorders (Key Ones)

MPS Eponym Deficient Enzyme Accumulated Key Differentiating Features

Type GAG(s)

MPS Hurler α-L-Iduronidase Heparan Sulfate, Severe form. All typical features
IH Dermatan Sulfate including corneal clouding and
intellectual disability. AR.

MPS Scheie α-L-Iduronidase (partial) Heparan Sulfate, Milder form. Corneal clouding, stiff
IS Dermatan Sulfate joints, normal lifespan, NORMAL
intelligence. AR.

MPS Hunter Iduronate-2-Sulfatase Heparan Sulfate, X-LINKED Recessive (males

II Dermatan Sulfate primarily affected). NO corneal
clouding. Variable severity (mild/
severe forms).
 MPS Eponym Deficient Enzyme Accumulated Key Differentiating Features
Type GAG(s)

MPS Sanfilippo 4 different enzymes involved in Heparan Sulfate Most common MPS. Severe
III A-D Heparan Sulfate degradation neurological regression often
predominates. Variable somatic
features. No corneal clouding. AR.

MPS Morquio A Galactosamine-6-Sulfatase (A) Keratan Sulfate, Severe skeletal dysplasia. NORMAL
IV &B or β-Galactosidase (B) Chondroitin-6- intelligence. NO visceromegaly.
Sulfate Corneal clouding present. AR.

MPS Maroteaux- N-Acetylgalactosamine-4- Dermatan Sulfate Hurler-like physical features, corneal

VI Lamy Sulfatase (Arylsulfatase B) clouding. NORMAL intelligence. AR.

MPS Natowicz Hyaluronidase Hyaluronic Acid Very rare, periarticular soft tissue
IX masses.

(Mnemonic Hint: Hunter lacks Haze (corneal clouding) and is X-linked. Scheie spares the Skull
(intelligence).)
Diagnosis & Findings

• Clinical presentation.
• Urinary GAG analysis (increased excretion).
• Enzyme activity assays in leukocytes or cultured fibroblasts (confirmatory).
• Genetic testing.
• X-rays: Show dysostosis multiplex.
• Leukocyte inclusions: Alder-Reilly bodies.
Treatment Modalities

• Enzyme Replacement Therapy (ERT):

◦ MPS I (Hurler, Scheie): Aldurazyme (Laronidase)
◦ MPS II (Hunter): Elaprase (Idursulfase)
◦ MPS VI (Maroteaux-Lamy): Naglazyme (Galsulfase)
◦ MPS IVA (Morquio A): Vimizim (Elosulfase alfa) - Text mentioned GALNS as under trial, Vimizim is
approved.
• Hematopoietic Stem Cell Transplantation (HSCT):
◦ Most established for severe MPS I (Hurler). Can improve neurocognitive outcome if done early.
◦ Considered for some other types (e.g., MPS VI).
• Substrate Reduction Therapy (SRT):
◦ Example: Genistein (a flavonoid) explored for MPS III (Sanfilippo) - aims to decrease GAG
synthesis. Text mentioned flavinoid. Still largely experimental for MPS.
• Supportive Care: Management of specific symptoms (orthopedic, cardiac, respiratory, neurological).
--- Key Points to Emphasize ---

• GAGs are negatively charged polysaccharides crucial for ECM structure and function.
• Know the key GAGs and their distinctive features/locations:
◦ Heparan Sulfate: Kidney basement membrane (charge selectivity), anchors LPL.
 ◦ Keratin Sulfate: Cornea transparency (KS I), contains galactose not uronic acid.
◦ Heparin: Intracellular anticoagulant.
◦ Hyaluronic Acid: Cell migration, no sulfate, not bound to protein core usually.
◦ Dermatan Sulfate: Atherogenic potential.
◦ Chondroitin Sulfate: Most abundant, cartilage compressibility.
• MPS are lysosomal storage disorders due to defective GAG degradation enzymes.
• Remember the general clinical features of MPS (coarse facies, short stature, skeletal issues, etc.).
• Focus on differentiating MPS I (Hurler/Scheie) and MPS II (Hunter):
◦ Hurler (IH): Severe, AR, Corneal Clouding +, Intellectual Disability +.
◦ Scheie (IS): Milder, AR, Corneal Clouding +, Normal Intelligence.
◦ Hunter (II): X-Linked, NO Corneal Clouding, variable Intellectual Disability.
• Know that MPS III (Sanfilippo) is the most common MPS, often with severe neurological issues.
• Know that MPS IV (Morquio) and MPS VI (Maroteaux-Lamy) have normal intelligence.
• Inheritance Pattern: All AR except Hunter (X-linked).
• Treatment: ERT and HSCT are mainstays for specific types.

Summary: Glucose Transporters Lecture

I. Introduction & Importance

• Need: Glucose is hydrophilic and cannot easily cross the hydrophobic plasma membrane. Transporters
(usually proteins) provide a hydrophilic pathway.
• Significance: Understanding transporters is crucial for pharmacology and medicine due to their specific
locations and functions, meticulously designed by the body.
• Key Questions Addressed: Why transporters are needed, SGLT2 inhibitors as Oral Hypoglycemic
Agents (OHAs), specific GLUT4 location, role of glycolipids/ORS in cholera.
II. Classification of Glucose Transporters

1. Sodium-Dependent Glucose Transporters (SGLT)

◦ Abbreviation: SGLT (Sodium-Glucose Transporter)
◦ Requires Sodium.
2. Sodium-Independent Glucose Transporters (GLUT)
◦ Abbreviation: GLUT
◦ Does not require Sodium.
III. Sodium-Dependent Glucose Transporters (SGLT)

• Types & Location:

◦ SGLT1: Luminal side of Intestine, Proximal tubules of Kidney.
◦ SGLT2: Proximal tubules of Kidney ONLY.
• Function: Absorb/Reabsorb glucose from the lumen into the cell.
• Characteristics:
◦ Sodium-Dependent: Requires Na+ for transport.
◦ Unidirectional: Transports glucose into the cell.
◦ Secondary Active Transport: Does not directly use ATP, but relies on the Na+ gradient maintained
by the Na+/K+ ATPase pump (which uses ATP).
 ◦ Symport: Transports Na+ and Glucose together in the same direction (into the cell).
◦ Mechanism:
▪ Na+ moves down its concentration gradient (into the cell). This gradient is actively maintained by
the Na+/K+ ATPase pump located on the basolateral/serosal membrane (pumps 3 Na+ out, 2 K+
in, using ATP).
▪ Glucose moves against its concentration gradient (into the cell), driven by the Na+ movement.
This ensures maximal absorption even when intracellular glucose is high.
• Clinical Applications:
◦ Oral Rehydration Salts (ORS): Contain Glucose + Sodium (Salt). SGLT1 co-transports both,
enhancing water and electrolyte absorption in the intestine, crucial for treating dehydration (e.g.,
Cholera). Adding amino acids does not aggravate the situation as they use different transporters.
◦ Renal Glycosuria: Caused by mutation in the SLC5A2 gene (codes for SGLT2). Leads to defective
SGLT2, reduced glucose reabsorption in the kidney, glucose excretion in urine, and a lowered renal
threshold (< 180 mg/dL).
◦ SGLT2 Inhibitors (e.g., Gliflozins): Used as Oral Hypoglycemic Agents (OHAs) for diabetes. They
block SGLT2 -> inhibit glucose reabsorption -> glucose excreted in urine (therapeutic glycosuria) ->
lower blood glucose.
▪ Side Effect: Increased risk of Urinary Tract Infections (UTIs) due to glucose in urine.
IV. Sodium-Independent Glucose Transporters (GLUT)

• Characteristics:
◦ Sodium-Independent.
◦ Facilitated, Carrier-Mediated Diffusion: Requires a protein carrier.
◦ Passive Process: Does not require metabolic energy (ATP).
◦ Bidirectional: Can transport glucose in or out of the cell depending on the gradient.
◦ Along Concentration Gradient: Moves glucose from high concentration to low concentration.
◦ Ping-Pong Mechanism: Transporter protein alternates conformation (Ping facing high concentration,
Pong facing low concentration).
◦ Saturable Kinetics: Shows a Vmax (maximum velocity) because the number of carriers is limited.
Plot of transport rate (V) vs. solute concentration yields a hyperbolic curve (unlike simple diffusion,
which is linear and non-saturable).
• Specific GLUT Transporters:
◦ GLUT1:
▪ Location: Brain (Blood-Brain Barrier - BBB), Kidney, Placenta, Colon, Retina, RBCs (Widely
distributed).
▪ Affinity: High Affinity (Low Km).
▪ Function: Basal glucose uptake. Ensures constant glucose supply even at low blood levels, crucial
for barrier tissues and RBCs (which rely solely on glucose).
◦ GLUT2:
▪ Location: Beta cells of Pancreas, Liver (sinusoidal membrane), Basolateral side of Intestine,
Basolateral side of Proximal Renal Tubules. (Mnemonic: BLIP - Beta, Liver, Intestine, Proximal
tubule)
▪ Affinity: Low Affinity (High Km).
 ▪ Function: Transports glucose only when blood levels are high. Acts as a "sensor" in pancreas (for
insulin release) and liver (for uptake/storage). Transports absorbed/reabsorbed glucose into blood
from intestine/kidney cells. Insulin-independent.
◦ GLUT3:
▪ Location: Neurons (within the brain), Placenta, Kidney.
▪ Affinity: Highest Affinity (Very Low Km).
▪ Function: Ensures glucose uptake for high-demand tissues even at very low concentrations.
◦ GLUT4:
▪ Location: Heart, Skeletal Muscle, Adipose Tissue. (Mnemonic: HAS - Heart, Adipose, Skeletal
muscle)
▪ Affinity: (Not specified, likely moderate and responsive).
▪ Function: Insulin-Dependent/Inducible. Insulin promotes translocation of GLUT4 from
intracellular vesicles to the plasma membrane, increasing glucose uptake after a meal. These
tissues can use alternative fuels (e.g., fatty acids), so they uptake glucose mainly when it's
abundant (high insulin).
◦ GLUT5:
▪ Location: Luminal side of Intestine, Spermatozoa.
▪ Function: Primarily a Fructose Transporter. (Mnemonic: F for Five, F for Fructose). Important
for dietary fructose absorption and providing energy for sperm motility.
◦ GLUT6:
▪ Location: Spleen, Leukocytes.
▪ Function: Pseudogene (gene exists but lacks transporter function).
◦ GLUT7:
▪ Location: Liver Smooth Endoplasmic Reticulum (SER).
▪ Function: Transports glucose from the SER lumen into the cytoplasm (relevant after glucose-6-
phosphatase action during gluconeogenesis/glycogenolysis).
◦ GLUT8:
▪ Location: Blastocyst.
◦ GLUT9:
▪ Function: Primarily a Urate Transporter. Mutation linked to Gout.
V. Integrated Glucose Handling & Glycemic Index

• Intestinal Absorption:
◦ Lumen -> Cell: SGLT1 (Glucose, Galactose + Na+), GLUT5 (Fructose).
◦ Cell -> Blood (Basolateral): GLUT2 (Glucose, Galactose, Fructose).
• Post-Meal Response:
◦ High blood glucose sensed by Pancreatic Beta Cells (via GLUT2) -> Insulin Secretion.
◦ Liver uptakes glucose (via GLUT2) for storage.
◦ Insulin stimulates GLUT4 translocation in Heart, Skeletal Muscle, Adipose tissue -> increased
glucose uptake.
• Glycemic Index (GI):
◦ Glucose/Galactose (via SGLT1 - active uptake): High GI (100%). Sugars like Maltose, Lactose also
have high GI.
 ◦ Fructose (via GLUT5 - facilitated diffusion, saturable): Lower GI (<100%). Sucrose has lower GI
than maltose/lactose. Reason: SGLT1 actively pulls glucose against gradient; GLUT5 is passive and
saturable.
◦ Dietary Fiber: GI = 0.
VI. Cholera Context

• Glycolipid Role: GM1 Ganglioside (a glycolipid) acts as the receptor for Cholera toxin on intestinal
cells.
• ORS Efficacy: Works because SGLT1 couples Na+ and Glucose uptake, driving electrolyte and water
absorption despite toxin-induced secretion.
VII. Important Points to Remember

• SGLT vs GLUT: SGLT is Sodium-Dependent, Secondary Active, Against Gradient (for glucose),
Unidirectional. GLUT is Sodium-Independent, Passive Facilitated Diffusion, Along Gradient,
Bidirectional.
• Affinity & Km: High Affinity = Low Km (transports well at low concentrations, e.g., GLUT1, GLUT3).
Low Affinity = High Km (transports well only at high concentrations, e.g., GLUT2).
• Insulin Dependence: ONLY GLUT4 (Heart, Adipose, Skeletal Muscle) is Insulin-Dependent. GLUT2
(Pancreas, Liver) is crucially Insulin-Independent.
• SGLT2 Inhibitors: Clinically important OHAs (Gliflozins) acting in the kidney. Cause glycosuria. Risk
of UTIs.
• GLUT Locations Dictate Function: e.g., GLUT1/3 in brain/RBCs for constant supply, GLUT2 in
pancreas/liver as sensor/overflow, GLUT4 in muscle/fat for post-meal uptake.
• Fructose Transport: Primarily via GLUT5 (facilitated diffusion).
• ORS Mechanism: Relies on SGLT1 co-transport.

Glycolysis: The Prototype Metabolic Pathway of Carbohydrates

I. Core Concept & Overview

• Definition: Glycolysis (from Greek glykys = sweet/sugar, lysis = splitting) is the pathway of sugar
splitting. Also known as the Embden-Meyerhof-Parnas (EMP) pathway.
• State: Primarily active in the well-fed state (high insulin:glucagon ratio).
• Process: Insulin promotes glucose uptake via GLUT4 (insulin-dependent transporter). Glucose enters the
cell and is trapped by phosphorylation.
◦ Glucose -> Glucose 6-Phosphate (G6P)
◦ G6P Fates: Glycogen synthesis, HMP Shunt, or Glycolysis.
◦ Glycolysis Path: G6P -> Pyruvate.
◦ Pyruvate Fates:
▪ Aerobic: -> Acetyl-CoA -> TCA Cycle (requires O2).
▪ Anaerobic: -> Lactate (low O2 or no mitochondria).
▪ Excess Acetyl-CoA -> Fatty Acid Synthesis -> Triacylglycerol storage.
• Location: Occurs in the cytoplasm of all cells/organs.
• Overall Reaction: One 6-carbon Glucose is split into two 3-carbon Pyruvate molecules.
◦ Generates ATP (via substrate-level phosphorylation).
 ◦ Generates NADH (reducing equivalents).
◦ Anaerobic conditions: NADH is used to convert Pyruvate to Lactate, regenerating NAD+.
II. Significance of Glycolysis

• Universality: The only pathway functioning both aerobically and anaerobically.

• Red Blood Cells (RBCs):
◦ Sole energy source: Relies exclusively on anaerobic glycolysis as it lacks mitochondria.
◦ ATP Production: Essential for maintaining ion gradients (e.g., Na+/K+ ATPase) and cell shape.
◦ Clinical Link: Defects in glycolytic enzymes lead to insufficient ATP -> RBC swelling and
hemolysis.
• Skeletal Muscle:
◦ High Capacity: Possesses enormous glycolytic capacity.
◦ Hypoxia Tolerance: Can derive significant energy via anaerobic glycolysis during intense exercise
(low O2).
◦ Clinical Link: Enzyme defects can cause muscle fatigue and exercise intolerance.
• Cardiac Muscle (Heart):
◦ Low Capacity: Very low glycolytic capacity.
◦ Hypoxia Sensitivity: Highly dependent on aerobic metabolism; highly susceptible to ischemia (lack
of O2) leading to conditions like angina and myocardial infarction.
• Cancer Cells: Exhibit high rates of glycolysis (even aerobically - Warburg effect), contributing to
hypermetabolism and cachexia (reason detailed in a later part, not fully explained here).
• Brain: Primarily relies on aerobic oxidation of glucose. Highly sensitive to hypoxia, leading to loss of
consciousness if aerobic pathways are compromised.
III. Phases and Steps of Glycolysis
(A) Preparatory Phase (Energy Investment Phase) * Goal: Phosphorylate glucose and convert it to two
molecules of Glyceraldehyde 3-Phosphate (G3P). * Investment: 2 ATP consumed.

1. Glucose -> Glucose 6-Phosphate (G6P)

◦ Enzyme: Hexokinase (most tissues) or Glucokinase (Hexokinase IV - liver, pancreas).
◦ ATP Consumed: 1 ATP -> 1 ADP.
◦ Key Features:
▪ Irreversible.
▪ Regulatory Step.
▪ Flux-generating step: Traps glucose inside the cell.
2. G6P -> Fructose 6-Phosphate (F6P)
◦ Enzyme: Phosphohexose Isomerase.
◦ Key Features:
▪ Reversible.
▪ Isomerization (aldose to ketose) prepares C1 for phosphorylation.
3. F6P -> Fructose 1,6-Bisphosphate (F1,6BP)
◦ Enzyme: Phosphofructokinase-1 (PFK-1).
◦ ATP Consumed: 1 ATP -> 1 ADP.
◦ Key Features:
▪ Irreversible.
 ▪ Rate-limiting step of glycolysis.
▪ Committed step: F1,6BP must proceed through glycolysis.
▪ Major Regulatory Point.
4. F1,6BP -> Dihydroxyacetone Phosphate (DHAP) + Glyceraldehyde 3-Phosphate (G3P)
◦ Enzyme: Aldolase.
◦ Key Features:
▪ Reversible.
▪ Splits the 6-carbon sugar into two 3-carbon triose phosphates.
5. DHAP -> Glyceraldehyde 3-Phosphate (G3P)
◦ Enzyme: Phosphotriose Isomerase (or Triose Phosphate Isomerase).
◦ Key Features:
▪ Reversible.
▪ Ensures both triose phosphates proceed through the payoff phase as G3P.
▪ Result: Two molecules of G3P are formed from one glucose.
(B) Payoff Phase (Energy Generation Phase) * Goal: Convert two G3P molecules to two Pyruvate
molecules, generating ATP and NADH. * Yield: 4 ATP and 2 NADH per glucose (remember everything is
doubled from here).

1. Glyceraldehyde 3-Phosphate (G3P) -> 1,3-Bisphosphoglycerate (1,3-BPG) (x2)

◦ Enzyme: Glyceraldehyde 3-Phosphate Dehydrogenase (G3PDH).
◦ Generated: 1 NADH + H+ (from NAD+).
◦ Inorganic Phosphate (Pi) Added.
◦ Key Features:
▪ Reversible.
▪ Generates high-energy phosphate bond in 1,3-BPG.
▪ NADH produced here yields ATP via oxidative phosphorylation (aerobic).
2. 1,3-Bisphosphoglycerate (1,3-BPG) -> 3-Phosphoglycerate (3-PG) (x2)
◦ Enzyme: Phosphoglycerate Kinase (PGK) or BPG Kinase.
◦ ATP Generated: 1 ATP (from ADP).
◦ Key Features:
▪ Reversible (unique among kinases in glycolysis).
▪ Substrate-Level Phosphorylation (SLP): ATP synthesis directly from a high-energy substrate.
3. 3-Phosphoglycerate (3-PG) -> 2-Phosphoglycerate (2-PG) (x2)
◦ Enzyme: Phosphoglycerate Mutase.
◦ Key Features:
▪ Reversible.
▪ Relocates the phosphate group.
4. 2-Phosphoglycerate (2-PG) -> Phosphoenolpyruvate (PEP) (x2)
◦ Enzyme: Enolase.
◦ Removes: 1 molecule of H2O.
◦ Requires: Mg2+ or Mn2+.
◦ Key Features:
▪ Reversible.
 ▪ Creates a high-energy enol phosphate bond in PEP.
5. Phosphoenolpyruvate (PEP) -> Pyruvate (x2)
◦ Enzyme: Pyruvate Kinase (PK).
◦ ATP Generated: 1 ATP (from ADP).
◦ Key Features:
▪ Irreversible.
▪ Regulatory Step.
▪ Substrate-Level Phosphorylation (SLP).
IV. Key Enzyme Details

• Hexokinase vs. Glucokinase (Hexokinase IV): | Feature | Hexokinase (I-III) | Glucokinase (IV) |
| :--------------- | :---------------------- | :---------------------- | | Location | Most tissues | Liver, Pancreatic β-
cells | | Affinity (Km)| High (Low Km) | Low (High Km) | | Vmax | Low | High | | Induction | No
(Housekeeping) | Yes (by Insulin) | | Inhibition | By G6P (product) | Not by G6P |
◦ Significance: Glucokinase functions effectively only at high glucose levels (post-meal), suitable for
liver storage (glycogen) and pancreatic insulin release. Hexokinase ensures glucose utilization even at
low concentrations.
• Phosphofructokinase-1 (PFK-1):
◦ The rate-limiting enzyme and major control point.
◦ Allosteric Regulation:
▪ Activators: AMP, Fructose 2,6-Bisphosphate (F2,6BP - most potent).
▪ Inhibitors: ATP, Citrate, Low pH (H+).
◦ Inducible: By insulin.
• Phosphofructokinase-2 (PFK-2) / Fructose 2,6-Bisphosphatase: (Bifunctional Enzyme, involved in
regulation)
◦ Synthesizes Fructose 2,6-Bisphosphate (F2,6BP) from F6P when dephosphorylated (active kinase
domain).
◦ F2,6BP is a powerful allosteric activator of PFK-1.
V. Anaerobic Glycolysis

• Reaction: Pyruvate -> Lactate

• Enzyme: Lactate Dehydrogenase (LDH).
• Coenzyme: Uses NADH, regenerates NAD+.
• Significance:
◦ Allows glycolysis to continue in absence of O2 or mitochondria by providing NAD+ for the G3PDH
step.
◦ No net NADH production in anaerobic glycolysis.
◦ Crucial for RBCs and exercising muscle.
VI. Energetics (ATP Yield per Glucose)

• Aerobic Glycolysis (Glucose -> 2 Pyruvate):

◦ G3PDH: 2 NADH * 2.5 ATP/NADH = 5 ATP (assuming mitochondrial oxidation)
◦ PGK (SLP): 2 * 1 ATP = 2 ATP
◦ PK (SLP): 2 * 1 ATP = 2 ATP
 ◦ Consumed (HK, PFK-1): -2 ATP
◦ Net = 7 ATP
• Anaerobic Glycolysis (Glucose -> 2 Lactate):
◦ G3PDH NADH used by LDH: 0 net ATP from NADH.
◦ PGK (SLP): 2 * 1 ATP = 2 ATP
◦ PK (SLP): 2 * 1 ATP = 2 ATP
◦ Consumed (HK, PFK-1): -2 ATP
◦ Net = 2 ATP (Crucial for RBCs)
• Complete Aerobic Oxidation (Glucose -> CO2 + H2O):
◦ Aerobic Glycolysis = 7 ATP
◦ Pyruvate Dehydrogenase (2 Pyruvate -> 2 Acetyl-CoA): 2 NADH * 2.5 ATP/NADH = 5 ATP
◦ TCA Cycle (2 Acetyl-CoA): 2 * 10 ATP/cycle = 20 ATP
◦ Total Net = 32 ATP
VII. Inhibitors of Glycolysis

• Arsenate: Competes with Pi at G3PDH step. Forms unstable 1-arseno-3-phosphoglycerate which

spontaneously hydrolyzes to 3-PG, bypassing ATP generation at the PGK step.
• Iodoacetate: Inhibits G3PDH.
• Fluoride: Inhibits Enolase by binding Mg2+/Mn2+ cofactors.
◦ Clinical Use: Sodium fluoride (+ oxalate as anticoagulant) is used in blood collection tubes to
prevent glycolysis by RBCs, ensuring accurate glucose measurement.
VIII. Rapoport-Luebering Shunt (2,3-BPG Shunt)

• Location: RBCs only.

• Pathway: A bypass of the PGK step (~10-20% of glycolytic flux in RBCs).
◦ 1,3-BPG -> 2,3-Bisphosphoglycerate (2,3-BPG)
▪ Enzyme: BPG Mutase
◦ 2,3-BPG -> 3-Phosphoglycerate (3-PG) + Pi
▪ Enzyme: BPG Phosphatase
• Significance:
◦ Produces 2,3-BPG, which decreases hemoglobin's affinity for oxygen (shifts ODC curve to the right),
facilitating O2 release to tissues.
◦ Energetic Cost: Bypasses the PGK ATP-generating step. Glycolysis via this shunt yields 0 net ATP.
IX. Regulation of Glycolysis

• Key Regulatory Enzymes (Irreversible Steps):

1. Hexokinase/Glucokinase
2. PFK-1 (Rate-limiting)
3. Pyruvate Kinase
4. Mnemonic: "How Glycolysis Pushes Forward" (Hexokinase, Glucokinase, PFK-1, Pyruvate Kinase)
• (A) Hormonal Regulation (via Covalent Modification of PFK-2):
◦ Fed State (High Insulin/Low Glucagon):
▪ Insulin promotes dephosphorylation.
▪ PFK-2 is dephosphorylated & active (kinase activity dominant).
 ▪ High F2,6BP produced.
▪ PFK-1 is strongly activated.
▪ Glycolysis ON.
◦ Fasting State (Low Insulin/High Glucagon):
▪ Glucagon (via cAMP/PKA) promotes phosphorylation.
▪ PFK-2 is phosphorylated & inactive (phosphatase activity dominant).
▪ Low F2,6BP.
▪ PFK-1 activity decreases.
▪ Glycolysis OFF.
• (B) Allosteric Regulation:
◦ Hexokinase: Inhibited by product G6P.
◦ PFK-1:
▪ Activators: F2,6BP (most important!), AMP (signals low energy), F6P (substrate).
▪ Inhibitors: ATP (signals high energy), Citrate (signals TCA cycle is full), Low pH (H+, signals
lactate buildup).
▪ Mnemonic: "AMP Up, ATP/Citrate Down" for PFK-1 activity.
◦ Pyruvate Kinase:
▪ Inhibitors: ATP (signals high energy). (Feed-forward activation by F1,6BP also occurs but wasn't
detailed in the lecture text).
X. Clinical Correlations Recap

• Hemolysis: Enzyme defects -> Low ATP in RBCs -> Membrane instability.
• Muscle Fatigue: Enzyme defects -> Low ATP during exercise.
• Cardiac Ischemia Sensitivity: Low glycolytic capacity.
• Hypoxia & Consciousness: Brain's reliance on aerobic glucose oxidation.
• HIF-1: Hypoxia Inducible Factor 1 increases glycolytic enzyme transcription during low O2.
XI. Important Points to Remember

• Glycolysis occurs in the cytoplasm of all cells.

• It functions aerobically (-> Pyruvate -> Acetyl-CoA) and anaerobically (-> Lactate).
• Irreversible / Regulatory Steps: Hexokinase/Glucokinase, PFK-1, Pyruvate Kinase.
• PFK-1 is the rate-limiting and committed step.
• ATP Generation:
◦ Substrate-Level Phosphorylation (SLP): PGK and PK steps.
◦ Oxidative Phosphorylation: From NADH produced at G3PDH step (aerobic only).
• Net ATP: 7 (Aerobic), 2 (Anaerobic). Complete Oxidation: 32.
• Anaerobic Glycolysis is vital for RBCs (2 ATP net) and regenerates NAD+ via LDH.
• 2,3-BPG shunt in RBCs facilitates O2 unloading but yields no net ATP.
• Key Regulators: Insulin/Glucagon ratio (via F2,6BP), ATP/AMP ratio, Citrate.
• Inhibitors: Fluoride (Enolase - clinical use), Arsenate (bypasses PGK ATP generation).
Applied Aspects of Glycolysis Summary
I. Glycolysis and Cancer

1. Warburg Hypothesis (Otto Warburg, 1924):

◦ Cancer cells exhibit increased glucose uptake compared to normal cells.
◦ Mechanism:
▪ Normal Aerobic Path: Glucose -> Pyruvate -> Acetyl-CoA -> TCA Cycle -> ~32 ATP.
▪ Cancer Cells (Even with O2): Preferentially convert Glucose -> Lactate (Aerobic Glycolysis).
Yields only 2 ATP.
◦ Consequences:
▪ High Glucose Demand: To compensate for low ATP yield (2 vs 32 ATP), cancer cells
dramatically increase glucose uptake.
▪ Acidic Microenvironment: Lactate accumulation around cancer cells creates acidity.
▪ Hypermetabolic State & Cancer Cachexia:
▪ Lactate needs conversion back to glucose via gluconeogenesis, which requires 6 ATP.
▪ This cycle (low ATP production + high ATP consumption for gluconeogenesis/growth) leads
to a net energy deficit and a hypermetabolic state, contributing to cancer cachexia.
(Mnemonic: Warburg = Wasteful Aerobic Route, Builds Up Rotten (Glucose to Lactate))
2. Metabolic Reprogramming (The "Why"):
◦ Explains the shift to lactate production in cancer cells.
◦ Key Enzyme: Pyruvate Kinase (PK)
▪ Normal Cells: Express Pyruvate Kinase M1 (PKM1).
▪ Tetrameric form.
▪ High catalytic activity.
▪ Favors conversion of phosphoenolpyruvate (PEP) to pyruvate, which then enters the TCA
cycle (as Acetyl-CoA).
▪ Cancer Cells: Express Pyruvate Kinase M2 (PKM2).
▪ Dimeric form.
▪ Low catalytic activity.
▪ Slows down the final step of glycolysis, leading to pyruvate being preferentially converted to
lactate (even in the presence of oxygen - hence aerobic glycolysis).
◦ End Product: The main end product of glucose metabolism in cancer cells is Lactate.
3. HIF-1 (Hypoxia-Inducible Factor 1):
◦ Induced by cancer.
◦ Increases transcription of:
▪ Glycolytic enzymes.
▪ Vascular Endothelial Growth Factor (VEGF), promoting angiogenesis (new blood vessel
formation).

II. PET Scanning & Fluorodeoxyglucose (FDG)

1. FDG (2-Fluoro-deoxyglucose):
◦ A harmless glucose analogue labeled with Fluorine-18 (a positron emitter).
2. Mechanism:
◦ Cancer cells avidly take up glucose, and thus also take up FDG via glucose transporters (GLUTs).
Concentration inside cancer cells can be ~10x higher than normal.
◦ Hexokinase phosphorylates FDG to Phospho-fluoro-deoxyglucose.
◦ Crucial Point: Phospho-fluoro-deoxyglucose cannot be further metabolized in glycolysis.
◦ Accumulation: It gets trapped and accumulates inside cancer cells.
3. Detection (PET Scan):
◦ The Fluorine-18 in accumulated FDG decays, emitting positrons.
◦ Positron Emission Tomography (PET) detects these emissions.
◦ Allows for pinpointing the location of cancer cells/tumors due to their high metabolic activity and
FDG uptake. (Mnemonic: FDG = Finds Disease (Glucose-hungry cells))

III. Glycolysis Inhibitors as Chemotherapeutics

1. Rationale: Cancer cells heavily depend on glycolysis for survival and energy.
2. Strategy: Inhibit key glycolytic enzymes to starve cancer cells.
3. Agents Under Trial:
◦ 2-Deoxyglucose (2-DG): Inhibits Hexokinase.
◦ Lonidamine
◦ 3-Bromopyruvate
4. Goal: Blocking glycolysis leads to cancer cell death.

IV. Metabolic Defects in Glycolysis (Enzyme Deficiencies)

1. Pyruvate Kinase (PK) Deficiency:

◦ Second most common enzyme defect causing hemolytic anemia (after G6PD deficiency - implied, not
explicitly stated as #1).
◦ Impact: Affects Red Blood Cells (RBCs) most severely, as they rely solely on anaerobic glycolysis
for ATP.
◦ Mechanism:
▪ Defective PK -> Decreased ATP production in RBCs.
▪ ATP needed for ion pumps (e.g., maintaining Na+/K+ gradient).
▪ Pump failure -> Ion imbalance (Ca2+ influx, K+ efflux mentioned) -> Cell swelling -> Hemolysis
-> Hemolytic Anemia.
◦ Compensation: Pathway shifts slightly, increasing 2,3-Bisphosphoglycerate (2,3-BPG) via the
Rapoport-Luebering shunt. Higher 2,3-BPG helps RBCs unload oxygen more efficiently to tissues,
partially compensating for the anemia. (Mnemonic: Low PK -> Low ATP -> RBCs Pop!)
2. Aldolase A Deficiency:
◦ Also results in hemolysis.
3. Muscle Phosphofructokinase-1 (PFK-1) Deficiency:
◦ Causes exercise intolerance. (Also known as Tarui Disease - additional info for context)

V. Maturity Onset Diabetes of the Young (MODY) Type 2

1. Enzyme Involved: Glucokinase (GCK) in pancreatic β-cells.

 2. Normal Function of GCK:
◦ Acts as a "glucose sensor" due to its high Km (low affinity) - only highly active when blood glucose
is high.
◦ In β-cells: High Glucose -> Enters via GLUT2 -> GCK phosphorylates Glucose -> Glycolysis ->
ATP levels rise.
◦ Increased ATP closes ATP-sensitive K+ channels -> Depolarization -> Ca2+ influx -> Insulin
secretion.
3. MODY 2 Defect:
◦ Mutation alters the kinetic properties of Glucokinase.
◦ Impaired glucose sensing: GCK doesn't respond appropriately to high glucose.
◦ Insufficient ATP generation in β-cells even during hyperglycemia.
◦ Result: Failure to secrete adequate insulin in response to high blood glucose.

Key Takeaways / Important Points to Remember:

• Cancer & Glycolysis: Cancer cells prefer aerobic glycolysis (Warburg effect), producing lactate even
with oxygen, leading to high glucose uptake, acidosis, and cachexia. This is due to PKM2 isoform
dominance.
• FDG-PET: Uses cancer's high glucose uptake. FDG is taken up, phosphorylated by hexokinase, then
trapped, allowing tumor localization via positron emission.
• Chemotherapy Target: Inhibiting glycolysis (e.g., with 2-DG targeting hexokinase) is a potential anti-
cancer strategy.
• PK Deficiency: Causes hemolytic anemia due to ATP depletion in RBCs. Compensated partly by
increased 2,3-BPG.
• MODY 2: Caused by defective Glucokinase kinetics in pancreatic β-cells, impairing glucose sensing
and insulin secretion.
• Aerobic Glycolysis vs Anaerobic Glycolysis: Remember cancer uses aerobic glycolysis (lactate
production despite oxygen). RBCs rely on anaerobic glycolysis (no mitochondria).

Pyruvate Dehydrogenase (PDH) Reaction Summary

1. Introduction & Core Reaction

• Function: Converts Pyruvate (3-carbon) to Acetyl-CoA (2-carbon).

• Nature: A crucial "Link Reaction" connecting Glycolysis and the TCA cycle.
• Key Questions Addressed:
◦ Why excess carbohydrate is stored as fat.
◦ Why fat generally cannot be converted back to glucose.
◦ Why ATP production is low in chronic alcoholics.
◦ Clinical case of PDH deficiency leading to metabolic acidosis and neurological issues.
2. Fates of Pyruvate (Metabolic Junction)

• Pyruvate can be converted to:

◦ Lactate: Enzyme: Lactate Dehydrogenase (LDH). Coenzyme: NADH -> NAD+. (Seen in
Glycolysis)
 ◦ Oxaloacetate (4C): Enzyme: Pyruvate Carboxylase. Requires: ATP, Biotin (Vitamin B7).
(Carboxylation)
◦ Alanine (Amino Acid): Enzyme: Alanine Aminotransferase (ALT). Requires: PLP (Pyridoxal
Phosphate - Vitamin B6). (Transamination)
◦ Acetyl-CoA (2C): Enzyme Complex: Pyruvate Dehydrogenase (PDH). Requires 5 coenzymes.
(Oxidative Decarboxylation - Focus of this lecture)
3. The Pyruvate Dehydrogenase (PDH) Complex

• Location: Mitochondrial Matrix.

• Pyruvate Transport: Pyruvate (formed in cytoplasm via Glycolysis) enters mitochondria via a Proton
Symporter in the inner mitochondrial membrane.
• Structure: A Multi-enzyme Complex containing:
◦ Three Enzymes:
▪ E1: Pyruvate Dehydrogenase (uses TPP)
▪ E2: Dihydrolipoyl Transacetylase (uses Lipoamide & Coenzyme A)
▪ E3: Dihydrolipoyl Dehydrogenase (uses FAD & NAD+)
◦ Five Coenzymes:
▪ Thiamine Pyrophosphate (TPP - from Vitamin B1)
▪ Lipoamide (derived from Lipoic Acid)
▪ Coenzyme A (CoA - contains Pantothenate/Vitamin B5)
▪ Flavin Adenine Dinucleotide (FAD - from Riboflavin/Vitamin B2)
▪ Nicotinamide Adenine Dinucleotide (NAD+ - from Niacin/Vitamin B3)
▪ Mnemonic for Coenzymes: Tender Loving Care For Nancy
• Similarity to Other Complexes:
◦ Alpha-ketoglutarate Dehydrogenase (TCA Cycle)
◦ Branched-Chain Keto Acid Dehydrogenase (Branched-Chain Amino Acid Catabolism)
◦ Common Features: Multi-enzyme structure, require the same 5 coenzymes, share common E2 & E3
subunits (a defect in E2/E3 affects all three!), perform Oxidative Decarboxylation.
4. PDH Reaction Mechanism Steps

1. Decarboxylation (E1): Pyruvate releases CO2. The remaining 2-carbon unit attaches to TPP, forming
Hydroxyethyl-TPP.
2. Oxidation & Transfer (E1/E2): The Hydroxyethyl group is oxidized and transferred from TPP to the
Oxidized Lipoamide (on E2), forming Acetyl-Lipoamide. TPP is regenerated.
3. Acetyl-CoA Formation (E2): The Acetyl group is transferred from Acetyl-Lipoamide to Coenzyme A,
forming Acetyl-CoA (the 2C product). Lipoamide is left in a Reduced state.
4. Lipoamide Regeneration (E3): Reduced Lipoamide is re-oxidized by transferring electrons to FAD
(bound to E3), forming FADH2. Oxidized Lipoamide is regenerated for the next cycle.
5. FAD Regeneration & NADH Production (E3): FADH2 (on E3) transfers electrons to NAD+, forming
NADH + H+ and regenerating FAD. NADH can enter the Electron Transport Chain.
5. Overall Net Reaction

• Pyruvate + CoA + NAD+ --> Acetyl-CoA + CO2 + NADH + H+

• This is an irreversible oxidative decarboxylation.
6. ATP Yield

• Each NADH produced yields ~2.5 ATP via the Electron Transport Chain.
• From 1 molecule of Glucose (yields 2 Pyruvate), the PDH reaction generates 2 NADH, contributing ~5
ATP.
7. Regulation of PDH

• Activation (Promotes Acetyl-CoA formation):

◦ Condition: Well-fed state, high Insulin/Glucagon ratio.
◦ Mechanism: PDH is DEPHOSPHORYLATED (Active form). This is facilitated by a phosphatase
enzyme (stimulated indirectly by insulin).
• Inhibition (Slows Acetyl-CoA formation):
◦ Condition: High energy state / Product Accumulation.
◦ Inhibitors:
▪ High ATP/ADP ratio
▪ High Acetyl-CoA/CoA ratio
▪ High NADH/NAD+ ratio
◦ Mechanism: These products activate a kinase enzyme that PHOSPHORYLATES PDH (Inactive
form).
8. Significance of PDH

• Biochemical Significance:
◦ Irreversible Step: Acts like a one-way valve, committing pyruvate carbon to Acetyl-CoA or
downstream pathways (TCA, fatty acid synthesis).
◦ Acetyl-CoA Cannot Form Glucose: Because PDH is irreversible, Acetyl-CoA (e.g., from fatty acid
breakdown) cannot be converted back to pyruvate for gluconeogenesis.
◦ Fat Cannot Be Converted to Glucose (Mostly): Direct consequence of the point above. Fatty acids
break down to Acetyl-CoA.
▪ Exceptions: Glycerol backbone of Triacylglycerols, Propionyl-CoA from odd-chain fatty acids
CAN contribute to gluconeogenesis.
◦ Excess Carbohydrate -> Fat: Glucose -> Pyruvate -> (irreversible PDH) -> Acetyl-CoA. In energy
surplus (high ATP, NADH), TCA cycle slows, excess Acetyl-CoA is directed to Fatty Acid Synthesis
-> Triacylglycerol storage in Adipose Tissue. (Lack of this reversal in animals explains adipose tissue;
plants have alternative pathways).
◦ Link Reaction: Irreversibly connects Glycolysis (cytoplasmic) to the TCA Cycle (mitochondrial).
• Clinical Significance:
◦ Chronic Alcoholism & ATP Depletion:
▪ Alcohol reduces Thiamine (B1) absorption -> Less TPP -> Decreased PDH & Alpha-ketoglutarate
Dehydrogenase activity -> Less NADH -> Less ATP.
▪ Associated general nutritional deficiencies (lack of B vitamins B1, B2, B3, B5 needed for PDH
coenzymes) exacerbate low ATP production.
◦ PDH Complex Deficiency:
▪ Genetic metabolic disorder, most commonly affecting the E1 subunit.
▪ Consequences:
▪ Less Pyruvate converted to Acetyl-CoA.
 ▪ Pyruvate accumulation -> shunted to Lactate -> Lactic Acidosis (especially after glucose
intake).
▪ Reduced Acetyl-CoA for TCA cycle -> Impaired oxidative metabolism -> Reduced ATP
production.
▪ Brain Affected: The brain relies heavily on oxidative metabolism; deficiency leads to
neurological problems (e.g., psychomotor disability).
9. Important Points to Remember:

• Irreversibility: The PDH reaction is physiologically irreversible. This is key to understanding why fat
cannot become glucose and why excess carbs become fat.
• Location: Mitochondria (requires pyruvate transport).
• Regulation: Activated by dephosphorylation (low energy signals/insulin), inhibited by phosphorylation
(high energy signals/products - ATP, Acetyl-CoA, NADH).
• Coenzymes: Requires 5 coenzymes derived from B vitamins (B1, B2, B3, B5) plus Lipoamide.
Deficiencies (e.g., alcoholism) impair function.
• Clinical Relevance: Deficiency leads to lactic acidosis and severe neurological defects due to impaired
energy metabolism in the brain.
• Link Role: Connects glycolysis output (pyruvate) to TCA cycle input (acetyl-CoA).

Glycogen Metabolism Summary

I. Introduction & Key Questions

1. Fate of Glucose Load: What happens when a large glucose load is ingested by a normal person in a
basal state? (Answer: Primarily Glycogen Synthesis)
2. Blood Glucose Maintenance: How is a steady state of blood glucose maintained to prevent
hypoglycemia, coma, and seizures? (Answer: Liver Glycogenolysis)
3. Glycogen Storage Rationale: Why is glucose stored as glycogen? (Answer: Compactness, Rapid
Mobilization, Osmotic Advantage)
II. Glycogen Structure & Storage

• Definition: Glycogen is a branched polymer of alpha-D-glucose.

• Linkages:
◦ Linear chain: alpha-1,4 glycosidic bonds. (Anomeric carbon 1 of one glucose linked to carbon 4 of
the next).
◦ Branch points: alpha-1,6 glycosidic bonds. (Occur typically every 8-12 residues).
• Storage Form: Stored in the cytoplasm as cytoplasmic granules (often visible as "alpha rosettes").
◦ Alpha Rosette: Composed of 20-40 "beta particles".
◦ Beta Particle:
▪ Diameter: ~21 nm.
▪ Contains ~55,000 glucose residues.
▪ Has ~2,000 non-reducing ends (ends without a free anomeric carbon, usually C4-OH available).
▪ Has only one reducing end (with a free anomeric carbon, C1-OH), typically attached to a protein
primer called Glycogenin.
 • Significance of Structure:
◦ Multiple Non-Reducing Ends: Allow enzymes (for synthesis and degradation) to act simultaneously
at many points, enabling rapid glucose release or storage.
• Reasons for Storing Glucose as Glycogen:
1. Compact: Efficient storage.
2. Rapid Mobilization: Due to multiple non-reducing ends.
3. Osmotic Pressure: Storing the equivalent amount of free glucose would result in a very high
intracellular concentration (~4 Molar according to the lecture), drawing excessive water into the cell
and causing lysis. Glycogen has a much lower molarity (~0.01 micromolar), preventing this osmotic
problem.
III. Glycogen Synthesis (Glycogenesis)

• Physiological State: Occurs during the fed state (postprandial state, ~4-6 hours after a meal) when blood
glucose is high.
• Hormonal Control: Stimulated by Insulin (High Insulin:Glucagon ratio).
• Primary Sites:
◦ Liver: Stores glycogen up to ~10% of its weight. Primarily for maintaining blood glucose.
◦ Muscle: Stores glycogen up to 1-2% of its weight. Primarily for its own energy needs during
exercise.
◦ Note: Muscle has the highest total glycogen content due to greater mass, but liver has the highest
percentage by tissue weight.
• Cellular Location: Cytoplasm.
• Steps:
1. Synthesis of UDP-Glucose (The Glucose Donor):
▪ Glucose + ATP → Glucose-6-Phosphate (G6P) + ADP (Enzyme: Hexokinase [muscle/most
tissues] or Glucokinase [liver])
▪ G6P ⇌ Glucose-1-Phosphate (G1P) (Enzyme: Phosphoglucomutase - an isomerase/mutase)
▪ G1P + UTP → UDP-Glucose + PPi (Pyrophosphate) (Enzyme: UDP-glucose
pyrophosphorylase)
▪ Note: Hydrolysis of PPi drives this reaction forward. UTP is the key nucleotide.
2. Action of Glycogen Synthase (Rate-Limiting Enzyme):
▪ Requires a primer. The protein Glycogenin acts as the primer by auto-glycosylating itself with
the first ~7-8 glucose residues (from UDP-glucose) onto a tyrosine residue (no enzyme needed for
initial attachment).
▪ Glycogen Synthase then elongates the chain by adding glucose units (from UDP-glucose) via
alpha-1,4 linkages to the non-reducing end of the primer or existing chain.
▪ Analogy: Like DNA Polymerase, Glycogen Synthase needs a primer.
3. Formation of Branches:
▪ Enzyme: Branching Enzyme (Amylo-α(1,4)→α(1,6)-transglucosylase or Glucan transferase)
▪ Action: Transfers a block of ~6 glucose residues from the non-reducing end of a chain (that is at
least 11 residues long) from an alpha-1,4 linkage to form an alpha-1,6 linkage further down the
same or another chain (must be at least 4 residues away from a pre-existing branch point).
▪ Result: Creates a branch point, increasing the number of non-reducing ends for faster synthesis/
degradation and increasing solubility.
IV. Glycogen Degradation (Glycogenolysis)

• Physiological State: Occurs during fasting (post-absorptive state, ~4-16 hours after a meal, "early
fasting"), stress, or exercise.
• Hormonal Control: Stimulated by Glucagon (liver only) and Epinephrine (liver and muscle). (Low
Insulin:Glucagon ratio).
• Primary Sites: Liver and Muscle.
◦ Liver: Provides free glucose to maintain blood glucose levels. (Considered "selfless").
◦ Muscle: Provides G6P for glycolysis within the muscle cell for ATP production. (Cannot release free
glucose into blood).
• Cellular Location: Primarily Cytoplasm. Minor pathway (~1-2%) in lysosomes. Role for Smooth
Endoplasmic Reticulum (SER) in the liver.
• Steps:
1. Action of Glycogen Phosphorylase (Rate-Limiting Enzyme):
▪ Requires Pyridoxal Phosphate (PLP / Vitamin B6) as a coenzyme.
▪ Action: Cleaves alpha-1,4 glycosidic bonds from the non-reducing ends by adding inorganic
phosphate (Pi) – a process called phosphorolysis.
▪ Product: Glucose-1-Phosphate (G1P). (Advantage: Glucose is already phosphorylated, saving 1
ATP in glycolysis).
▪ Stops cleaving 4 glucose residues away from an alpha-1,6 branch point. The remaining structure
is called a "limit dextrin".
2. Removal of Branches by Debranching Enzyme:
▪ A bifunctional enzyme with two activities:
▪ α(1→4) Glucanotransferase: Transfers 3 of the 4 glucose residues from the limit branch
(oligosaccharide) to the non-reducing end of another chain via an alpha-1,4 linkage.
▪ α(1→6) Glucosidase: Hydrolyzes the single remaining glucose residue attached at the
alpha-1,6 branch point.
▪ Product of glucosidase activity: Free Glucose (a minor fraction of the total glucose released from
glycogen).
3. Conversion of G1P:
▪ G1P ⇌ G6P (Enzyme: Phosphoglucomutase - same enzyme as in synthesis).
▪ Fate of G6P:
▪ In Liver:
▪ G6P is transported into the Smooth Endoplasmic Reticulum (SER) via transporter T1.
▪ Inside the SER, Glucose-6-Phosphatase hydrolyzes G6P to Free Glucose + Pi.
▪ Glucose exits the SER via transporter T2 (and potentially T3 is involved with Pi
transport).
▪ Free glucose exits the liver cell into the bloodstream via GLUT transporters.
▪ In Muscle (and Adipose Tissue):
▪ Lacks Glucose-6-Phosphatase.
▪ G6P cannot be converted to free glucose.
▪ G6P enters Glycolysis directly within the muscle cell to generate ATP for muscle activity.
▪ ATP Yield: Anaerobic glycolysis from glycogen-derived G6P yields 3 ATP (net), because
the ATP-consuming Hexokinase step is bypassed (compared to 2 ATP net from free
glucose).
V. Regulation of Glycogen Metabolism

• Coordinated Control: Synthesis and degradation pathways are reciprocally regulated to prevent futile
cycling.
• Mechanisms: Hormonal (via covalent modification - phosphorylation/dephosphorylation) and Allosteric.
• A. Hormonal Regulation:
◦ Fed State (High Glucose, High Insulin):
▪ Insulin dominates.
▪ Activates Phosphodiesterase (breaks down cAMP).
▪ Activates Protein Phosphatases (like PP1, implied).
▪ Result:
▪ Glycogen Synthase is DEphosphorylated → ACTIVE.
▪ Glycogen Phosphorylase is DEphosphorylated → INACTIVE.
▪ Favors: Glycogen SYNTHESIS.
◦ Fasting/Stress/Exercise (Low Glucose, High Glucagon/Epinephrine):
▪ Glucagon (liver) / Epinephrine (liver & muscle) dominate.
▪ Bind GPCRs → Activate Adenylyl Cyclase → ↑ cAMP.
▪ cAMP activates Protein Kinase A (PKA).
▪ PKA PHOSPHORYLATES target enzymes (directly or indirectly via phosphorylase kinase):
▪ Glycogen Synthase is PHOSPHORYLATED → INACTIVE.
▪ Glycogen Phosphorylase is PHOSPHORYLATED → ACTIVE.
▪ Result:
▪ Favors: Glycogen DEGRADATION (Glycogenolysis).
▪ Mnemonic Idea: Phosphorylation Promotes Phosphorylase (breakdown) activity when Problems
(fasting/stress) arise.
• B. Muscle-Specific Regulation:
◦ Neural Stimulation / Exercise:
▪ Ca2+ released from Sarcoplasmic Reticulum.
▪ Ca2+ binds Calmodulin, activating Ca2+/Calmodulin-dependent protein kinase.
▪ Ca2+-Calmodulin complex also directly activates Phosphorylase Kinase (even without PKA
phosphorylation).
▪ Result: Activates Glycogen Phosphorylase → promotes Glycogenolysis for muscle energy.
◦ High Energy Demand (Anoxia / Strenuous Exercise):
▪ ATP hydrolysis leads to increased AMP (5'-AMP).
▪ AMP acts as an allosteric activator of Muscle Glycogen Phosphorylase, activating it even
without phosphorylation.
• C. Allosteric Regulation: Fine-tuning based on cellular energy status and substrate availability.
◦ Liver:
▪ Glycogen Synthase (Synthesis): Activated (+) by G6P.
▪ Glycogen Phosphorylase (Breakdown): Inhibited (-) by ATP, G6P, Glucose.
◦ Muscle:
▪ Glycogen Synthase (Synthesis): Activated (+) by G6P.
 ▪ Glycogen Phosphorylase (Breakdown):
▪ Activated (+) by Ca2+, AMP.
▪ Inhibited (-) by ATP, G6P.
▪ (Note: Glucose is NOT an allosteric inhibitor in muscle).
VI. Summary of Initial Questions

• Glucose Load Fate: Stored as Glycogen (Glycogenesis stimulated by insulin).

• Blood Glucose Maintenance: Primarily by Liver Glycogenolysis (stimulated by glucagon/epinephrine,
releasing free glucose via Glucose-6-Phosphatase).
• Why Glycogen: Compact storage, allows rapid glucose mobilization (many non-reducing ends), avoids
osmotic lysis.

Key Points to Remember:

• Rate-Limiting Enzymes: Glycogen Synthase (Synthesis) and Glycogen Phosphorylase (Degradation).

• Hormonal Control: Insulin promotes synthesis (dephosphorylation makes synthase active). Glucagon/
Epinephrine promote degradation (phosphorylation makes phosphorylase active).
• Liver vs. Muscle:
◦ Liver maintains blood glucose (has Glucose-6-Phosphatase). Regulated by Glucagon & Epinephrine.
Allosterically inhibited by Glucose.
◦ Muscle provides glucose for its own energy (lacks Glucose-6-Phosphatase). Regulated by
Epinephrine & Ca2+/AMP. Not inhibited allosterically by Glucose.
• Key Enzyme Cofactor: Glycogen Phosphorylase requires PLP (Vitamin B6).
• Branching & Debranching: Branching enzyme creates α-1,6 links; Debranching enzyme breaks α-1,6
links (glucosidase activity) and transfers α-1,4 linked units (transferase activity).
• UDP-Glucose: The activated glucose donor for synthesis.
• G1P vs. G6P: Interconverted by Phosphoglucomutase. G1P is the product of phosphorylase; G6P is the
entry point to glycolysis (muscle) or substrate for G6Pase (liver).
• ATP Yield in Muscle: Glycogenolysis + Anaerobic Glycolysis = 3 ATP net per glucose residue.
• Osmotic Advantage: Crucial reason for storing glucose as a polymer.

Glycogen Storage Disorders (GSDs) Summary

I. Introduction

• Definition: A group of disorders associated with defects in the metabolism of glycogen.

• Classification:
◦ Liver GSDs: Primarily affect the liver's ability to maintain blood glucose.
◦ Muscle GSDs: Primarily affect the muscle's ability to use glycogen for energy during exercise.
II. Core Differences: Liver vs. Muscle GSDs

Feature Liver GSD Muscle GSD

Primary Inability to release glucose into blood Inability to utilize glycogen for muscle
Defect energy
 Feature Liver GSD Muscle GSD

Blood Fasting Hypoglycemia (Liver function: supply blood glucose Normoglycemia (Muscle glycogen not for
Glucose during early fasting, 4-16 hrs) blood glucose)

Exercise No exercise intolerance Exercise Intolerance (Muscle needs

glycogen for exercise)

III. Types of GSDs

• A. Liver GSDs:
◦ Type 1 (Von Gierke's Disease):
▪ Type 1a: Most common GSD. Defect in Glucose-6-Phosphatase.
▪ Type 1b: Features of Type 1a + Neutropenia (leading to recurrent infections).
◦ Type 3 (Cori's Disease / Forbes Disease / Limit Dextrinosis): Defect in Debranching Enzyme.
◦ Type 4 (Andersen's Disease / Amylopectinosis): Defect in Branching Enzyme.
◦ Type 6 (Hers' Disease): Defect in Hepatic Glycogen Phosphorylase.
• B. Muscle GSDs:
◦ With Hypertrophic Cardiomyopathy (HCM):
▪ Type 2 (Pompe's Disease): Defect in Lysosomal alpha-1,4-glucosidase (Acid Maltase).
▪ Danon Disease: Defect in Lysosomal Associated Membrane Protein 2 (LAMP2). (Less
common)
◦ Without Hypertrophic Cardiomyopathy:
▪ Type 5 (McArdle's Disease): Defect in Muscle Glycogen Phosphorylase. Most common
muscle GSD in adolescents.
▪ Type 7 (Tarui's Disease): Defect in Muscle & Erythrocyte Phosphofructokinase-1 (PFK-1).
• C. Other GSDs:
◦ Type 0: Defect in Glycogen Synthase. (Usually fatal early).
◦ Fanconi-Bickel Syndrome: Defect in Glucose Transporter 2 (GLUT2). (Recently added).
IV. Detailed GSD Profiles

• Type 1 (Von Gierke's Disease - G6Pase Defect):

◦ Mnemonic: Von ~ One.
◦ Biochemical Hallmarks: (Due to G6Pase block in both glycogenolysis & gluconeogenesis)
▪ Severe Fasting Hypoglycemia: Cannot release free glucose from liver.
▪ Lactic Acidosis: Excess Glucose-6-Phosphate (G6P) shunted to glycolysis -> pyruvate -> lactate.
▪ Hyperuricemia: G6P shunted to HMP pathway -> ↑ Pentoses -> ↑ Purine breakdown -> ↑ Uric
Acid.
▪ Hyperlipidemia: Acetyl-CoA (from pyruvate) cannot enter TCA (OAA depleted by
gluconeogenesis) -> diverted to Fatty Acid & Triacylglycerol synthesis.
▪ Ketosis: Acetyl-CoA diverted to ketone body synthesis (alternative fuel for brain).
◦ Clinical Features: Doll-like facies (chubby cheeks, thin extremities), Protruding abdomen
(Hepatomegaly + Renomegaly, No Splenomegaly), Hypoglycemia symptoms.
◦ Diagnosis: Labs reflect hallmarks (↓Glucose, ↑Lactate, ↑Uric Acid, ↑Ketones, ↑TAGs), USG shows
hepato/renomegaly. IV Glucagon challenge (see below).
 ◦ Type 1b: All Type 1a features + Neutropenia & recurrent infections.
• Type 3 (Cori's Disease - Debranching Enzyme Defect):
◦ Mnemonic: Cori's / Debranching.
◦ Biochemical Defect: Accumulation of Limit Dextrin (abnormally structured glycogen).
◦ Clinical Features: Fasting hypoglycemia (milder than Type 1 as gluconeogenesis intact & outer
branches usable), Hepatomegaly + Splenomegaly (No Renomegaly), Micronodular liver cirrhosis
(often reversible post-puberty).
◦ Diagnosis: Ketosis present, elevated liver enzymes. No significant ↑Lactate or ↑Uric Acid. IV
Glucagon challenge (see below).
• Type 4 (Andersen's Disease - Branching Enzyme Defect):
◦ Mnemonic: Andersen's / Branching.
◦ Biochemical Defect: Accumulation of abnormal, amylopectin-like glycogen (long, unbranched
chains).
◦ Clinical Features: Fatal condition. Hypoglycemia, Ketosis. Progressive micronodular cirrhosis ->
Portal hypertension, Esophageal varices, Liver failure & death (~5 years). Can have neurological
manifestations (brain, anterior horn cells).
◦ Diagnosis: Hypoglycemia, Ketosis, Elevated transaminases (ALT). No ↑Lactate or ↑Uric Acid.
Abnormal glycogen on electron microscopy. Enzyme studies.
• Type 6 (Hers' Disease - Hepatic Glycogen Phosphorylase Defect):
◦ Mnemonic: Hers' / Hepatic Phosphorylase.
◦ Clinical Features: Milder hypoglycemia (gluconeogenesis intact), Hepatomegaly.
• Type 2 (Pompe's Disease - Lysosomal Acid Maltase Defect):
◦ Classification: Muscle GSD with HCM; also a Lysosomal Storage Disorder.
◦ Clinical Features: Infantile onset. Feeding difficulties, Failure to thrive, Hypotonia (Floppy infant),
Massive Cardiomegaly (due to HCM) leading to cardiac failure & death (~2 years).
◦ Diagnosis: X-ray (Cardiomegaly), Elevated muscle enzymes (CK, AST, LDH).
• Type 5 (McArdle's Disease - Muscle Glycogen Phosphorylase Defect):
◦ Mnemonic: McArdle's / Muscle Phosphorylase.
◦ Clinical Features: Exercise intolerance (cramps, pain). Second Wind Phenomenon (initial pain ->
rest -> improved exercise). Rhabdomyolysis -> Myoglobinuria (Burgundy colored urine).
Normoglycemia.
• Type 7 (Tarui's Disease - Muscle & RBC PFK-1 Defect):
◦ Clinical Features: Exercise intolerance, Myoglobinuria (similar to Type 5). No Second Wind
Phenomenon. Additional feature: Hemolysis (due to RBC enzyme defect). Normoglycemia.
• Fanconi-Bickel Syndrome (GLUT2 Defect):
◦ Features: Proximal renal tubular acidosis, Impaired glucose utilization.
V. Diagnostic Workup & Differentiation

• Initial Clues:
◦ Fasting Hypoglycemia → Suspect Liver GSD.
◦ Exercise Intolerance → Suspect Muscle GSD.
 • Liver GSD Branch:
◦ Abnormal glycogen on EM → Type 3 or 4.
◦ No abnormal glycogen → Type 1 or 6.
◦ Severe symptoms (hypoglycemia, acidosis, hyperuricemia, hyperlipidemia) → Strongly suggest Type
1.
• Muscle GSD Branch:
◦ HCM present → Type 2 (Pompe's).
◦ HCM absent:
▪ Second Wind Phenomenon present → Type 5 (McArdle's).
▪ Second Wind Phenomenon absent (+/- hemolysis) → Type 7 (Tarui's).
• IV Glucagon Challenge (Differentiating Type 1 vs Type 3):
◦ Performed in Well-Fed State and Overnight Fast State.
◦ Well-Fed State:
▪ Normal: Glucagon causes ↑ Blood Glucose.
▪ Type 1 (G6Pase Defect): NO increase in blood glucose (final step blocked).
▪ Type 3 (Debranching Defect): YES, increase in blood glucose (outer branches released by
phosphorylase can yield glucose).
◦ Overnight Fast State:
▪ Type 1: NO increase in blood glucose.
▪ Type 3: NO significant increase (glycogenolysis already limited by defect, glucagon cannot
overcome this effectively in fasting).
◦ Key Use: Most useful in the well-fed state to distinguish Type 1 (no response) from Type 3
(response).
VI. Special Considerations

• Liver GSD with Myopathy: Type 3, Type 4.

• Liver GSD with Neurological Manifestations: Type 4 (Andersen's).
VII. Important Points & Mnemonics Summary

• Most Common GSD: Type 1a (Von Gierke's).

• Most Common Muscle GSD in Adolescents: Type 5 (McArdle's).
• Lysosomal Storage Disorder GSD: Type 2 (Pompe's).
• Severe Fasting Hypoglycemia + Lactic Acidosis + Hyperuricemia + Hyperlipidemia: Classic for
Type 1 (Von Gierke's).
• Exercise Intolerance + Second Wind Phenomenon + Myoglobinuria: Classic for Type 5 (McArdle's).
• Exercise Intolerance + Hemolysis + No Second Wind: Classic for Type 7 (Tarui's).
• Infantile Hypotonia + Massive Cardiomegaly: Classic for Type 2 (Pompe's).
• Fatal Progressive Cirrhosis: Type 4 (Andersen's).
• Mnemonic Recap:
◦ Von Gierke's = Type 1
◦ Andersen's = Branching enzyme defect (Type 4)
◦ Cori's = Debranching enzyme defect (Type 3)
◦ McArdle's = Muscle phosphorylase defect (Type 5)
 ◦ Hers' = Hepatic phosphorylase defect (Type 6)
◦ Pompe's = Predominantly affects the 'Pump' (Heart) & is lysosomal (Type 2)
• Key Enzyme Locations:
◦ Glucose-6-Phosphatase: Liver (affected in Type 1) - final step of glycogenolysis AND
gluconeogenesis.
◦ Debranching Enzyme: Liver & Muscle (affected in Type 3).
◦ Branching Enzyme: Liver & Muscle (affected in Type 4).
◦ Glycogen Phosphorylase: Separate Liver (Type 6) and Muscle (Type 5) isoforms.
◦ Acid Maltase (alpha-glucosidase): Lysosomes (affected in Type 2).
◦ PFK-1: Muscle & RBCs (affected in Type 7).

Gluconeogenesis Summary
1. Concept & Need

• What: Synthesis of glucose from non-carbohydrate precursors.

• Why: To maintain blood glucose levels during fasting, especially after glycogen stores are depleted.
◦ Glycogenolysis (Hepatic): Supplies glucose for the first ~14-18 hours of fasting.
◦ Gluconeogenesis: Becomes the primary source of blood glucose from ~16-18 hours onwards (during
prolonged fasting, e.g., 16-48 hours without food).
• Energy Source: Gluconeogenesis is an energy-requiring process. The ATP needed is supplied primarily
by fatty acid oxidation (beta-oxidation of fatty acids released from stored triacylglycerols).

2. Definition

• The process by which glucose is synthesized from non-carbohydrate substrates.

3. Substrates (Non-Carbohydrate Precursors)

• Glucogenic Amino Acids:

◦ Especially Alanine (the principal gluconeogenic amino acid).
◦ Supplied via the Cahill Cycle (Glucose-Alanine Cycle):
▪ Organs: Skeletal Muscle & Liver.
▪ Process: In muscle (during fasting), Glucose -> Pyruvate -> Alanine (via transamination). Alanine
travels to the liver. In liver, Alanine -> Pyruvate (via transamination) -> Glucose.
▪ Timing: Active during fasting.
• Lactate:
◦ Supplied via the Cori Cycle (Glucose-Lactate Cycle):
▪ Organs: Skeletal Muscle (exercising), RBCs & Liver.
▪ Process: In Muscle/RBCs, Glucose -> Pyruvate -> Lactate (anaerobic glycolysis). Lactate travels
to the liver. In liver, Lactate -> Pyruvate -> Glucose.
▪ Function: Prevents lactic acidosis in muscle/clears lactate from RBCs.
▪ Timing: Active during exercise (muscle) and continuously for RBCs.
• Glycerol:
◦ Released from triacylglycerol breakdown (lipolysis by hormone-sensitive lipase).
 ◦ Entry Pathway: Glycerol --(Glycerol Kinase, uses ATP)--> Glycerol 3-phosphate --(Glycerol 3-
phosphate Dehydrogenase)--> Dihydroxyacetone Phosphate (DHAP) -> enters gluconeogenesis/
glycolysis pathway.
• Propionyl CoA:
◦ Originates from odd-chain fatty acid oxidation.
◦ Entry Pathway:
1. Propionyl CoA (3C) + CO2 --(Propionyl CoA Carboxylase; requires Biotin, ATP)--> D-
Methylmalonyl CoA (4C).
2. D-Methylmalonyl CoA --(Racemase)--> L-Methylmalonyl CoA (4C).
3. L-Methylmalonyl CoA --(Methylmalonyl CoA Mutase; requires B12)--> Succinyl CoA (4C).
◦ Succinyl CoA enters the TCA cycle and can be converted to Oxaloacetate, which then enters
gluconeogenesis.
◦ Clinical Note: B12 deficiency impairs the mutase step, leading to accumulation of methylmalonic
acid in serum.
• Crucial Exclusion: Acetyl CoA is NEVER a substrate for gluconeogenesis. (While Oxaloacetate is an
intermediate, not a starting substrate, it is part of the pathway; Acetyl CoA cannot yield net glucose
synthesis in humans).

4. Site of Gluconeogenesis

• Organs: Primarily Liver (major), also Kidney (minor).

• Organelles: Cytoplasm and Mitochondria. The Smooth Endoplasmic Reticulum (SER) is also
involved (for the final step, similar to glycogenolysis).

5. Key Enzymes (Bypassing Irreversible Glycolysis Steps)

Gluconeogenesis largely reverses glycolysis, but requires specific enzymes to bypass the 3 irreversible steps
of glycolysis.

• Bypass 1: Pyruvate -> Phosphoenolpyruvate (PEP) (Circumvents Pyruvate Kinase)

◦ Requires two enzymes and occurs in Mitochondria + Cytoplasm.
◦ Pyruvate Carboxylase:
▪ Reaction: Pyruvate (3C) + CO2 -> Oxaloacetate (OAA) (4C)
▪ Location: Mitochondria
▪ Requirements: ATP, Biotin
▪ Regulation: First regulatory step. Allosterically activated by Acetyl CoA.
◦ Malate-Aspartate Shuttle: OAA cannot exit mitochondria directly. It's converted to Malate (using
NADH) or Aspartate (via AST), transported to the cytoplasm, and converted back to OAA
(regenerating NADH or via AST).
◦ PEP Carboxykinase (PEPCK):
▪ Reaction: Oxaloacetate (4C) -> Phosphoenolpyruvate (PEP) (3C) + CO2
▪ Location: Cytoplasm
▪ Requirements: GTP (energetically equivalent to ATP)
▪ Process: Involves decarboxylation and phosphorylation.
 • Bypass 2: Fructose 1,6-bisphosphate -> Fructose 6-phosphate (Circumvents PFK-1)
◦ Fructose 1,6-bisphosphatase:
▪ Reaction: Fructose 1,6-bisphosphate + H2O -> Fructose 6-phosphate + Pi
▪ Location: Cytoplasm
▪ Process: Hydrolysis (phosphatase, not kinase).
▪ Regulation: Regulatory step. Allosterically inhibited by Fructose 2,6-bisphosphate.
• Bypass 3: Glucose 6-phosphate -> Glucose (Circumvents Hexokinase/Glucokinase)
◦ Glucose 6-phosphatase:
▪ Reaction: Glucose 6-phosphate + H2O -> Glucose + Pi
▪ Location: Smooth Endoplasmic Reticulum (SER)
▪ Process: Hydrolysis.
▪ Note: This enzyme is also crucial for releasing free glucose from glycogenolysis in the liver.

6. Energy Requirement

• Synthesizing 1 molecule of Glucose from 2 molecules of Lactate (or Pyruvate) requires:

◦ 2 ATP at Pyruvate Carboxylase step (1 ATP per Pyruvate)
◦ 2 GTP (equivalent to 2 ATP) at PEPCK step (1 GTP per OAA)
◦ 2 ATP at 3-Phosphoglycerate Kinase step (reverse reaction, 1 ATP per 3-PG)
• Total: 6 high-energy phosphate bonds (4 ATP + 2 GTP) are consumed.
• Also requires 2 NADH (at Glyceraldehyde 3-phosphate Dehydrogenase step, reverse reaction).

7. Regulation

• Hormonal:
◦ Active under conditions of low Insulin : Glucagon ratio (fasting state).
◦ Glucagon promotes phosphorylation of regulatory enzymes, generally activating gluconeogenesis
(and inhibiting glycolysis).
• Allosteric:
◦ Activator: Acetyl CoA activates Pyruvate Carboxylase (signals availability of energy from fat
breakdown).
◦ Inhibitor: Fructose 2,6-bisphosphate inhibits Fructose 1,6-bisphosphatase (signals high glucose
availability/fed state).

8. Clinical Correlations / Applied Aspects

• Biguanides (e.g., Metformin):

◦ Use: Oral hypoglycemic agent.
◦ Mechanism: Inhibits Pyruvate Carboxylase -> decreases gluconeogenesis -> lowers blood glucose.
◦ Theoretical Concern: Lactic Acidosis. Inhibition of Pyruvate Carboxylase causes Pyruvate
accumulation, which is shunted to Lactate. OAA depletion also slows the TCA cycle, further
increasing Pyruvate -> Lactate conversion.
• Chronic Alcoholism + Fasting:
◦ Presentation: Hypoglycemia, confusion, sweating, seizures (due to severe hypoglycemia).
 ◦ Mechanism: Alcohol metabolism (Ethanol -> Acetaldehyde -> Acetate) generates excess NADH,
leading to a high NADH/NAD+ ratio. This inhibits gluconeogenesis by:
1. Shifting Pyruvate -> Lactate (uses NADH), depleting Pyruvate substrate.
2. Shifting mitochondrial OAA -> Malate (uses NADH).
3. Inhibiting cytoplasmic Malate -> OAA (requires NAD+), depleting OAA substrate for PEPCK.
◦ Result: Impaired glucose production during fasting -> severe hypoglycemia.
• Glucocorticoids (e.g., Prednisolone, Methylprednisolone):
◦ Use: Anti-inflammatory (e.g., Asthma exacerbation).
◦ Side Effect: Hyperglycemia (can present as polyuria, polydipsia, muscle weakness).
◦ Mechanism:
1. Increase the synthesis of gluconeogenic enzymes (e.g., PEPCK).
2. Promote muscle protein breakdown, releasing glucogenic amino acids (substrates).
◦ Result: Increased rate of gluconeogenesis -> hyperglycemia.
• Raw Egg Consumption:
◦ Issue: Raw egg white contains Avidin, which strongly binds Biotin.
◦ Result: Biotin deficiency inhibits Biotin-dependent carboxylases:
▪ Pyruvate Carboxylase
▪ Propionyl CoA Carboxylase
◦ Effect: Decreased gluconeogenesis.

Key Points to Remember:

• Purpose: Make glucose from non-carbs during fasting.

• Main Site: Liver.
• Key Substrates: Lactate, Alanine (and other glucogenic AAs), Glycerol, Propionyl CoA. (LAG-P).
• NEVER a Substrate: Acetyl CoA.
• Energy Cost: 6 ATP/GTP per glucose from pyruvate/lactate. Energy comes from Fatty Acid Oxidation.
• Key Enzymes (Bypass steps):
1. Pyruvate Carboxylase (Mitochondria, needs Biotin, ATP; activated by Acetyl CoA)
2. PEPCK (Cytoplasm, needs GTP)
3. Fructose 1,6-bisphosphatase (Cytoplasm; inhibited by Fructose 2,6-bisphosphate)
4. Glucose 6-phosphatase (SER)
• Regulation: Stimulated by Glucagon (low I:G ratio) & Acetyl CoA. Inhibited by Insulin (high I:G
ratio) & Fructose 2,6-bisphosphate.
• Clinical Links:
◦ Alcohol inhibits via high NADH/NAD+ ratio -> Hypoglycemia.
◦ Glucocorticoids induce enzymes & provide substrates -> Hyperglycemia.
◦ Biguanides inhibit Pyruvate Carboxylase -> Hypoglycemia (risk of Lactic Acidosis).
◦ Avidin (raw eggs) inhibits Biotin-dependent enzymes.
◦ B12 deficiency blocks Propionyl CoA entry (via Methylmalonyl CoA Mutase).
Summary: Minor Metabolic Pathways
Overall Importance: Though termed "minor," these pathways (Galactose, Fructose, HMP Shunt, Uronic
Acid, Polyol) are clinically significant.

1. Galactose Metabolism

• Source: Lactose (milk sugar).

• Functions:
◦ Convert to Glucose.
◦ Synthesis of Lactose (during breastfeeding).
◦ Synthesis of Glycosaminoglycans (GAGs), Proteoglycans, Glycolipids.
• Site: Primarily Liver (minor: erythrocytes, fibroblasts).
• Pathway:
1. Galactose + ATP → Galactose 1-Phosphate + ADP (Enzyme: Galactokinase)
2. Galactose 1-Phosphate + UDP-Glucose ⇌ Glucose 1-Phosphate + UDP-Galactose (Enzyme:
Galactose 1-phosphate uridyltransferase - GALT)
3. UDP-Galactose ⇌ UDP-Glucose (Enzyme: UDP-hexose 4-epimerase)
4. UDP-Galactose → Used for synthesis (Lactose, GAGs, etc.)
5. Glucose 1-Phosphate ⇌ Glucose 6-Phosphate → Glucose / Glycolysis / Glycogen synthesis.
• Disorders (Galactosemia):
◦ Classic Galactosemia:
▪ Defect: GALT deficiency.
▪ Accumulation: Galactose 1-Phosphate (toxic).
▪ Mechanism: Traps inorganic phosphate (Pi) → Inhibits Glycogen Phosphorylase &
Gluconeogenesis → Fasting hypoglycemia.
▪ Onset: First 2 weeks of life (due to breast milk).
▪ Clinical Features: Feeding difficulties, vomiting, jaundice, failure to regain birth weight,
hepatomegaly, liver failure, mental retardation, oil drop cataract, resembles neonatal sepsis
(often E. coli).
▪ Mnemonic Hint: GALACTOSE - Growth failure, Abnormal feeding/vomiting, Liver damage/
jaundice, Cataract (oil drop), Toxic accumulation (Gal-1-P), Onset early, Sepsis risk (E.coli),
Enzyme (GALT) defect.
▪ Lab Dx: Urine reducing substance (+, Benedict's), Glucose oxidase (-), Muscic acid test (+) for
galactose, Enzyme assays, Genetic mutation studies.
▪ Treatment: Strict lactose restriction (contraindication for breastfeeding). Diet needed until ~4
years (alternative pathway via Galactose 1-phosphate pyrophosphorylase develops).
◦ Non-Classic Galactosemia:
▪ Defect: Galactokinase deficiency.
▪ Accumulation: Galactose.
▪ Clinical Feature: Cataract only.
▪ Defect: UDP-hexose 4-epimerase deficiency.
▪ Variable clinical features.
2. Fructose Metabolism

• Sources: Dietary Sucrose, Free Fructose (honey, fruits), Polyol pathway.

• Site: Primarily Liver.
• Pathway:
1. Fructose + ATP → Fructose 1-Phosphate + ADP (Enzyme: Fructokinase)
2. Fructose 1-Phosphate ⇌ Dihydroxyacetone Phosphate (DHAP) + Glyceraldehyde (Enzyme:
Aldolase B - liver isoform).
3. DHAP → Enters Glycolysis.
4. Glyceraldehyde + ATP → Glyceraldehyde 3-Phosphate + ADP (Enzyme: Triose kinase) → Enters
Glycolysis.
5. Note: Bypasses the major regulatory step (PFK-1) of glycolysis.
• Disorders:
◦ Essential Fructosuria:
▪ Defect: Fructokinase deficiency.
▪ Accumulation: Fructose (excreted in urine).
▪ Benign condition.
▪ Reason for Fructosuria (not Fructosemia): Fructose has no renal threshold, readily excreted.
▪ Lab Dx: Benedict's test (+), specific tests for fructose needed to differentiate.
◦ Hereditary Fructose Intolerance (HFI):
▪ Defect: Aldolase B deficiency.
▪ Accumulation: Fructose 1-Phosphate (toxic).
▪ Mechanism: Traps Pi → Inhibits Glycogen Phosphorylase & Gluconeogenesis → Fasting
hypoglycemia.
▪ Onset: ~6 months (when fructose introduced in diet).
▪ Clinical Features: Similar to Classic Galactosemia (feeding issues, vomiting, jaundice,
hepatomegaly, liver failure, mental retardation) BUT NO CATARACT.
▪ Lab Dx: Benedict's test (+), Glucose oxidase (-), Positive Seliwanoff's test or Rapid furfural
test (for ketoses), Enzyme assays, Genetic studies.
▪ Treatment: Lifelong restriction of sucrose and fructose.
• Harmful Effects of Fructose:
◦ Insulin-independent metabolism.
◦ Bypasses PFK-1 regulation → rapid glycolysis → increased Pyruvate → Acetyl-CoA.
◦ Leads to increased Fatty Acid & Triacylglycerol (TAG) synthesis → increased VLDL & LDL →
Dyslipidemia. Particularly problematic in diabetics and contributes to lifestyle disorders (high
fructose corn syrup in beverages).

3. Hexose Monophosphate Pathway (HMP Shunt / Pentose Phosphate

Pathway - PPP)

• Other Names: Dicken-Horecker pathway, Phosphogluconate pathway.

• Site: Cytoplasm.
 • Phases:
◦ Oxidative Phase (Irreversible):
1. Glucose 6-P + NADP⁺ → 6-Phosphogluconolactone + NADPH (Enzyme: Glucose 6-phosphate
dehydrogenase - G6PD - Rate-limiting)
2. 6-Phosphogluconolactone → 6-Phosphogluconate (Enzyme: Lactonase)
3. 6-Phosphogluconate + NADP⁺ → Ribulose 5-P + NADPH + CO₂ (Enzyme: 6-
Phosphogluconate dehydrogenase)
4. Primary Goal: Generate NADPH.
◦ Non-Oxidative Phase (Reversible):
▪ Interconverts pentoses (Ribulose 5-P, Ribose 5-P, Xylulose 5-P) and glycolytic intermediates
(Fructose 6-P, Glyceraldehyde 3-P).
▪ Key Enzymes: Transketolase (requires Thiamine Pyrophosphate - TPP & Mg²⁺),
Transaldolase.
▪ Primary Goal: Produce Pentoses (e.g., Ribose 5-P for nucleotide synthesis) and allow
interconversion based on cell needs.
• Overall Significance:
◦ Provides NADPH for:
▪ Reductive Biosynthesis: Fatty acids (Liver, Adipose, Lactating Mammary Gland), Steroids
(Adrenal cortex, Gonads).
▪ Antioxidant Defense: Regenerates reduced Glutathione (GSH) via Glutathione Reductase.
Essential for RBCs and Lens to combat oxidative stress.
▪ Keeping Hemoglobin iron in Fe²⁺ state (prevents Methemoglobinemia).
◦ Provides Pentoses (Ribose 5-P) for nucleotide/nucleic acid synthesis (important in rapidly dividing
cells: Bone marrow, Skin, Intestine).
• Does NOT produce ATP.
• Disorder: G6PD Deficiency:
◦ Most common human enzyme deficiency.
◦ X-linked recessive (mostly affects males).
◦ Mechanism: ↓G6PD → ↓NADPH → ↓Reduced Glutathione → Increased susceptibility to oxidative
stress → RBC membrane damage → Hemolysis.
◦ Clinical Features: Hemolytic Anemia, Hemolytic Jaundice, potentially Methemoglobinemia. Often
triggered by oxidative stress.
◦ Triggers: Infections, Fava beans (Favism), Drugs (Sulfonamides, Primaquine, other antimalarials,
high-dose aspirin).
◦ Protective against Plasmodium falciparum malaria (prevalent in Mediterranean, Middle East)
because the parasite's life cycle is disrupted by premature RBC lysis.
▪ Mnemonic Hint: G6PD - Glutathione regeneration hampered, 6-Phosphate Dehydrogenase
defect, Protection vs malaria, Drug/Diet/Disease triggers Hemolysis.

4. Uronic Acid Pathway

• Site: Liver cytoplasm.

• Function: An oxidative pathway for glucose.
 • Significance:
◦ Produces UDP-Glucuronic Acid (a uronic acid) required for:
▪ Conjugation of bilirubin, steroids, drugs.
▪ Synthesis of GAGs / Proteoglycans.
◦ Minor source of Pentoses (L-Xylulose).
◦ Synthesis of Ascorbic Acid (Vitamin C) - occurs in most animals but NOT in humans, higher
primates, guinea pigs due to deficiency of L-gulonolactone oxidase.
• Disorder: Essential Pentosuria:
◦ Benign condition.
◦ Defect: Xylitol Dehydrogenase (or L-xylulose reductase).
◦ Accumulation/Excretion: L-Xylulose in urine.
◦ Lab Dx: Benedict's test (+), Bial's test (+) for pentoses.

5. Polyol Pathway (Sorbitol Pathway)

• Function: Converts Glucose → Fructose.

• Pathway:
1. Glucose + NADPH + H⁺ ⇌ Sorbitol + NADP⁺ (Enzyme: Aldose Reductase)
2. Sorbitol + NAD⁺ ⇌ Fructose + NADH + H⁺ (Enzyme: Sorbitol Dehydrogenase)
• Organ Variation & Significance:
◦ Lens, Nerves, Kidney: Have Aldose Reductase but low/absent Sorbitol Dehydrogenase.
▪ In hyperglycemia (Diabetes), Glucose → Sorbitol. Sorbitol accumulates.
▪ Sorbitol is osmotically active → attracts water → cell swelling/damage → Diabetic
complications (Cataract, Neuropathy, Nephropathy).
◦ Liver, Ovary, Seminal Vesicles: Have both enzymes. Pathway converts Glucose → Fructose.
▪ Seminal vesicles: Provides fructose as the primary energy source for spermatozoa in seminal
fluid.
• Cataract Formation:
◦ Diabetes Mellitus: High glucose enters lens (via insulin-independent GLUT1) → Aldose Reductase
(low affinity, active at high glucose) converts Glucose → Sorbitol → Sorbitol accumulation →
Osmotic damage → Cataract.
◦ Galactosemia: High galactose enters lens → Aldose Reductase converts Galactose → Galactitol
(Dulcitol) → Galactitol accumulation → Osmotic damage → Cataract (often 'oil drop' type).
◦ Not seen in Fructosuria because fructose has a low renal threshold and doesn't accumulate to high
levels in tissues like the lens.

Key Points to Remember:

• Galactosemia (Classic): GALT defect, Gal-1-P accumulation, early onset, involves liver/brain/eyes (oil
drop cataract). Requires immediate lactose restriction.
• HFI: Aldolase B defect, Fructose-1-P accumulation, onset with fructose introduction (~6 months),
involves liver/hypoglycemia, NO cataracts. Requires lifelong fructose restriction.
• HMP Shunt: Main source of NADPH (antioxidant, biosynthesis) and Pentoses (nucleotides). G6PD is
rate-limiting.
• G6PD Deficiency: X-linked, causes drug/infection/fava-induced hemolytic anemia due to ↓NADPH.
Commonest enzyme defect.
 • Uronic Acid Pathway: Forms UDP-glucuronate (conjugation, GAGs). Humans cannot make Vitamin C
via this pathway (lack L-gulonolactone oxidase).
• Polyol Pathway: Links glucose to fructose. Sorbitol accumulation (due to Aldose Reductase activity
exceeding Sorbitol Dehydrogenase activity in certain tissues) is implicated in diabetic cataracts,
neuropathy, and nephropathy. Aldose reductase also causes galactitol accumulation in galactosemia,
leading to cataracts.
• Enzyme Cofactors: Transketolase requires TPP (Thiamine). G6PD and 6-PG Dehydrogenase require
NADP⁺. Aldose Reductase requires NADPH. Sorbitol Dehydrogenase requires NAD⁺.

Self-Correction/Additions based on context for clarity: * Corrected enzyme names and spelling throughout. *
Clarified Aldolase B as liver isoform. * Added GLUT1 transporter context for lens glucose uptake. * Noted
Aldose Reductase's low affinity. * Clarified the specific pentose L-Xylulose in Essential Pentosuria. * Added
rate-limiting enzyme for HMP Shunt. * Specified cofactors for key enzymes. * Added Lactating Mammary
Gland as a site for HMP/NADPH use.

Chemistry and Properties of Amino Acids Summary

I. Introduction & Importance

• Clinical Relevance Examples:

◦ Hemoglobin S (Sickle Cell Anemia): Glutamate (6th position, beta-globin) replaced by Valine.
Understanding the nature of this mutation (polar to non-polar) requires knowledge of amino acid
properties.
◦ Protein Structure: Leucine (non-polar) found in the interior of albumin (globular protein) illustrates
the role of amino acid characteristics in protein folding.
• The chemistry and properties of amino acids are fundamental to understanding protein structure,
function, and related diseases.

II. General Structure of Amino Acids

• Components: Central alpha-carbon (Cα) bonded to:

1. Amino group (-NH2)
2. Carboxyl group (-COOH)
3. Hydrogen atom (-H)
4. Variable side chain (-R group)
• Alpha-Amino Acids: Most biologically relevant amino acids have the amino and carboxyl groups
attached to the alpha-carbon.
• R-Group Significance: The R-group varies between amino acids, defining their unique identity and
properties.

III. Non-Alpha Amino Acids

• Amino group is attached to a carbon other than the alpha-carbon.

• Examples: Beta-alanine, Beta-aminoisobutyrate, Gamma-aminobutyrate (GABA).
IV. Classification of Amino Acids
Classified based on four main criteria:

1. Side Chain (R-group) Structure

2. Side Chain Characteristics (Polarity)
3. Metabolic Fate
4. Nutritional Requirements

A. Classification Based on Side Chain Structure

• Importance: The R-groups determine the properties of a polypeptide chain, as the backbone amino and
carboxyl groups are involved in peptide bonds (CO-NH linkage formed by dehydration). Peptide bonds
themselves primarily participate in hydrogen bonding.
• Categories:
◦ Aliphatic:
▪ Simple: Glycine (Gly, G), Alanine (Ala, A)
▪ Branched-Chain (BCAA): (Mnemonic: LIVE) Leucine (Leu, L), Isoleucine (Ile, I), Valine (Val,
V)
◦ Hydroxyl (-OH) Containing: Serine (Ser, S), Threonine (Thr, T), Tyrosine (Tyr, Y)
◦ Sulfur Containing: Cysteine (Cys, C), Methionine (Met, M)
◦ Acidic (contain extra -COOH in R group): Aspartic Acid (Asp, D), Glutamic Acid (Glu, E)
◦ Amides (contain amide group -CONH2 in R group): Asparagine (Asn, N), Glutamine (Gln, Q)
(Mnemonic: Q-tamine)
◦ Basic (contain extra basic N in R group): (Mnemonic: HAL) Histidine (His, H), Arginine (Arg,
R), Lysine (Lys, K)
◦ Aromatic (contain ring structures): Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp, W)
▪ Ring Structures:
▪ Phenylalanine: Benzene ring
▪ Tyrosine: Phenol ring (Benzene + OH)
▪ Tryptophan: Indole ring (Benzopyrrole)
▪ Histidine: Imidazole ring
◦ Imino Acid: Proline (Pro, P) - Contains a Pyrrolidine ring where the alpha-amino group is part of the
ring structure.
• Specific R-Group Features:
◦ Arginine: Guanidinium group
◦ Lysine: Epsilon (ε) amino group

B. Classification Based on Side Chain Characteristics (Polarity)

• Importance: Determines protein folding (hydrophobic core, hydrophilic surface) and the nature of
mutations (conservative vs. non-conservative).
• Categories:
◦ Polar (Hydrophilic - soluble in water):
▪ Charged:
▪ Acidic: Aspartate (D), Glutamate (E)
▪ Basic: Lysine (K), Arginine (R), Histidine (H)
 ▪ Uncharged:
▪ Hydroxyl: Serine (S), Threonine (T)
▪ Amides: Asparagine (N), Glutamine (Q)
▪ Sulfur: Cysteine (C) (due to polar -SH group)
▪ Simple: Glycine (G) (Least polar)
▪ Note: Arginine (Arg) is the most polar.
◦ Non-Polar (Hydrophobic - insoluble in water):
▪ Aliphatic (Branched): Leucine (L), Isoleucine (I), Valine (V)
▪ Aliphatic (Simple): Alanine (A)
▪ Aromatic: Phenylalanine (F), Tryptophan (W), Tyrosine (Y)
▪ Sulfur: Methionine (Met)
▪ Imino: Proline (P)
• Mutations:
◦ Conservative: Replacement with an amino acid of similar polarity (e.g., polar replaced by polar).
◦ Non-conservative: Replacement with an amino acid of different polarity (e.g., polar replaced by non-
polar).
◦ Example: Hemoglobin S (Glu [polar, acidic] -> Val [non-polar]) is a non-conservative mutation.

C. Classification Based on Metabolic Fate

• Importance: Determines whether the carbon skeleton can be used for glucose synthesis
(gluconeogenesis) or ketone body synthesis.
• Categories:
◦ Ketogenic: Carbon skeleton yields ketone bodies or their precursors (acetyl-CoA or acetoacetyl-
CoA).
▪ Leucine (L), Lysine (K) (Mnemonic: the pure L's)
◦ Both Ketogenic & Glucogenic: Carbon skeleton yields precursors for both pathways.
▪ Mnemonic: PITT Phenylalanine (F), Isoleucine (I), Tryptophan (W), Tyrosine (Y)
◦ Glucogenic: Carbon skeleton yields pyruvate or Krebs cycle intermediates, can be used for glucose
synthesis.
▪ All other amino acids.
▪ Principal Glucogenic AA: Alanine (A).

D. Classification Based on Nutritional Requirements

• Importance: Relates to dietary needs, nitrogen balance, and protein quality (complete vs. incomplete
proteins).
• Categories:
◦ Nutritionally Essential: Cannot be synthesized by the body in sufficient amounts (or synthesis
pathway is too long/complex >7 steps); must be obtained from the diet. Deficiency leads to negative
nitrogen balance.
▪ Mnemonic: MATT VIL PHLy (sometimes PVT TIM HALL)
▪ Methionine (M)
▪ Arginine (R)*
▪ Threonine (T)
▪ Tryptophan (W)
 ▪ Valine (V)
▪ Isoleucine (I)
▪ Leucine (L)
▪ Phenylalanine (P)
▪ Histidine (H)*
▪ Lysine (L)
▪ *Semi-essential: Arginine and Histidine are synthesized but may be insufficient during growth or
certain conditions. Arginine is often specifically cited as semi-essential.
◦ Nutritionally Non-Essential: Can be synthesized by the body (usually simple pathways, 1-2 steps).
▪ All other amino acids (Alanine, Aspartate, Asparagine, Cysteine, Glutamate, Glutamine, Glycine,
Proline, Serine, Tyrosine). Note: Tyrosine is synthesized from Phenylalanine (essential).

V. Derived Amino Acids

• Modified from standard amino acids after protein synthesis (post-translational modification) or during
metabolism.
• Standard vs. Derived:
◦ Standard AAs: Have genetic codons, incorporated during translation (co-translational).
◦ Derived AAs: Do not have direct codons, formed by modifying standard AAs (post-translational).
• Examples Seen in Proteins:
◦ Hydroxylysine, Hydroxyproline: Found in Collagen. Requires Vitamin C and α-ketoglutarate for
hydroxylation enzymes (prolyl/lysyl hydroxylase).
◦ Gamma-carboxyglutamate (Gla): Found in clotting factors (II, VII, IX, X), Protein C, Protein S,
Osteocalcin. Requires Vitamin K for gamma-carboxylation.
◦ Desmosin: Found in Elastin. Derived from Lysine.
◦ Methyllysine: Found in Myosin (skeletal muscle protein).
• Examples Not Seen in Proteins (Metabolic Intermediates):
◦ Ornithine, Citrulline, Argininosuccinate (Urea cycle intermediates).
◦ Homoserine, Homocysteine (Sulfur amino acid metabolism intermediates).

VI. Special Amino Acids (21st & 22nd)

• Incorporated during translation despite unusual codons (recoding). Considered standard amino acids.
• Selenocysteine (Sec, U) - The 21st AA:
◦ Codon: UGA (normally a stop codon) - requires specific mRNA secondary structure (SECIS
element).
◦ Incorporation: Co-translational modification of Serine attached to a special tRNA (tRNA[Ser]Sec).
◦ Found in enzymes: Glutathione peroxidase, Thioredoxin reductase, Deiodinases, Selenoprotein P.
• Pyrrolysine (Pyl, O) - The 22nd AA:
◦ Codon: UAG (normally a stop codon).
◦ Precursor: Lysine.
◦ Found primarily in methanogenic archaea.
VII. Properties of Amino Acids
A. Absorption of UV Light

• Amino acids are colorless (do not absorb visible light).

• Aromatic Amino Acids (Phe, Tyr, Trp) absorb UV light due to their conjugated ring structures.
◦ Absorption range: 250-290 nm.
◦ Maximum absorption typically near 280 nm.
◦ Absorption intensity: Trp > Tyr > Phe.
• Application: Basis for quantifying protein concentration using UV spectrophotometry at 280 nm.

B. Isomerism (Stereoisomerism)

• Due to the chiral (asymmetric) alpha-carbon (bonded to 4 different groups).

• Amino acids exist as stereoisomers (enantiomers): D-form and L-form, which are mirror images.
• Exception: Glycine (R-group is H) is achiral and optically inactive.
• Biological Significance: Proteins in humans (and most organisms) exclusively contain L-amino acids.
Enzymes are stereospecific for L-isomers.
• Enzyme Exception: Racemases can interconvert D and L forms.
• D-Amino Acids in Humans: Small amounts exist, e.g., D-Aspartate and D-Serine in the brain.

C. Existence in Different Charged States (Amphoteric Nature)

• Amino acids contain both acidic (-COOH) and basic (-NH2) groups.
• Ionization State: Varies with the pH of the surrounding medium.
◦ Low pH (acidic): -COOH is neutral (COOH), -NH2 is protonated (NH3+) -> Net positive charge.
◦ High pH (basic): -COOH is deprotonated (COO-), -NH2 is neutral (NH2) -> Net negative charge.
◦ Intermediate pH: Can exist as Zwitterion (dipolar ion) with both COO- and NH3+.
• Isoelectric Point (pI):
◦ The specific pH at which an amino acid (or protein) has no net electrical charge (positive charges =
negative charges).
◦ Calculated as the average of the pKa values flanking the zwitterionic form.
◦ Properties at pI:
▪ Net charge = 0
▪ No migration in an electric field (used in electrophoresis separations).
▪ Minimum solubility (molecules aggregate as electrostatic repulsion is minimal).
▪ Maximum precipitability.
▪ Least buffering capacity.
• Charge Dependence on pH vs pI:
◦ pH < pI: Molecule has a net positive charge (protonated).
◦ pH > pI: Molecule has a net negative charge (deprotonated).
◦ Example: Albumin (pI ≈ 4.7) is negatively charged at blood pH (7.4) because pH > pI.

D. Buffering Capacity

• Amino acids can act as buffers, resisting changes in pH, due to their ionizable groups (-COOH, -NH2,
and some R-groups).
 • Henderson-Hasselbalch Equation: pH = pKa + log ([conjugate base]/[weak acid]) describes the
relationship.
• Maximum Buffering: Occurs when the pH ≈ pKa of the ionizable group. At this point, the
concentrations of the weak acid and its conjugate base are approximately equal.
• Physiological Significance: The imidazole group of Histidine (R-group pKa ≈ 6.0 - 7.0, close to
physiological pH 7.4) is a crucial buffer in blood and tissues (e.g., in Hemoglobin).

VIII. Important Points to Remember

• Structure: Know the basic alpha-amino acid structure and the significance of the R-group.
• Classification: Understand the four main ways to classify AAs and know key examples in each
(especially essential AAs, polar/non-polar, ketogenic/glucogenic). Mnemonics (LIVE, HAL, PITT,
MATT VIL PHLy) can help.
• Codes: Be familiar with the three-letter and one-letter codes for common amino acids.
• Polarity: Crucial for protein structure (hydrophobic core) and mutation effects (conservative vs. non-
conservative). HbS mutation (Glu->Val) is a key non-conservative example.
• Essential AAs: Must be from diet; deficiency causes negative nitrogen balance.
• Derived AAs: Formed post-translationally (e.g., Hydroxyproline in collagen, Gla in clotting factors);
know key examples and required vitamins (C, K).
• 21st/22nd AAs: Selenocysteine (UGA, Ser precursor) and Pyrrolysine (UAG, Lys precursor)
incorporated via stop codon recoding. Know enzymes containing Selenocysteine.
• Properties:
◦ UV Absorption: Aromatic AAs (Trp > Tyr > Phe) absorb at ~280 nm.
◦ Isomerism: L-forms predominate; Glycine is achiral.
◦ Charged State/pI: Understand zwitterions, pI, and how charge changes with pH relative to pI.
Proteins are least soluble at their pI.
◦ Buffering: Max buffering at pH ≈ pKa; Histidine's imidazole group is key physiological buffer.

Fibrous Proteins Summary

I. Introduction to Fibrous Proteins

• Fibrous proteins are primarily structural proteins.

• Examples: Collagen, Elastin, Keratin, Fibrillin, Laminin.
• Collagen is the main focus:
◦ Most abundant protein in the body.
◦ Ubiquitous (present in almost all organs).
◦ High-yield topic relevant across medical specialties.

II. Collagen

• General Characteristics:
◦ Most abundant fibrous protein in the extracellular matrix (ECM).
◦ Highest density found in the Cornea, followed by the Skin.
• Structure:
◦ Triple Helix:
▪ Composed of 3 polypeptide alpha chains (Note: transcript calls them polyproline alpha chains,
but they are polypeptide chains rich in proline).
▪ Each individual alpha chain twists in a left-handed direction.
▪ The three chains together twist in a right-handed direction, providing tensile strength.
◦ Alpha Chain Composition:
▪ Approximately 1000 amino acids long.
▪ Characterized by a repeating Gly-X-Y sequence.
▪ Glycine (Gly) is every 3rd residue (most abundant, ~33%, recurring amino acid).
▪ X is often Proline.
▪ Y is often Hydroxyproline or Hydroxylysine.
◦ Quarter-Staggered Arrangement:
▪ Triple helices (fibrils) align in parallel rows.
▪ Each row is staggered by approximately 1/4th of its length relative to the adjacent row.
Contributes to strength and banding pattern seen on electron microscopy.
◦ Cross-Links:
▪ Covalent Aldol Cross-Links form between fibrils, further strengthening the structure.
• Synthesis: Occurs in two main stages: Intracellular and Extracellular.
◦ Intracellular Events (within Fibroblasts - RER & Golgi):
1. Synthesis of Pre-pro-alpha Chains: Occurs on ribosomes of the RER. Contain signal sequences.
2. Hydroxylation: Specific Proline and Lysine residues are hydroxylated by prolyl hydroxylase and
lysyl hydroxylase.
▪ These enzymes require Vitamin C (Ascorbic Acid) and alpha-ketoglutarate. (Mnemonic: C
for Collagen needs Vitamin C for Hydroxylation).
▪ Deficiency of Vitamin C leads to Scurvy, impairing collagen synthesis.
3. Glycosylation: Specific Hydroxylysine residues are glycosylated (carbohydrates added).
Linkage is Type 3 O-glycosidic. Collagen is a glycoprotein.
4. Disulfide Bond Formation: Interchain disulfide bonds form at the C-terminal propeptide region,
helping to align the three alpha chains.
5. Triple Helix Formation: The three hydroxylated and glycosylated alpha chains spontaneously
fold into a triple helix, forming Procollagen.
6. Secretion: Procollagen is packaged into secretory vesicles by the Golgi apparatus and secreted
into the ECM.
◦ Extracellular Events (within the ECM):
1. Cleavage of Propeptides: N-terminal and C-terminal propeptides are cleaved from procollagen
by procollagen peptidases (e.g., ADAMTS2 for N-terminal). This forms Tropocollagen.
2. Fibril Assembly: Tropocollagen molecules spontaneously self-assemble into collagen fibrils,
aligning in the characteristic quarter-staggered fashion.
3. Cross-Linking: Covalent cross-links (Aldol type) are formed between Lysine and Hydroxylysine
residues of adjacent tropocollagen molecules. This reaction is catalyzed by Lysyl Oxidase, which
requires Copper as a cofactor. Cross-linking provides tensile strength and stability.
 • Distribution of Collagen Types (Important Examples):
◦ Type I: Most abundant (90% in bone). Found in Bone, Skin, Tendons, Ligaments, Cornea, Dentin,
Wound healing. (Mnemonic: Type One = Bone).
◦ Type II: Main collagen of Cartilage (hyaline & elastic), Vitreous humor. Absent in Bone.
(Mnemonic: Type Two = Cartwolage).
◦ Type III: Found in extensible connective tissues like Skin, Blood Vessels (esp. aorta), Lungs, Uterus,
Fetal tissues. (Often found with Type I). (Mnemonic: Type Three = Rethreeculin fibers are flexible).
◦ Type IV: Forms networks in Basement Membranes. Does not form fibrils. (Mnemonic: Type Four
= Floor/Basement membrane).
◦ Type V: Widespread, often associated with Type I. Involved in classic EDS.
◦ Type VII: Forms Anchoring Fibrils that link the epidermis to the dermis (basement membrane to
underlying stroma). (Mnemonic: Type Seven anchors Heaven [skin layers] to Earth).
◦ Type X: Found in Hypertrophic Cartilage (mineralization zone).
◦ Type XVII: Found in Hemidesmosomes.
◦ Type XIX: Found in Rhabdomyosarcoma cells.
• Disorders Associated with Collagen:
◦ Osteogenesis Imperfecta (OI): Primarily Type I collagen defect (COL1A1, COL1A2 genes). Brittle
bones.
◦ Ehlers-Danlos Syndrome (EDS): Heterogeneous group.
▪ Classic EDS: Type V (COL5A1/A2) and sometimes Type I defects. Skin hyperextensibility, joint
hypermobility.
▪ Vascular EDS (Type IV EDS): Type III collagen defect (COL3A1). Most serious type due to risk
of arterial/organ rupture.
▪ Kyphoscoliotic EDS (Type VI EDS): Deficiency of Lysyl Hydroxylase.
▪ Arthrochalasia EDS (Type VIIA/B EDS): Type I collagen defect (failure to cleave N-propeptide).
▪ Dermatosparaxis EDS (Type VIIC EDS): Deficiency of Procollagen N-proteinase (ADAMTS2).
◦ Alport Syndrome: Type IV collagen defect (COL4A3/A4/A5/A6 genes). Affects basement
membranes of kidneys, ears, eyes.
◦ Scurvy: Vitamin C deficiency leading to poor hydroxylation (Lysyl/Prolyl Hydroxylase
dysfunction). Weak collagen, bleeding gums, poor wound healing.
◦ Menkes Disease: Defect in copper transport (ATP7A gene). Leads to Lysyl Oxidase dysfunction
(copper-dependent enzyme needed for cross-linking). Kinky hair, neurodegeneration.
◦ Dystrophic Epidermolysis Bullosa (DEB): Type VII collagen defect (COL7A1 gene). Blistering
due to separation at the dermo-epidermal junction.
◦ Schmid Metaphyseal Chondrodysplasia: Type X collagen defect.
◦ Osteoarthritis: Degradation involves Type II collagen in cartilage.

III. Elastin

• Structure & Features:

◦ Only one type of elastin.
◦ No triple helix structure.
◦ No Gly-X-Y repeating sequence.
◦ Contains Proline and Glycine, but very little Hydroxyproline and NO Hydroxylysine.
 ◦ Not glycosylated.
◦ Unique cross-links called Desmosine (and Isodesmosine).
▪ Formed from four Lysine residues.
▪ Reaction catalyzed by Lysyl Oxidase (copper-dependent).
• Function & Location: Provides elasticity and recoil to tissues like Lungs, Large Arteries (Aorta), Skin,
Elastic Ligaments.
• Disorders:
◦ Williams-Beuren Syndrome: Deletion involving the elastin gene. Supravalvular aortic stenosis,
characteristic facial features, intellectual disability.
◦ Cutis Laxa: Can be caused by elastin mutations; results in loose, sagging skin.

IV. Keratin

• Structure & Features:

◦ Forms intermediate filaments.
◦ Structure is primarily alpha-helix.
◦ Rich in Cysteine residues, which form disulfide bonds.
◦ Hardness of keratin is proportional to the number of disulfide bonds (e.g., hair vs. nails).
• Function & Location: Provides strength and protection to Hair, Nails, Outer layers of the Skin
(epidermis).
• Disorders:
◦ Epidermolysis Bullosa Simplex: Defects in Keratin 5 and Keratin 14. Causes blistering within the
epidermis upon minor trauma. (Note: Transcript mentioned 'Classic EB' affecting Keratin 5, but this
is usually associated with EB Simplex).

V. Fibrillin

• Structure & Function:

◦ Glycoprotein essential for forming microfibrils in the ECM.
◦ Microfibrils act as a scaffold for elastin deposition.
• Types & Disorders:
◦ Fibrillin-1 (FBN1 gene):
▪ Defect causes Marfan Syndrome: Skeletal abnormalities (arachnodactyly, tall stature), ocular
problems (ectopia lentis), cardiovascular issues (aortic aneurysm/dissection).
▪ Also associated with Acromicric Dysplasia and Geleophysic Dysplasia.
◦ Fibrillin-2 (FBN2 gene):
▪ Defect causes Congenital Contractural Arachnodactyly (CCA): Characterized by contractures,
arachnodactyly, abnormal ears.

Key Points to Remember:

1. Collagen Structure: Gly-X-Y repeat, Triple Helix (Left-handed chains -> Right-handed helix), Quarter-
staggered fibrils.
2. Collagen Synthesis: Requires Vitamin C (hydroxylation) and Copper (lysyl oxidase for cross-linking).
Intracellular (hydroxylation, glycosylation, helix formation) vs. Extracellular (cleavage, fibril assembly,
cross-linking).
 3. Major Collagen Types & Locations:
◦ I: Bone, Skin, Tendon (Most common)
◦ II: Cartilage (Absent in bone)
◦ III: Vessels, Skin (Extensible)
◦ IV: Basement Membrane
◦ VII: Anchoring Fibrils
4. Key Collagen Diseases:
◦ OI: Type I defect.
◦ Alport: Type IV defect.
◦ Vascular EDS: Type III defect (most severe EDS).
◦ Scurvy: Vit C deficiency (Hydroxylation↓).
◦ Menkes: Copper deficiency (Lysyl Oxidase↓, Cross-linking↓).
◦ DEB: Type VII defect.
5. Elastin: Elastic recoil, Desmosine cross-links (from Lysine via Lysyl Oxidase), NO hydroxylysine, NO
glycosylation. Disease: Williams-Beuren Syndrome.
6. Keratin: Intermediate filaments, Alpha-helix, Cysteine-rich (Disulfide bonds = hardness). Disease:
Epidermolysis Bullosa Simplex (K5/K14).
7. Fibrillin-1: Microfibrils, Elastin scaffold. Disease: Marfan Syndrome.
8. Fibrillin-2: Disease: Congenital Contractural Arachnodactyly.

Summary: General Amino Acid Metabolism

I. Introduction & Importance

• This topic is fundamental to understanding all amino acid (AA) metabolism reactions.
• It helps answer key questions:
◦ Why do glutamine levels rise in hyperammonemia?
◦ What does "all amino acids are getting concentrated as glutamate" mean, and why?
◦ Why is ammonia (NH3) toxic to the brain?
II. General Reactions of Amino Acids

• Structure: Alpha-carbon bonded to COOH (carboxyl), NH2 (amino), H, and an R group.

• Decarboxylation:
◦ Reaction: Removal of CO2 from the COOH group.
◦ Product: Biologically important amine.
◦ Coenzyme: PLP (Pyridoxal Phosphate).
• Deamination:
◦ Reaction: Removal of the NH2 group (as NH3).
◦ Product: Corresponding alpha-keto acid. (The carbon where NH2 was removed forms a keto group
C=O).
III. Transamination

• Definition: Transfer of an amino group (NH2) from an alpha-amino acid to an alpha-keto acid, forming a
new alpha-amino acid and a new alpha-keto acid.
 • Mechanism Analogy: Swapping caps between an amino acid bottle and a keto acid bottle. AA becomes
KA; KA becomes AA.
• Key Properties:
◦ Location: Occurs in all organs.
◦ Ammonia Handling: No free NH3 is released. The amino group is carefully transferred.
◦ Reversibility: Freely reversible.
◦ Coenzyme: PLP (Pyridoxal Phosphate).
◦ Organelle: Cytoplasm.
• Examples & Specificity:
◦ ALT (Alanine Aminotransferase) / SGPT:
▪ Alanine + α-Ketoglutarate <=> Pyruvate + Glutamate
◦ AST (Aspartate Aminotransferase) / SGOT:
▪ Aspartate + α-Ketoglutarate <=> Oxaloacetate + Glutamate
◦ Specificity: Enzymes are specific for one AA/KA pair (e.g., ALT for Ala/Pyr; AST for Asp/OAA) but
often non-specific for the other pair, frequently using the α-Ketoglutarate/Glutamate pair.
• Funnel Concept:
◦ Most amino acids transfer their alpha-amino group to α-Ketoglutarate, converting it to Glutamate.
◦ Therefore, the amino groups from various AAs are concentrated into Glutamate.
◦ Why Glutamate? It's the primary amino acid that undergoes oxidative deamination to release free
ammonia for disposal (mainly in the liver).
• Mechanism Type:
◦ Ping-Pong Mechanism (or Bi-Bi Reaction / Two Substrate-Two Product):
1. Substrate 1 (AA) binds, Product 1 (KA) leaves.
2. Substrate 2 (α-KG) binds, Product 2 (Glutamate) leaves.
• Role in Synthesis: Used for the biosynthesis of non-essential amino acids.
◦ α-Ketoglutarate -> Glutamate
◦ Pyruvate -> Alanine
◦ Oxaloacetate -> Aspartate
• Non-Alpha Amino Group Transamination:
◦ Example: Delta-amino group of Ornithine.
◦ Enzyme: Ornithine Delta-Aminotransferase (uses PLP).
◦ Clinical Correlation: Deficiency causes Gyrate Atrophy of retina and choroid.
◦ Treatment: Restrict dietary Arginine (precursor) and Ornithine; supplement with PLP.
IV. Ammonia (NH3) Handling

• Sources of Ammonia:
◦ Major: Alpha-amino groups of amino acids (via deamination, primarily from glutamate).
◦ Minor: Non-protein nitrogenous substances, amino sugars, gut bacteria activity.
• Ammonia Transport (Detoxification during transit):
◦ From Most Organs (including Brain):
1. Free NH3 is toxic.
2. Glutamate + NH3 + ATP → Glutamine + ADP + Pi
3. Enzyme: Glutamine Synthetase (Mitochondrial, requires ATP - a ligase).
 4. Called the "First line trapping" / "Savior mechanism".
5. Glutamine is the non-toxic transport form from most tissues to the liver.
6. Mnemonic: Synthetase Stuffs NH3 onto Glutamate to make Glutamine (using ATP).
◦ From Muscle:
1. Amino groups transferred to Glutamate (via transamination).
2. Glutamate + Pyruvate → α-Ketoglutarate + Alanine.
3. Alanine is the major transport form from muscle to the liver (part of Glucose-Alanine Cycle).
• Destination for Transport Forms (Glutamine/Alanine): Liver, for processing via the Urea Cycle.
• Ammonia Toxicity (Especially to Brain):
◦ Mechanism:
1. Excess NH3 (uncharged) crosses the Blood-Brain Barrier (BBB); NH4+ (charged) cannot easily
cross.
2. In the brain, NH3 is detoxified: Glutamate + NH3 → Glutamine (via Glutamine Synthetase).
3. Consequences of Excess NH3 Detoxification:
▪ ↑ Glutamine: Osmotically active → attracts water → Cerebral Edema.
▪ Depletion of α-Ketoglutarate: α-KG is used to regenerate Glutamate → TCA cycle slows
down → ↓ ATP production (energy crisis).
▪ ↑ Glutamate (potentially) / Altered Neurotransmitters: Glutamate conversion to GABA
(Gamma-Aminobutyric Acid - inhibitory neurotransmitter) may increase → CNS Depression.
(Lecture mentioned GABA production from glutamate decarboxylation as a consequence).
V. Oxidative Deamination

• Process: The removal of an amino group from an amino acid (primarily Glutamate) as free NH3, coupled
with oxidation.
• Key Reaction:
◦ Glutamate + NAD+ (or NADP+) <=> α-Ketoglutarate + NH3 + NADH (or NADPH) + H+
• Enzyme: Glutamate Dehydrogenase (GDH).
• Location: Primarily Liver (for urea cycle) and Kidney.
• Organelle: Mitochondria.
• Reversibility: Reversible.
• Coenzyme: Unique - can use either NAD+ or NADP+.
• Regulation (Allosteric):
◦ Activators: ADP (signals low energy).
◦ Inhibitors: ATP, GTP, NADH (signal high energy / product inhibition).
◦ Mnemonic: ADP Activates; ATP/GTP/NADH Go Negative (Inhibit).
VI. Transdeamination

• Definition: The functional coupling of Transamination (concentrating amino groups onto Glutamate in
various tissues) and Oxidative Deamination (releasing NH3 from Glutamate in the liver mitochondria).
• Overall Flow: AA (in peripheral tissue) → (Transamination) → Glutamate → (Transport as Gln/Ala) →
Liver → Glutamate → (Oxidative Deamination by GDH) → NH3 → Urea Cycle.
• Although occurring in different locations/compartments, these processes work together for safe handling
and disposal of amino groups.
VII. Summary & Revisiting Initial Questions

• Hyperammonemia & ↑Glutamine: Glutamine is the transport form of NH3 from most tissues (incl.
brain); excess NH3 drives Glutamine synthesis.
• AAs concentrated as Glutamate: Transamination funnels most AA's amino groups onto α-KG, forming
Glutamate, the substrate for GDH.
• NH3 Brain Toxicity: Due to cerebral edema (via glutamine), ↓ATP (via α-KG depletion), and CNS
depression (via neurotransmitter imbalance/GABA).
VIII. Important Points to Remember:

• PLP is the key coenzyme for Transamination and Decarboxylation.

• Transamination transfers NH2 groups, does not release free NH3, and concentrates amino groups onto
Glutamate.
• Glutamine is the main NH3 transporter from most tissues (via Glutamine Synthetase, uses ATP).
• Alanine is the main NH3 transporter from muscle.
• Liver is the primary site for Urea synthesis.
• Glutamate Dehydrogenase (GDH) performs oxidative deamination of Glutamate in liver mitochondria,
releasing free NH3 for the urea cycle. GDH uniquely uses NAD+ or NADP+ and is regulated by energy
charge (ADP activates, ATP/GTP inhibit).
• Ammonia toxicity in the brain involves cerebral edema, energy depletion (↓ATP), and CNS
depression.
• Transdeamination = Transamination + Oxidative Deamination working together.
(Final motivational quote omitted as per instruction to provide summary only)

Here is a detailed summary of the Urea Cycle and its Disorders lecture:
I. Urea Cycle Fundamentals

• Other Names:
◦ Krebs-Henseleit Cycle: Named after its discoverers. (Note: Different from Krebs Cycle/TCA Cycle).
◦ Ornithine Cycle: Because ornithine is regenerated.
◦ Urea Bicycle / Krebs Bicycle: Links Urea Cycle and TCA Cycle.
▪ Urea Cycle provides Fumarate to TCA Cycle.
▪ TCA Cycle provides Aspartate to Urea Cycle.
• Urea Formula & Sources: NH₂-CO-NH₂
◦ 1st Nitrogen (NH₂): From free Ammonia (NH₃).
◦ Carbon (C): From Carbon Dioxide (CO₂).
◦ 2nd Nitrogen (NH₂): From Aspartate.
◦ Compounds Consumed: NH₃, CO₂, Aspartate.
• Location:
◦ Organ: Exclusively in the Liver (only organ with all enzymes).
◦ Cellular: Mitochondria and Cytoplasm.
◦ Mnemonic for dual-location pathways: HUG-P
▪ Heme Synthesis
▪ Urea Cycle
 ▪ Gluconeogenesis
▪ Pyrimidine Synthesis
• Rate-Limiting Enzyme: Carbamoyl Phosphate Synthetase 1 (CPS1).
II. Urea Cycle Steps

1. Mitochondria:
◦ Step 1: CO₂ + NH₃ + 2 ATP → Carbamoyl Phosphate + 2 ADP + Pi
▪ Enzyme: Carbamoyl Phosphate Synthetase 1 (CPS1)
▪ Rate-limiting step.
▪ Requires 2 ATP (2 high-energy phosphates).
▪ Allosteric Activator: N-Acetylglutamate (NAG) (Obligate activator).
◦ Step 2: Carbamoyl Phosphate + Ornithine → Citrulline + Pi
▪ Enzyme: Ornithine Transcarbamoylase (OTC).
▪ Ornithine enters mitochondria via Ornithine Transporter (Permease).
2. Cytoplasm:
◦ Citrulline exits mitochondria.
◦ Aspartate transported out of mitochondria via Citrin (Aspartate-Glutamate Transporter).
◦ Step 3: Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi
▪ Enzyme: Argininosuccinate Synthetase (ASS).
▪ Requires 1 ATP (equivalent to 2 high-energy phosphates as ATP → AMP).
◦ Step 4: Argininosuccinate → Arginine + Fumarate
▪ Enzyme: Argininosuccinate Lyase (ASL).
▪ Fumarate enters TCA cycle.
◦ Step 5: Arginine + H₂O → Urea + Ornithine
▪ Enzyme: Arginase (ARG1) (a Hydrolase).
▪ Releases Urea.
▪ Regenerates Ornithine, which is transported back into mitochondria.
III. Energetics

• High-Energy Phosphates Used: 4 per cycle (2 from CPS1, 2 from ASS).

• ATP Molecules Used Directly: 3 (2 ATP → 2 ADP by CPS1; 1 ATP → 1 AMP by ASS).
• ATP Equivalents: 4.
IV. Regulation

1. Diet: High protein intake → Increased ammonia → Induces urea cycle enzymes.
2. Allosteric Regulation: N-Acetylglutamate (NAG) activates CPS1.
3. Compartmentation: Separation of reactions between mitochondria and cytoplasm acts as regulation.
V. Urea Cycle Disorders (UCDs)

• General Presentation:
◦ Encephalopathy: Due to ammonia toxicity.
◦ Respiratory Alkalosis: High NH₃ stimulates respiratory center → hyperventilation → CO₂ washout.
◦ Hyperammonemia: Defective cycle cannot process ammonia.
 • Neonatal Presentation: Feeding difficulty, lethargy, vomiting, failure to thrive, convulsions, tachypnea
(rapid breathing - a key sign).
• Enzyme Deficiencies & Associated Disorders:
◦ CPS1 Deficiency: Hyperammonemia Type 1.
◦ OTC Deficiency: Hyperammonemia Type 2.
◦ ASS Deficiency: Citrullinemia Type 1 (High blood citrulline).
◦ ASL Deficiency: Argininosuccinic Aciduria (High blood/urine argininosuccinate).
◦ ARG1 Deficiency: Argininemia (High blood arginine).
• Transporter Deficiencies & Associated Disorders:
◦ Ornithine Transporter Deficiency: HHH Syndrome (Hyperornithinemia, Hyperammonemia,
Homocitrullinuria).
◦ Citrin Deficiency: Citrullinemia Type 2.
VI. Specific Disorder Details

• Hyperammonemia & Glutamine: High NH₃ leads to increased synthesis of Glutamine (transporter
form of ammonia) → High plasma Glutamine. Plasma ammonia and glutamine levels are typically higher
in Type 1 than Type 2 deficiency.
• Hyperammonemia Type 2 (OTC Deficiency):
◦ Most common UCD (approx. 40%).
◦ Inheritance: X-linked partially dominant (mainly affects males).
◦ Increased Orotic Acid in urine (Orotic Aciduria): Mitochondrial carbamoyl phosphate accumulates,
leaks to cytoplasm, enters pyrimidine synthesis pathway → excess orotic acid production.
◦ Presents with high ammonia, low citrulline, high orotic acid.
• Argininosuccinic Aciduria (ASL Deficiency):
◦ Characteristic finding: Trichorrhexis nodosa (brittle, tufted hair).
• HHH Syndrome:
◦ Defect in mitochondrial ornithine transporter.
◦ Hyperornithinemia: Ornithine accumulates in cytoplasm.
◦ Hyperammonemia: Lack of ornithine in mitochondria halts cycle.
◦ Homocitrullinuria: Excess carbamoyl phosphate reacts with lysine to form homocitrulline.
• Argininemia (ARG1 Deficiency):
◦ Often least severe hyperammonemia (last step; alternative Arginase isoform may exist).
◦ Distinct features: Progressive spastic diplegia, scissoring gait.
VII. Diagnosis of UCDs

1. Measure Blood Ammonia and Blood pH (often alkalosis).

2. If high ammonia, check Blood/Urine Specific Amino Acids:
◦ High Citrulline → Citrullinemia Type 1 (ASS Defect).
◦ High Argininosuccinate → Argininosuccinic Aciduria (ASL Defect).
◦ High Arginine → Argininemia (ARG1 Defect).
◦ High Ornithine → HHH Syndrome (Ornithine Transporter Defect).
 3. If specific amino acids (citrulline, argininosuccinate etc.) are normal/low but ammonia is high:
◦ Check Urine/Blood Orotic Acid:
▪ High Orotic Acid → Hyperammonemia Type 2 (OTC Defect).
▪ Normal/Low Orotic Acid → Hyperammonemia Type 1 (CPS1 Defect) or NAGS deficiency (not
explicitly covered but implied).
VIII. Treatment

1. Arginine Supplementation (First-line):

◦ Provides essential amino acid.
◦ Provides Ornithine (via Arginase action if present) to help drive the cycle.
◦ Stimulates NAG synthase, increasing NAG levels and activating CPS1.
◦ Contraindicated in Argininemia (ARG1 deficiency).
2. Acetylation Therapy (Ammonia Scavenging): Alternative nitrogen excretion pathways.
◦ Sodium Phenylbutyrate: Prodrug metabolized to Phenylacetate. Phenylacetate + Glutamine →
Phenylacetylglutamine (excreted in urine, removing ammonia stored in glutamine).
◦ Sodium Benzoate: Sodium Benzoate + Glycine → Benzoyl Glycine (Hippurate) (excreted in urine,
removing nitrogen via glycine).

Key Points to Remember:

• Rate-limiting Enzyme: CPS1, activated by NAG.

• Location: Liver (Mitochondria + Cytoplasm).
• Urea Sources: 1 N from NH₃, 1 N from Aspartate, C from CO₂.
• Energy Cost: 4 high-energy phosphates (3 ATP molecules).
• OTC Deficiency: Most common, X-linked, causes Orotic Aciduria.
• ASL Deficiency: Associated with Trichorrhexis Nodosa.
• Argininemia: Least hyperammonemia, spastic diplegia.
• HHH Syndrome: Ornithine transporter defect, Homocitrullinuria.
• Diagnosis: Check Ammonia, pH, specific Amino Acids, and Orotic Acid.
• Treatment: Arginine (except in Argininemia), Nitrogen Scavengers (Phenylbutyrate, Benzoate).
• Clinical Clue: Neonate with tachypnea and neurological symptoms should raise suspicion for UCD.

Summary: Aromatic Amino Acid Metabolism

(Phenylalanine & Tyrosine)
This summary details the metabolism of Phenylalanine (Phe) and Tyrosine (Tyr), focusing on pathways,
associated disorders, clinical features, diagnosis, and treatment, based strictly on the provided lecture
transcript.
I. Introduction & Importance

• Aromatic Amino Acid Metabolism carries maximum weightage for exams.

• Focus: Phenylalanine (Phe) & Tyrosine (Tyr) metabolism.
 • Clinical Relevance Examples:
◦ Parkinson's Disease: Symptoms (pin-rolling tremor, cogwheel rigidity, bradykinesia, speech
difficulties, shuffling gait) suggest a lack of a chemical derivative of an aromatic amino acid (later
identified as Dopamine from Tyrosine).
◦ Phenylketonuria (PKU): Infant case (twitching, mousy body odor, positive Guthrie's test). Linked to
Phe/Tyr metabolism.
II. Phenylalanine (Phe) & Tyrosine (Tyr): Basics

• Structures:
◦ Phe: Alanine backbone + CH2 + Benzene Ring (Phenyl group).
◦ Tyr: Alanine backbone + CH2 + Benzene Ring + Hydroxyl (-OH) group. (Tyr = Hydroxylated Phe).
• Properties: | Feature | Phenylalanine (Phe) | Tyrosine (Tyr) | | :---------------- | :-------------------------
| :--------------------------------- | | Essentiality | Essential | Non-essential (from Phe) | | Metabolic Fate |
Ketogenic & Glucogenic | Ketogenic & Glucogenic | | Polarity | Non-polar | Non-polar (Least non-
polar among non-polar aromatic AAs due to -OH). |
• Note: Histidine is an aromatic AA but is classified as polar.
III. Metabolism Overview: Phe & Tyr are "Yoked Together"

• Phe -> Tyr Conversion: Phe must be converted to Tyr to enter metabolic fates (catabolic or anabolic).
• Tyrosine Fates:
◦ Catabolic: Via Tyrosine Transaminase -> Leads to Ketogenic (Acetoacetate) and Glucogenic
(Fumarate) products.
◦ Anabolic:
▪ Via Tyrosine Hydroxylase -> Catecholamines (Dopamine, NE, Epi).
▪ Via Tyrosinase -> Melanin (Pigment).
▪ Involved in Thyroxine (Thyroid Hormone) synthesis.
IV. Catabolic Pathway: Phenylalanine -> Fumarate + Acetoacetate

1. Phe -> Tyr:

◦ Enzyme: Phenylalanine Hydroxylase (PAH)
◦ Cofactor: Tetrahydrobiopterin (BH4) (derived from GTP). BH4 is oxidized to Dihydrobiopterin
(BH2).
◦ Regeneration: Dihydrobiopterin Reductase converts BH2 back to BH4 (requires NADPH).
◦ Characteristics: Irreversible reaction, Monooxygenase. Acts as a one-way "valve".
2. Tyr -> p-Hydroxyphenylpyruvate:
◦ Enzyme: Tyrosine Transaminase (TT)
◦ Cofactor: PLP (Pyridoxal Phosphate).
◦ Reaction: Transamination (α-ketoglutarate -> Glutamate).
3. p-Hydroxyphenylpyruvate -> Homogentisate:
◦ Enzyme: p-Hydroxyphenylpyruvate (PHPP) Hydroxylase
◦ Cofactor: Vitamin C (Ascorbate).
◦ Characteristics: Dioxygenase.
4. Homogentisate -> Maleylacetoacetate (MAA):
◦ Enzyme: Homogentisate Oxidase
 ◦ Cofactor: Iron (Fe).
5. MAA -> Fumarylacetoacetate (FAA):
◦ Enzyme: MAA cis-trans Isomerase
◦ Cofactor: GSH (Glutathione).
6. FAA -> Fumarate + Acetoacetate:
◦ Enzyme: FAA Hydrolase
◦ Products: Fumarate (Glucogenic), Acetoacetate (Ketogenic).
V. Disorders of Catabolism (Mapped to Pathway Steps)

• PAH Defect: Classic Phenylketonuria (PKU).

• BH4 Reductase Defect: Non-classic PKU (Type 2 & 3).
• BH4 Synthesis Defect (from GTP): Non-classic PKU (Type 4 & 5); Segawa Syndrome.
• Tyrosine Transaminase Defect: Tyrosinemia Type 2.
• PHPP Hydroxylase Defect: Tyrosinemia Type 3.
• Mutant/Partially Active PHPP Hydroxylase: Hawkinsinuria.
• Homogentisate Oxidase Defect: Alkaptonuria.
• FAA Hydrolase Defect: Tyrosinemia Type 1.
VI. Phenylketonuria (PKU) In-Depth

• Biochemical Defect: PAH deficiency. Leads to ↑ Phe, ↓ Tyr.

• Alternate Phe Metabolism: Phe enters minor pathways:
◦ Phe -> Phenylpyruvate (Transamination) -> Phenyl lactate (Reduction) / Phenylacetate (Oxidation).
◦ Phenylacetate -> Conjugation with Glutamine -> Phenylacetylglutamine.
• Key Features:
◦ Phenylketonuria Name: Due to excretion of Phenylpyruvate (a ketoacid).
◦ Mousy/Musty Body Odor: Due to Phenylacetate.
◦ Hypopigmentation: ↓ Tyr -> ↓ Melanin (fair skin, blonde hair, blue eyes). Not complete absence due
to dietary Tyr.
◦ Neurological Deficits: Intellectual disability/delay, hyperactivity, agitation, seizures.
▪ Mechanism 1: ↓ Tyr -> ↓ Catecholamines (neurotransmitters) & Thyroxine.
▪ Mechanism 2: Blood-Brain Barrier (BBB) Competition: High blood Phe saturates the Large
Neutral Amino Acid (LNAA) transporter, blocking entry of dietary Tyr and Tryptophan (Trp) into
the brain -> ↓ brain Tyr & Trp -> ↓ synthesis of Dopamine, NE, Epi (from Tyr) and Serotonin
(from Trp).
◦ Infancy Presentation: Intractable vomiting (may mimic Congenital Hypertrophic Pyloric Stenosis -
CHPS).
◦ Eczematous rash.
• Lab Diagnosis:
◦ Guthrie Test (Bacterial Inhibition Assay): Newborn screening (heel prick blood). Uses Bacillus
subtilis (requires Phe). Growth indicates ↑ Phe.
◦ Ferric Chloride Test (Urine): Add FeCl3 -> Transient Blue-Green color.
◦ Blood Phenylalanine Levels: Quantitative. Normal: 2-6 mg/dL. PKU: > 20 mg/dL (indicates poor
prognosis if untreated).
 ◦ Tandem Mass Spectrometry (TMS): Gold standard neonatal screening for multiple IEMs.
◦ Enzyme assays/Genetic testing.
• Treatment:
◦ Dietary Phenylalanine Restriction: Lifelong. Special formulas, low-Phe foods (e.g., Cassava-
based).
◦ Large Neutral Amino Acid (LNAA) Concentrate: Supplement provides high Tyr, Trp etc. to
compete with Phe at BBB, improving neurotransmitter synthesis.
◦ Sapropterin Dihydrochloride (Kuvan): Synthetic BH4. Effective for BH4-responsive PKU.
◦ Enzyme Replacement Therapy (Under Trial): Phenylalanine Ammonia Lyase (PAL).
VII. Alkaptonuria (Black Urine Disease / Ochronosis) In-Depth

• Biochemical Defect: Homogentisate Oxidase deficiency. Leads to ↑ Homogentisate.

• Pathophysiology: Homogentisate accumulates -> Auto-oxidizes to Benzoquinone Acetate ->
Polymerizes into Alkapton Bodies (dark pigment).
• Clinical Features:
◦ Urine Darkening: On standing/exposure to air or alkali (freshly voided urine is normal). May cause
black/reddish diaper stains; worsens when washing with soap (alkaline).
◦ Ochronosis: Bluish-black pigmentation deposition in connective tissues (sclera, ear cartilage, nose).
◦ Arthritis: Pigment deposition in articular cartilage -> severe, early-onset osteoarthritis, particularly
in the spine and large joints (back pain due to IV disc involvement).
◦ NO Mental Retardation.
◦ Usually presents in middle age.
• Garrod's Tetrad: One of the first 4 IEMs described by Garrod (Mnemonic: CAAP - Cystinuria,
Alkaptonuria, Albinism, Pentosuria).
• Lab Diagnosis:
◦ Urine darkens upon alkalinization (e.g., adding NaOH).
◦ Ferric Chloride Test: Positive.
◦ Silver Nitrate Test: Positive (Homogentisate reduces Ag+).
◦ X-ray Spine: Shows calcification, characteristic changes ("parrot beak" appearance - not explicitly
mentioned, but classic).
• Treatment:
◦ Nitisinone (NTBC): Inhibits PHPP Hydroxylase (enzyme upstream of the defect), reducing
homogentisate production.
◦ Symptomatic management of arthritis.
VIII. Segawa Syndrome (Dopa-Responsive Dystonia)

• Biochemical Defect: GTP Cyclohydrolase deficiency (involved in BH4 synthesis).

• Inheritance: Autosomal Dominant.
• Clinical Features: Dystonia with Marked Diurnal Variation (worse in evening, improves with sleep/
morning). Females > Males.
• Key Biochemical Finding: ↓ BH4 levels BUT Normal Blood Phenylalanine.
• Treatment: Responds well to low-dose L-DOPA.
IX. Tyrosinemias In-Depth

• Tyrosinemia Type 1 (Hepatorenal Tyrosinemia): Most Common Type.

◦ Defect: FAA Hydrolase.
◦ Accumulation: Succinylacetone (toxic metabolite).
◦ Features: Severe Liver Failure, Renal tubular disease, high risk of hepatocellular carcinoma,
neurological crises (resembling porphyria).
◦ Odor: Boiled Cabbage (due to disturbed methionine metabolism).
◦ Treatment: Nitisinone (NTBC), low Phe/Tyr diet, liver transplant.
• Tyrosinemia Type 2 (Oculocutaneous / Richner-Hanhart Syndrome):
◦ Defect: Tyrosine Transaminase (TT).
◦ Features: Ocular (corneal ulcers/plaques), Cutaneous (painful, non-pruritic Hyperkeratotic Plaques
on palms/soles). Possible intellectual disability.
◦ Treatment: Low Phe/Tyr diet.
• Tyrosinemia Type 3: Least Common Type.
◦ Defect: PHPP Hydroxylase.
◦ Features: Usually milder; neurological issues (ataxia, seizures).
◦ Treatment: Low Phe/Tyr diet.
X. Hawkinsinuria

• Biochemical Defect: Mutant, Partially Active PHPP Hydroxylase.

• Accumulation: Hawkinsin.
• Features: Metabolic acidosis, ketosis, failure to thrive (often transient, in infancy).
• Odor: Swimming Pool odor in urine.
XI. Anabolic Fates of Tyrosine & Related Disorders

• A. Catecholamine Synthesis (Dopamine, Norepinephrine, Epinephrine):

◦ Site: Chromaffin cells (Adrenal Medulla - mostly Epi; Extra-adrenal sites/Sympathetic nerves -
mostly NE).
◦ Pathway:
1. Tyr -> DOPA (Enzyme: Tyrosine Hydroxylase, requires BH4, Rate-limiting).
2. DOPA -> Dopamine (Enzyme: DOPA Decarboxylase / Aromatic L-AA Decarboxylase, requires
PLP). Dopamine = First Catecholamine.
3. Dopamine -> Norepinephrine (NE) (Enzyme: Dopamine β-Hydroxylase, requires Vit C, Cu).
4. NE -> Epinephrine (Epi) (Enzyme: PNMT - Phenylethanolamine N-Methyltransferase, requires
SAM from Methionine). Methylation step.
◦ Catabolism: Via MAO (Monoamine Oxidase) & COMT (Catechol-O-Methyltransferase). End
products: HVA (Homovanillic Acid) from Dopamine, VMA (Vanillylmandelic Acid) from NE/Epi.
◦ Disorder: Pheochromocytoma:
▪ Tumor of Adrenal Medulla -> Excess catecholamine secretion.
▪ Symptoms (Classic Triad): Palpitations, Headache, Episodic Sweating (PHEo). Hypertension.
▪ Diagnosis (Biochemical): ↑ Urinary/Plasma Metanephrines (fractionated/free - HIGHLY
sensitive), ↑ VMA, ↑ Catecholamines.
 • B. Melanin Synthesis:
◦ Site: Melanosomes in Melanocytes.
◦ Pathway:
1. Tyr -> DOPA (Enzyme: Tyrosinase, requires Cu).
2. DOPA -> Dopaquinone (Enzyme: Tyrosinase).
3. Dopaquinone -> -> Melanin.
◦ Function: Pigmentation (skin, hair, eyes).
◦ Disorder: Albinism (Oculocutaneous Albinism - OCA):
▪ Defect: Tyrosinase deficiency (most common form).
▪ Features: Complete lack of melanin (Depigmentation): white hair, milky skin, red eyes,
photophobia, nystagmus, vision problems.
▪ Contrast: PKU = Hypopigmentation; Albinism = Depigmentation.
• C. Thyroxine (Thyroid Hormone) Synthesis:
◦ Site: Thyroid follicles, on Thyroglobulin protein (contains ~115 Tyr residues).
◦ Process:
1. Iodination of Tyr residues -> MIT (Monoiodotyrosine) & DIT (Diiodotyrosine).
2. Coupling: MIT + DIT -> T3 (Triiodothyronine); DIT + DIT -> T4 (Thyroxine).
XII. Key Points to Remember

• Phe is essential; Tyr is non-essential (made from Phe via PAH).

• PAH is irreversible and requires BH4 (GTP-derived, regenerated by BH4 Reductase + NADPH).
• PKU: PAH defect -> ↑Phe, ↓Tyr. Mousy odor (phenylacetate), Neurotoxicity (BBB competition),
Hypopigmentation. Dx: Guthrie/TMS. Tx: Diet restriction, LNAA, Kuvan.
• Alkaptonuria: Homogentisate Oxidase defect -> ↑Homogentisate. Ochronosis (pigment deposition),
Arthritis, Urine darkens on standing. Tx: Nitisinone.
• Tyrosinemia Type 1: FAA Hydrolase defect -> Succinylacetone. Liver/Kidney failure, Cabbage odor.
Tx: Nitisinone.
• Albinism: Tyrosinase defect -> No melanin. Complete depigmentation.
• Pheochromocytoma: Adrenal tumor -> Excess catecholamines. PHE symptoms (Palpitations, Headache,
Episodic sweating), HTN. Dx: Metanephrines.
• Key Cofactors: BH4 (PAH, Tyr Hydroxylase), PLP (Transaminases, DOPA Decarboxylase), Vit C
(PHPP Hydroxylase, Dopamine β-Hydroxylase), Copper (Dopamine β-Hydroxylase, Tyrosinase), SAM
(PNMT), GSH (MAA Isomerase), Iron (Homogentisate Oxidase).
• LNAA Transporter: Crucial for BBB transport of Phe, Tyr, Trp; competition is key in PKU
pathophysiology and treatment.

Glycine & Serine Metabolism Summary

I. Introduction & Motivation * Core Message: Practice medicine for the love of it, not solely for money.
Ethics, attitude, and communication are crucial soft skills alongside clinical knowledge. * Importance of
Glycine/Serine: Despite being simple, these amino acids are vital precursors for many essential
biomolecules. * Key Questions Addressed: * Which amino acid is needed for Heme synthesis? (Glycine) *
Can amino acids conjugate xenobiotics? (Yes, Glycine) * Why oxalate stones in Vitamin B6 deficiency?
(Link via Glycine metabolism) * Which amino acid is fully incorporated into the Purine ring? (Glycine)
II. Glycine Metabolism

• A. Overview (Bird's Eye View)

◦ Nature: Non-essential, purely Glucogenic amino acid.
◦ Synthesis Sources:
1. Serine (major)
2. Glyoxalate (its keto acid)
3. Amphibolic intermediates (via Glycine Synthase)
4. Threonine
◦ Catabolic Fates:
1. Conversion to Serine -> Pyruvate (Glucogenic path)
2. Glycine Cleavage System (GCS): Breaks down into smaller units (CO2, NH3, one-carbon unit).
◦ Anabolic Fates (Products Derived):
1. Purines (C4, C5, N7 atoms)
2. Creatine / Creatinine
3. Heme
4. Glutathione
5. Conjugation agent (bile acids, xenobiotics)
• B. Chemistry
◦ Structure: Simplest amino acid (H atom as R-group: NH2-CH2-COOH).
◦ Unique Feature: No asymmetric (chiral) carbon, hence optically inactive.
◦ Classification: Non-essential, Glucogenic, generally classified as Polar (though has some non-polar
character).
• C. Synthesis Details
1. From Serine:
▪ Enzyme: Serine Hydroxymethyltransferase (SHMT)
▪ Cofactors: PLP (Vitamin B6) and Tetrahydrofolate (THFA - Folic Acid derivative)
▪ Reaction: Serine <=> Glycine + N5,N10-Methylene THFA
▪ Significance: Reversible. The N5,N10-Methylene THFA produced is a primary entry point for
the one-carbon pool. The beta-carbon of Serine is transferred.
2. From Glyoxylate:
▪ Reaction: Transamination (Glyoxylate + Amino Donor -> Glycine + Keto Acid)
▪ Primary Enzyme: Alanine-Glyoxylate Aminotransferase (AGT)
▪ Amino Donor: Alanine (-> Pyruvate)
▪ Cofactor: PLP (Vitamin B6)
▪ Other Donor: Glutamate (-> alpha-Ketoglutarate)
3. From Threonine:
▪ Enzyme: Threonine Aldolase
4. From Amphibolic Intermediates:
▪ Enzyme System: Glycine Synthase (reverse of GCS)
• D. Catabolism Details
1. Conversion to Serine -> Pyruvate: Standard pathway leading to glucose synthesis potential.
2. Glycine Cleavage System (GCS):
▪ Nature: Multi-enzyme complex located in mitochondria. Contains H-protein carrier.
▪ Function: Splits glycine into CO2, NH3 (implied), and transfers a one-carbon unit to THFA.
▪ Components/Steps:
▪ Glycine Dehydrogenase (Decarboxylating): Removes -COOH as CO2. Requires PLP.
▪ Amino Methyltransferase: Handles the remaining C-N part, transferring the methylene carbon.
▪ One-carbon unit transferred -> N5,N10-Methylene THFA.
▪ Dihydrolipoamide Dehydrogenase: Involved in cofactor regeneration (part of the complex).
▪ Reversibility: GCS running in reverse is Glycine Synthase.
• E. Anabolic Fates (Specialized Products)
1. Creatine/Creatinine Synthesis: Involves 3 amino acids: Glycine, Arginine, Methionine (as SAM).
Mnemonic: GAM creates Creatine.
▪ Step 1 (Kidney): Glycine + Arginine -> Guanidinoacetate. Enzyme: Arginine:Glycine
Amidinotransferase (AGAT).
▪ Step 2 (Liver): Guanidinoacetate + SAM -> Creatine. Enzyme: Guanidinoacetate
Methyltransferase (GAMT). (SAM -> SAH).
▪ Step 3 (Muscle): Creatine --(ATP->ADP, via Creatine Kinase*)--> Creatine Phosphate (CP).
▪ Creatine Phosphate: High-energy compound (Phosphagen), immediate ATP buffer in muscle
(first few seconds of exercise).
▪ Creatinine Formation (Muscle): Creatine Phosphate -> Creatinine (spontaneous, non-enzymatic
cyclization). Excreted by kidneys. Lohmann's Reaction refers to the reversible phosphorylation of
Creatine by ATP. (*Creatine Kinase not explicitly named but implied by ATP/ADP cycle and
Lohmann's Reaction mention).
2. Heme Synthesis: Glycine + Succinyl CoA are the initial substrates.
3. Glutathione Synthesis: Glycine is the C-terminal amino acid (Gamma-glutamyl-cysteinyl-glycine).
4. Purine Synthesis: Glycine contributes atoms C4, C5, and N7 to the purine ring. Mnemonic: Glycine
sits in the middle C4-C5-N7. (Note: Glycine is NOT involved in Pyrimidine synthesis).
5. Neurotransmitter: Acts as both inhibitory (spinal cord) and excitatory neurotransmitter.
6. Conjugation:
▪ Bile Acids: Forms conjugates like Glycocholic acid.
▪ Xenobiotics: e.g., Benzoic acid (as Benzoyl CoA) + Glycine -> Hippuric Acid (detoxification
product, excreted in urine).
7. Collagen Structure: Most abundant amino acid in collagen, creates bends/kinks in the protein
structure.
• F. Clinical Correlations (Glycine)
1. Hyperoxaluria (Oxalate Stones): Excess oxalate excretion.
▪ Primary Hyperoxaluria (Genetic):
▪ Type 1: Defect in Alanine:Glyoxylate Aminotransferase (AGT) (PLP-dependent).
Glyoxalate accumulates -> converted to Oxalate. Peroxisomal targeting defect.
▪ Type 2: Defect in Glyoxylate Reductase / Glycerate Dehydrogenase. Glyoxalate
accumulates -> Oxalate.
 ▪ Secondary Hyperoxaluria (Acquired):
▪ Vitamin B6 Deficiency: Impairs AGT function -> Glyoxalate buildup.
▪ Vitamin C Toxicity (Excess intake).
▪ Methoxyflurane anesthesia toxicity.
▪ Ethylene Glycol Poisoning (Antifreeze). Metabolized to oxalate.
▪ Enteric Hyperoxaluria (e.g., after intestinal resection, fat malabsorption leads to increased
oxalate absorption).
2. Non-Ketotic Hyperglycinemia (NKH):
▪ Cause: Defect in the Glycine Cleavage System (GCS).
▪ Result: Accumulation of Glycine in body fluids (blood, CSF), leading to severe neurological
symptoms.
III. Serine Metabolism

• A. Chemistry
◦ Structure: Contains a hydroxyl (-OH) group on the beta-carbon (NH2-CH(CH2OH)-COOH).
◦ Classification: Polar, uncharged, Non-essential, purely Glucogenic.
◦ Significance: Hydroxyl group is a key site for phosphorylation and glycosylation.
• B. Synthesis
1. From Glycine:
▪ Reverse reaction of Glycine synthesis from Serine.
▪ Enzyme: Serine Hydroxymethyltransferase (SHMT).
▪ Requires: N5,N10-Methylene THFA, PLP.
2. From 3-Phosphoglycerate (3-PG):
▪ Source: Intermediate of Glycolysis. (Pathway details not covered).
• C. Anabolic Fates (Specialized Products)
1. One-Carbon Metabolism: Primary donor of one-carbon units via conversion to Glycine (releases
N5,N10-Methylene THFA).
2. Cysteine Synthesis: Serine condenses with Homocysteine (from Methionine metabolism) in the
transsulfuration pathway. (Serine + Homocysteine -> Cystathionine -> Cysteine).
3. Phospholipid Synthesis: Precursor for Phosphatidylserine.
4. Sphingolipid Synthesis: Serine + Palmitoyl CoA -> Sphingosine (the backbone alcohol of
sphingolipids like sphingomyelin and glycosphingolipids). Mnemonic: Serene Sphynx sits on Palm
trees.
5. Selenocysteine Synthesis: Serine is the carbon backbone precursor for this "21st" amino acid.
Mnemonic: Sister Selena (Serine -> Selenocysteine).
6. Ethanolamine, Choline, Betaine Synthesis:
▪ Serine --(Decarboxylation, PLP)--> Ethanolamine.
▪ Ethanolamine --(Methylation x3 via SAM)--> Choline (Trimethyl-ethanolamine).
▪ Choline --(Oxidation)--> Betaine (Trimethylglycine).
7. Glycoprotein Synthesis: Serine residues (along with Threonine) are sites for O-linked glycosylation
(sugar attachment via the -OH group).
8. Protein Phosphorylation: Serine's -OH group is a major site for reversible phosphorylation, a key
mechanism for regulating enzyme activity (covalent modification).
IV. Important Points to Remember

• Glycine: Simplest, optically inactive, Glucogenic, Non-essential. Key roles in Heme, Purines
(C4,C5,N7), Creatine, Glutathione, Conjugation (Hippuric acid).
• Serine: Hydroxyl-containing, Polar, Glucogenic, Non-essential. Key roles as primary 1-C donor,
Cysteine synthesis, Phospholipids (Phosphatidylserine), Sphingolipids (Sphingosine), Ethanolamine/
Choline, Selenocysteine precursor, O-linked glycosylation, Phosphorylation site.
• Interconversion: Glycine <=> Serine via SHMT (PLP, THFA dependent) is central, linking their
metabolism and the one-carbon pool.
• Glycine Cleavage System (GCS): Major catabolic route for Glycine; defect causes Non-Ketotic
Hyperglycinemia (NKH).
• Hyperoxaluria: Linked to Glycine metabolism via Glyoxalate. Caused by enzyme defects (Primary Type
1 AGT, Type 2 GR/GDH) or secondary factors (Vit B6 deficiency, Vit C toxicity, Ethylene Glycol).
• PLP (Vitamin B6): Crucial cofactor for SHMT, AGT, Serine Decarboxylase, and many other amino acid
reactions. Deficiency impacts multiple pathways, including causing secondary hyperoxaluria.
• THFA (Folate): Essential for accepting/donating one-carbon units in the Glycine-Serine interconversion.
• Creatine Synthesis: Requires Glycine, Arginine, Methionine (SAM) across Kidney and Liver. Creatine
Phosphate is muscle's energy buffer.
• Purine vs. Pyrimidine: Glycine is heavily involved in Purine synthesis, but NOT in Pyrimidine
synthesis.

Summary: Sulfur-Containing Amino Acids Metabolism &

Pathology
I. Introduction

• Topic: Sulfur-containing amino acids (Cysteine & Methionine).

• Importance: Clinically relevant, bridging core and clinical biochemistry.
• Approach: Learn basics, then clinical correlations, starting with a clinical problem (Homocystinuria).
II. Clinical Case Presentation (Classic Homocystinuria)

• Initial Symptoms: Often normal at birth, slight developmental delay. Around 3 years: severe myopia,
quivering of the iris (Iridodonesis).
• Key Ocular Finding: Downward and medial dislocation of the lens (Ectopia Lentis).
• Systemic Features:
◦ Skeletal: Waddling gait, decreased bone mineralization, increased length of long bones
(Arachnodactyly), scoliosis (curvature of spine), Pectus excavatum (inward sternum) or Pectus
carinatum (outward sternum), Genu valgum/varum (knee deformities), high arched palate.
◦ Neurological/Cognitive: Poor scholastic performance, potential mental retardation.
◦ Vascular: Risk of thromboembolism (leading to CVA, CAD).
• Resembles: Marfan Syndrome, but lens dislocation is different (Mnemonic: Homocysteine Down,
Marfan's Up).
III. Chemistry of Sulfur-Containing Amino Acids

• Methionine (Met):
◦ Structure: Contains a thioether linkage (-S-CH3).
 ◦ Properties: Essential, purely glucogenic, non-polar.
◦ Sulfur Test: Negative (Sulfur not easily liberated).
• Cysteine (Cys):
◦ Structure: Contains a sulfhydryl group (-SH) (also called thiol or thioalcohol).
◦ Properties: Non-essential, purely glucogenic, polar (ionizable SH group).
◦ Sulfur Test: Positive (SH group reacts to form lead sulfide).
IV. Methionine Metabolism

1. Methionine → S-Adenosyl Methionine (SAM)

◦ Enzyme: Methionine Adenosyltransferase (MAT) (Isoforms: MAT1/3 hepatic, MAT2
extrahepatic).
◦ Process: Activation of methionine. The S-CH3 bond in Met is stable; SAM formation makes the
methyl group labile and ready for donation.
◦ SAM: The principal methyl donor.
◦ Clinical Correlation (MAT defect): Primary Hypermethioninemia (Characteristic "boiled
cabbage smell").
◦ Clinical Correlation (Transport defect): Defect in intestinal methionine transport → Oasthouse
Syndrome (Smith-Strang Disease).
2. SAM → S-Adenosyl Homocysteine (SAH)
◦ Process: SAM donates its methyl group (Transmethylation).
◦ Enzyme: Various Methyltransferases.
◦ Significance of SAM/Transmethylation:
▪ Methylation Reactions: Formation of Creatine (from Guanidinoacetate), Epinephrine (from
Norepinephrine), Melatonin (from Acetylserotonin), Choline (from Ethanolamine), Anserine
(from Carnosine).
▪ Polyamine Synthesis:
▪ Polyamines (e.g., Putrescine, Spermidine, Spermine) are positively charged molecules
involved in DNA interaction and gene regulation.
▪ Synthesized from Ornithine (via Ornithine Decarboxylase - Rate-limiting step) and
Methionine (as decarboxylated SAM).
▪ Note: Cadaverine is from Lysine.
▪ Precursors can be traced back to Arginine (source of Ornithine) and Methionine.
▪ DNA Methylation: Epigenetic regulation.
3. SAH → Homocysteine
◦ Enzyme: Adenosylhomocysteinase.
◦ Process: Removal of adenosine.
V. Fates of Homocysteine

1. Remethylation to Methionine:
◦ Methyl Donor: N5-Methyl-THF (Tetrahydrofolate).
◦ Coenzyme: Vitamin B12 (Cobalamin) - acts as an intermediate carrier (B12 → Methyl-B12 → B12).
◦ Enzyme: Methionine Synthase.
 ◦ Clinical Significance (B12/Folate Deficiency):
▪ Folate Trap: N5-Methyl-THF accumulates, functional deficiency of other THF forms.
▪ Impaired DNA synthesis → Megaloblastic Anemia.
▪ Homocysteine Accumulation: Leads to hyperhomocysteinemia and homocystinuria → Increased
risk of thrombosis (CAD, CVA).
2. Transsulfuration Pathway to Cysteine:
◦ Step 1: Homocysteine + Serine → Cystathionine
▪ Enzyme: Cystathionine Beta-Synthase (CBS).
▪ Coenzyme: PLP (Vitamin B6).
▪ Clinical Correlation (CBS Defect): Classic Homocystinuria.
◦ Step 2: Cystathionine → Cysteine + Homoserine
▪ Enzyme: Cystathionase.
▪ Coenzyme: PLP (Vitamin B6).
▪ Clinical Correlation (Cystathionase Defect): Cystathioninuria.
◦ Fate of Homoserine: Converted to Propionyl-CoA → Succinyl-CoA (enters TCA cycle) →
Glucogenic.
◦ Clinical Significance (B6 Deficiency): Can also cause Homocysteine accumulation and thrombosis
risk.
◦ Summary: Cysteine synthesis requires Methionine (as Homocysteine) and Serine.
VI. Homocystinurias

• Classic Homocystinuria:
◦ Defect: CBS deficiency.
◦ Biochemistry: ↑ Homocysteine, ↓ Cysteine, Normal/↑ Methionine.
◦ Clinical Features: As described in section II.
◦ Diagnosis: Cyanide Nitroprusside Test (Positive - magenta color), Tandem Mass Spectrometry
(Screening), Enzyme Assay.
◦ Treatment: High dose Vitamin B6 (pyridoxine) if responsive, Methionine restriction, Cysteine
supplementation, Betaine (remethylates homocysteine), Vitamin C.
• Non-Classic Homocystinuria:
◦ Defects:
1. MTHFR (Methylene Tetrahydrofolate Reductase) deficiency (impaired N5-Methyl-THF
synthesis).
2. Defect in Methylcobalamin (B12) formation/metabolism.
◦ Biochemistry: ↑ Homocysteine, ↓ Methionine, Normal Cysteine.
◦ Megaloblastic Anemia: Present in Methyl-B12 defects, Absent in MTHFR deficiency and Classic
Homocystinuria.
VII. Other Related Disorders

• Cystathioninuria:
◦ Defect: Cystathionase.
◦ Diagnosis: Cyanide Nitroprusside test is Negative.
 • Cystinuria:
◦ Defect: Impaired transport of dibasic amino acids (Cystine, Ornithine, Lysine, Arginine -
Mnemonic: C.O.L.A.) in kidney tubules and intestine.
◦ Features: Excretion of C.O.L.A. in urine, risk of cystine kidney stones.
◦ Diagnosis: Cyanide Nitroprusside test Positive (due to Cystine).
◦ Part of Garrod's Tetrad (Cystinuria, Alkaptonuria, Albinism, Pentosuria).
• Cystinosis:
◦ Type: Lysosomal Storage Disorder.
◦ Defect: Defective lysosomal cystine transporter (Cystinosin, encoded by CTNS gene).
◦ Features: Accumulation of cystine crystals in lysosomes → multi-organ damage (Kidney - renal
failure, Liver, Cornea - opacities, Bone marrow).
◦ Treatment: Cysteamine (helps remove cystine from lysosomes).
VIII. Cysteine - Specialized Products & Functions

• Beta-mercaptoethylamine: From Cysteine decarboxylation.

• Coenzyme A (CoA): Contains cysteine residue; the SH group is the active site.
• Taurine: Involved in bile acid conjugation.
• Cystine: Formed by linking two Cysteine molecules via a disulfide bond (-S-S-).
• Glutathione (GSH): See below.
IX. Glutathione (GSH)

• Structure: Tripeptide (Gamma-Glutamyl-Cysteinyl-Glycine). A "pseudo-peptide" due to the gamma-

glutamyl linkage.
• Active Group: The -SH group of the Cysteine residue.
• Forms: Reduced (GSH), Oxidized (GSSG).
• Functions:
◦ Antioxidant / Free Radical Scavenger: Detoxifies H2O2 and other reactive oxygen species.
◦ Maintains RBC membrane integrity.
◦ Keeps Hemoglobin iron in Fe2+ state (prevents Methemoglobin formation).
◦ Amino Acid Transport (Meister Cycle / Gamma-glutamyl cycle).
◦ Phase II Xenobiotic conjugation.
◦ Coenzyme (e.g., Maleylacetoacetate isomerase).
• Key Enzymes in GSH Redox Cycle:
◦ Glutathione Peroxidase: (GSH → GSSG while reducing H2O2). Requires Selenium.
◦ Glutathione Reductase: (GSSG → GSH). Requires NADPH (from HMP Shunt) and FAD
(Riboflavin/Vitamin B2).
◦ Clinical Note: Erythrocyte Glutathione Reductase activity assesses Vitamin B2 status.
X. Important Points to Remember

• Essential vs Non-Essential: Methionine is Essential, Cysteine is Non-Essential (can be synthesized from

Met + Ser).
• Key Cofactors: B12 and Folate for remethylation; B6 (PLP) for transsulfuration. Deficiencies lead to
↑Homocysteine.
• Homocysteine: High levels are a risk factor for thrombosis.
 • Classic Homocystinuria: CBS defect, ↓Cys, Normal Met, downward lens dislocation, B6/Betaine
treatment.
• Cystinuria: C.O.L.A. transporter defect, cystine stones, +ve Nitroprusside.
• Cystinosis: Lysosomal storage, Cystinosin defect, multi-organ damage.
• Glutathione (GSH): Key antioxidant, requires Cysteine, Glycine, Glutamate. Needs Selenium
(Peroxidase) and Riboflavin/B2 (Reductase).
• SAM: Universal methyl donor.
• Diagnostic Tests: Cyanide Nitroprusside (detects -SH/-S-S-, positive in Homocystinuria, Cystinuria;
negative in Cystathioninuria). Tandem mass spectrometry for screening.

Tryptophan Metabolism Summary

Introduction & Importance

• Tryptophan is a key aromatic amino acid to study after Phenylalanine and Tyrosine.
• Understanding its metabolism helps solve clinical problems related to Pellagra (Niacin deficiency),
Carcinoid Syndrome, and Hartnup Disease.

Tryptophan: Basic Chemistry

• Structure: Contains an alpha-amino group (NH2), a carboxyl group (COOH), and a unique side chain.
◦ Side Chain: Indole ring (formed by fusion of a Benzene ring and a Pyrrole ring) attached via a CH2
group.
• Properties:
◦ Aromatic amino acid.
◦ Essential amino acid (must be obtained from diet).
◦ Both Ketogenic and Glucogenic.

Overview of Tryptophan Metabolism

Tryptophan metabolism follows two main fates:

1. Catabolic Fate:
◦ Leads to Ketogenic and Glucogenic products.
◦ Key enzyme channeling towards catabolism: Tryptophan Pyrrolase (also called Tryptophan
Oxygenase).
2. Anabolic Fate:
◦ Synthesis of important compounds:
▪ Niacin (Vitamin B3): Rate-limiting enzyme is QPRTase (Quinolinate
Phosphoribosyltransferase).
▪ Serotonin: Rate-limiting enzyme is Tryptophan Hydroxylase.
▪ Melatonin (derived from Serotonin): Initial channeling enzyme is also Tryptophan
Hydroxylase.

Catabolic Pathway (Kynurenine-Anthranilate Pathway)

• Alternate Name: Kynurenine-Anthranilate Pathway (due to key intermediates).

 • Steps & Enzymes:
1. Tryptophan → N-Formylkynurenine
▪ Enzyme: Tryptophan Pyrrolase (or Tryptophan Oxygenase).
▪ Requires Heme (contains Iron).
2. N-Formylkynurenine → Kynurenine
▪ Enzyme: Formamidase (implicitly, via Formyltransferase action).
▪ The formyl group is transferred to Tetrahydrofolate (THF), forming Formyl-THFA. (Tryptophan
donates formyl group to one-carbon metabolism).
3. Kynurenine → 3-Hydroxykynurenine
▪ Enzyme: Kynurenine Hydroxylase.
▪ Requires NADPH.
4. 3-Hydroxykynurenine → 3-Hydroxyanthranilate
▪ Enzyme: Kynureninase.
▪ Requires PLP (Vitamin B6).
▪ Releases Alanine (contributing to the Glucogenic fate).
5. 3-Hydroxyanthranilate has two fates:
▪ Further catabolism (leading to Ketogenic products - details not provided in lecture).
▪ Conversion to NAD+ (Niacin) via multiple steps.
▪ Rate-limiting enzyme for Niacin synthesis branch: QPRTase (Quinolinate
Phosphoribosyltransferase).
• Clinical Relevance of B6 (PLP) Deficiency:
◦ Kynureninase activity decreases.
◦ Pathway intermediates accumulate: 3-Hydroxykynurenine is shunted towards Xanthurenic acid,
which is excreted in urine (Xanthurenic aciduria).
◦ Decreased Niacin (NAD+) synthesis, leading to Pellagra-like symptoms. This explains why B6
deficiency can cause Pellagra.
◦ Mnemonic for Pellagra: Remember the 4 D's - Dermatitis (photosensitive), Diarrhea, Dementia,
Death.
• Tryptophan to Niacin Conversion:
◦ 60 mg of Tryptophan yields 1 mg of Niacin.

Anabolic Pathways
1. Serotonin (5-Hydroxytryptamine / 5-HT) Synthesis

• Steps & Enzymes:

1. Tryptophan → 5-Hydroxytryptophan
▪ Enzyme: Tryptophan Hydroxylase.
▪ Requires Tetrahydrobiopterin (BH4) and NADPH. (This is the rate-limiting step).
2. 5-Hydroxytryptophan → 5-Hydroxytryptamine (Serotonin)
▪ Enzyme: Aromatic Amino Acid Decarboxylase (removes CO2).
▪ Requires PLP (Vitamin B6) - Note: Lecture only mentions PLP for Kynureninase, but this
enzyme also requires it. Adding for completeness.
 • Degradation:
◦ Serotonin → 5-Hydroxyindoleacetic Acid (5-HIAA) (excreted in urine).
• Functions of Serotonin:
◦ Neurotransmitter.
◦ Vasoconstrictor (primary view mentioned, though vasodilation noted as an alternative view/context-
dependent effect).
◦ Mood elevation. (Target for SSRIs in depression treatment).
◦ Temperature regulation.
◦ Regulates Gastrointestinal (GI) tract motility.
• Synthesis Sites:
◦ Argentaffin cells (also called Enterochromaffin cells) in the Intestine.
◦ Mast cells.
◦ Platelets.
◦ Brain.

2. Melatonin Synthesis

• Site: Pineal Gland.

• Pathway (starts from Serotonin):
1. Serotonin (5-HT) → N-Acetylserotonin (Acetylation).
2. N-Acetylserotonin → Melatonin (Methylation).
▪ Requires S-Adenosyl Methionine (SAM) as the methyl donor (becomes S-Adenosyl
Homocysteine - SAH).
▪ Indirectly requires Methionine (to regenerate SAM).
• Functions of Melatonin:
◦ Regulates Biological Rhythms (Sleep-wake cycle).
◦ Acts as a Neurotransmitter.

Clinical Correlations
1. Carcinoid Syndrome

• Cause: Tumor of Argentaffin cells (Argentaffinoma), a type of Neuroendocrine Tumor.

• Biochemical Defect:
◦ Increased Serotonin synthesis by the tumor.
◦ Tryptophan is shunted towards serotonin, leading to potential Decreased Niacin synthesis.
• Clinical Features:
◦ Intermittent Diarrhea (due to ↑ Serotonin stimulating GI motility).
◦ Cutaneous Flushing (deep red/violaceous erythema, warmth - potentially due to Serotonin's
vasoactive effects or associated ↑ Tachykinins).
◦ Sweating.
◦ Fluctuating Hypertension.
◦ Bronchoconstriction (mentioned in Q&A section).
◦ Pruritus, Lacrimation, Facial Edema (associated features).
◦ Pellagra-like symptoms (due to ↓ Niacin).
 • Diagnosis:
◦ Increased Serum Serotonin.
◦ Increased Urinary 5-HIAA (24-hour collection; normal < 5 mg/day).
• Neuroendocrine Markers:
◦ Serum Chromogranin A.
◦ Serum Synaptophysin.
◦ Serum Neuron-Specific Enolase (NSE).

2. Hartnup Disease

• Cause: Inherited defect in the transport/absorption of Tryptophan and other Neutral Amino Acids from
the Intestine and Renal Tubules.
• Genetic Defect: Mutation in the SLC6A19 gene, which codes for the B0AT1 transporter (Sodium-
dependent Neutral Amino acid Transporter).
• Pathophysiology & Features:
◦ Intestinal Malabsorption: Unabsorbed Tryptophan acted upon by gut bacteria → Indoxyl
compounds (e.g., Indican, Indigo blue). These are absorbed systemically and excreted in urine.
▪ Leads to Blue Diaper appearance (Indicanuria).
◦ Systemic Tryptophan Deficiency:
▪ Decreased Niacin synthesis → Pellagra-like symptoms, especially Cutaneous Photosensitivity
(rashes in sun-exposed areas - "Casal's necklace"). This is the most common symptom.
▪ Decreased Serotonin synthesis → Neurological Manifestations (e.g., Intermittent Ataxia,
wide-based gait).
◦ Often asymptomatic.
• Diagnosis:
◦ Aminoaciduria (Ninhydrin test positive).
◦ Presence of Indican in urine (Obermaier test positive - detects indoxyl compounds). Aldehyde test
mentioned likely refers to this or similar tests for indican.
• Treatment:
◦ Nicotinamide (Niacin) supplementation (NAD+).
◦ High protein diet (implied need, not explicitly stated).
◦ Possibly Lipid-soluble esters of Tryptophan to aid absorption.

3. Drummond Syndrome (Blue Diaper Syndrome)

• Similar defect involving the B0AT1 transporter but localized only to the Intestine (renal transport is
normal).
• Also presents with Indicanuria (hence the name "Blue Diaper Syndrome").

Important Points / High-Yield Facts to Remember:

• Tryptophan is essential, aromatic, and both ketogenic/glucogenic. Contains an Indole ring.

• Catabolism: Via Kynurenine pathway. Key enzyme Tryptophan Pyrrolase (Heme/Fe dependent).
Kynureninase requires PLP (B6) and produces Alanine (glucogenic).
• B6 Deficiency: Causes Xanthurenic aciduria and Pellagra-like symptoms (due to ↓ Niacin synthesis
via Kynureninase block).
• Niacin Synthesis: From Tryptophan. 60mg Trp → 1mg Niacin. Rate-limiting enzyme QPRTase.
 • Anabolism: Produces Serotonin (via Tryptophan Hydroxylase - BH4 dependent) and Melatonin (from
Serotonin, needs SAM).
• Serotonin (5-HT): Neurotransmitter, mood, GI motility, vasoconstrictor. Made in Argentaffin cells,
brain, platelets. Degraded to 5-HIAA.
• Carcinoid Syndrome: Tumor of Argentaffin cells → ↑↑ Serotonin → Flushing, Diarrhea, ↑Urinary 5-
HIAA. Markers: Chromogranin A.
• Hartnup Disease: Defective B0AT1 transporter (SLC6A19 gene) → ↓ Neutral AA (incl. Trp)
absorption → Pellagra-like rash (photosensitivity), Ataxia, Indicanuria (Obermaier test +ve).
• Drummond Syndrome: Intestinal-only B0AT1 defect, "Blue Diaper Syndrome".

Summary: Branched-Chain Amino Acid (BCAA) Metabolism & Pathology

I. Introduction & Importance

• Focus: Overview and clinical correlation of BCAA metabolism, specifically the first 3 reactions as per
syllabus requirements. The pathways are complex, so understanding the basics and clinical aspects is key.
• Motivation: Act today for future benefit; don't postpone learning.
• Clinical Case Teaser: A 3-week-old neonate with hypotonia, poor sucking, whose refrigerated urine
developed a sweet-smelling oily layer and tested positive with DNPH test – points towards a BCAA
disorder (MSUD).
II. Branched-Chain Amino Acids (BCAAs)

• Identity: Leucine (L), Isoleucine (I), Valine (V). (Mnemonic: LIVe)

• Structure: Have branched aliphatic side chains (structures not required for learning).
• Properties:
◦ Essential: Cannot be synthesized by the body (requires >7 enzymes); must be obtained from the diet.
Body avoids complex synthesis energy expenditure.
◦ Nonpolar.
• Catabolic Fates: Metabolism branches out:
◦ Leucine (L): Purely Ketogenic.
◦ Isoleucine (I): Both Ketogenic and Glucogenic.
◦ Valine (V): Purely Glucogenic.
III. BCAA Metabolism Overview (First 3 Common Steps)

1. Transamination:
◦ Reaction: BCAA → Branched-Chain Keto Acid (BCKA)
◦ Enzyme: Transaminase (Aminotransferase)
◦ Coenzyme: PLP (Pyridoxal Phosphate - Vit B6)
◦ Specific BCKAs formed:
▪ Leucine → α-ketoisocaproic acid
▪ Valine → α-ketoisovaleric acid
▪ Isoleucine → α-keto-β-methylvaleric acid
2. Oxidative Decarboxylation: (Crucial Step)
◦ Reaction: BCKA → Branched-Chain Acyl-CoA (releases CO2)
◦ Enzyme: Branched-Chain Keto Acid Dehydrogenase (BCKD) Complex
 ◦ Coenzymes: Requires 5 (details below)
◦ Specific Acyl-CoAs formed initially (undergo further steps): Isovaleryl-CoA (from Leucine),
Isobutyryl-CoA (from Valine), Tiglyl-CoA (from Isoleucine leading to Acetyl-CoA & Propionyl-
CoA)
3. Dehydrogenation:
◦ Reaction: Branched-Chain Acyl-CoA → Various products (via double bond formation)
◦ Enzyme: Acyl-CoA Dehydrogenase (specific names vary, e.g., Isovaleryl-CoA Dehydrogenase)
◦ Coenzyme: FAD (Flavin Adenine Dinucleotide - Vit B2/Riboflavin)
IV. Branched-Chain Keto Acid Dehydrogenase (BCKD) Complex

• Nature: Multi-enzyme complex, similar to Pyruvate Dehydrogenase (PDH).

• Enzymes & Genes:
◦ E1: Branched-chain keto acid decarboxylase (Product of E1α and E1β genes)
◦ E2: Dihydrolipoyl transacylase (Product of E2 gene)
◦ E3: Dihydrolipoamide dehydrogenase (Product of E3 gene)
• Coenzymes (5): (Mnemonic: Tender Loving Care For Nancy)
1. TPP (Thiamine Pyrophosphate - Vit B1) - Used by E1
2. Lipoamide (Lipoic Acid)
3. Coenzyme A (CoA - Pantothenic Acid/Vit B5)
4. FAD (Flavin Adenine Dinucleotide - Vit B2) - Used by E3
5. NAD+ (Nicotinamide Adenine Dinucleotide - Niacin/Vit B3) - Used by E3
V. Maple Syrup Urine Disease (MSUD)

• Biochemical Defect: Deficiency of the BCKD complex.

• Genetic Basis & Types: Defects in specific genes cause different types:
◦ Type 1a: E1α gene defect (Most common type)
◦ Type 1b: E1β gene defect
◦ Type 2: E2 gene defect
◦ Type 3: E3 gene defect
• Pathophysiology: Impaired oxidative decarboxylation (Step 2) leads to accumulation of BCAAs (L, I,
V) and BCKAs in blood, tissues, and urine.
• Clinical Features:
◦ Onset: Usually neonatal period.
◦ Symptoms: Feeding difficulties, failure to thrive, lethargy, vomiting, neurological signs (hypotonia
often, sometimes with bouts of hypertonia, convulsions), characteristic "boxing" or "bicycling"
movements.
◦ Urine Odor: Distinctive sweet smell like maple syrup, burnt sugar, or caramel, especially
noticeable after refrigeration.
• Diagnosis:
◦ Elevated levels of BCAAs and BCKAs in plasma and urine.
◦ DNPH (2,4-Dinitrophenylhydrazine) Test: Positive - produces a yellow precipitate with the keto
acids in urine.
◦ Rothera's Test: May be positive - detects ketones (purple ring).
 • Treatment:
◦ Dietary Restriction: Lifelong restriction of BCAA intake (Leucine, Isoleucine, Valine).
◦ Thiamine (Vitamin B1) Supplementation: May be effective in some cases (Thiamine-responsive
MSUD), particularly those involving the E1 enzyme which uses TPP.
VI. Isovaleric Acidemia (IVA)

• Biochemical Defect: Deficiency in Leucine catabolism pathway (Step 3 derivative).

• Enzyme Defect: Isovaleryl-CoA Dehydrogenase (FAD-dependent).
• Result: Accumulation of isovaleric acid.
• Characteristic Feature: Urine and body odor described as "sweaty feet".
VII. MCQ Analysis Example

• Assertion: Thiamine is used in MSUD treatment. (TRUE)

• Reason: Thiamine (as TPP) is a coenzyme for the first step (Transamination) of BCAA catabolism.
(FALSE - TPP is for the second step, oxidative decarboxylation via BCKD; PLP is for the first step).
• Conclusion: Assertion is true, Reason is false.

Key Points to Remember:

• BCAAs are Essential (L, I, V).

• Know their fates: L (Keto), V (Gluco), I (Both).
• Master the first 3 metabolic steps: Transamination (PLP), Oxidative Decarboxylation (BCKD + 5
coenzymes), Dehydrogenation (FAD).
• BCKD Complex is central: Know its components (E1, E2, E3), 5 coenzymes (TPP crucial for E1), and
similarity to PDH.
• MSUD: Caused by BCKD deficiency (often E1). Leads to BCAA/BCKA accumulation. Presents in
neonates with neuro symptoms and sweet/maple syrup urine odor. Diagnose with DNPH test. Treat
with diet and possibly Thiamine.
• IVA: Defect in Leucine path (Isovaleryl-CoA DH). Odor of "sweaty feet".
• Clinical Correlation is vital for understanding these disorders (e.g., the neonate case).

Summary: Acidic & Basic Amino Acids

I. Introduction

• Focus: This lecture covers the 5 acidic and basic amino acids, concentrating on their applications,
associated metabolic disorders, and functional importance, rather than their complete metabolic
pathways.
• Goal: To understand the link between lysine and fatty acid transport, why histones are positive, the
mechanism of sildenafil, the basis of the FIGLU test for folate deficiency, and a specific metabolic
disorder case (Canavan disease).
II. Identification of Amino Acids

• Acidic: Aspartic Acid (Asp), Glutamic Acid (Glu)

• Corresponding Amides: Asparagine (Asn), Glutamine (Gln)
 • Basic: Histidine (His), Arginine (Arg), Lysine (Lys)
◦ Mnemonic: HAL is Basic ( Histidine, Arginine, Lysine)
III. Basic Amino Acids (His, Arg, Lys)

• General Characteristics:
◦ Essentiality: His (Essential), Arg (Semi-essential), Lys (Essential). Primarily dietary essential, so
synthesis details are omitted.
◦ Polarity: All are polar (positively charged at physiological pH).
◦ Most Polar & Basic: Arginine.
◦ Special Groups:
▪ Histidine: Imidazole ring
▪ Arginine: Guanidinium group
▪ Lysine: Epsilon (ε) amino group
◦ Metabolic Fate:
▪ Histidine: Glucogenic
▪ Arginine: Glucogenic
▪ Lysine: Purely Ketogenic
• A. Histidine (His)
◦ Functions:
1. Histamine Synthesis: Histidine --(Decarboxylation)--> Histamine (vasodilator, important
physiological functions).
2. Metabolism & FIGLU:
▪ Pathway: Histidine -> (Histidase, releases NH3) -> Urocanate -> Imidazolone propionate ->
FIGLU (Formiminoglutamic acid) -> (Requires Tetrahydrofolate, THF) -> Glutamic acid ->
α-Ketoglutarate (Glucogenic).
▪ FIGLU Test: In folic acid (THF) deficiency, the conversion of FIGLU to Glutamate is
blocked. FIGLU accumulates and is excreted in urine.
▪ Histidine Load Test: Administering a large dose of histidine can help assess folate status by
measuring urinary FIGLU excretion.
3. Component of Dipeptides/Related Compounds:
▪ Carnosine = Beta-alanine + Histidine
▪ Anserine = Methylcarnosine
▪ Homocarnosine = GABA + Histidine
• B. Arginine (Arg)
◦ Functions (Precursor for Synthesis):
1. Agmatine: An anti-hypertensive compound. (Note: "acrimantin" in text likely meant Agmatine)
2. Creatine: Synthesized from Glycine + Arginine + Methionine (as SAM).
3. Urea: Arginine --(Arginase)--> Urea + Ornithine.
4. Ornithine: See Urea synthesis.
5. Nitric Oxide (NO):
▪ Also known as EDRF (Endothelium Derived Relaxing Factor).
▪ Properties: Free radical, gaseous, very short half-life (~0.1 sec).
 ▪ Synthesis: Arginine + O2 + NADPH --(Nitric Oxide Synthase, NOS)--> NO + Citrulline +
NADP+.
▪ Nitric Oxide Synthase (NOS):
▪ Type: Monooxygenase.
▪ Cofactors (5): Heme, FAD, FMN, NADPH, Tetrahydrobiopterin (BH4).
▪ Isoforms:
▪ nNOS (neuronal)
▪ iNOS (inducible, in macrophages) - Calcium-independent.
▪ eNOS (endothelial) - nNOS and eNOS are Calcium-dependent.
▪ NO Functions: Vasodilation, Penile erection, Neurotransmission.
▪ Pharmacological Relevance:
▪ Used to treat pulmonary hypertension.
▪ Sildenafil (Viagra): Treats impotence by inhibiting cGMP phosphodiesterase. This
increases levels of cGMP (NO's second messenger), enhancing vasodilation.
▪ Glycerol Trinitrate: Treats angina by releasing NO.
• C. Lysine (Lys)
◦ Single Letter Code: K
◦ Functions:
1. Histone Structure: Histones are rich in Lysine and Arginine, contributing to their positive charge
(binds negative DNA).
2. Cadaverine: Produced by bacterial putrefaction of lysine (a polyamine).
3. Carnitine Synthesis: Synthesized from Lysine + Methionine (as SAM).
▪ Mnemonic: Need Lots of Meat for Cars ( Lysine + Methionine -> Carnitine).
▪ Carnitine Role: Essential for transporting long-chain fatty acids into mitochondria for beta-
oxidation. Deficiency impairs fatty acid transport.
IV. Acidic Amino Acids (Asp, Glu) & Their Amides (Asn, Gln)

• General Characteristics:
◦ Structure: Asp (4C), Glu (5C). Have an extra carboxyl group (acidic, negatively charged). Asn &
Gln have an amide group instead (uncharged polar).
◦ Essentiality: All are Non-essential.
◦ Metabolic Fate: All are Glucogenic.
◦ Polarity: Asp, Glu (Charged Polar); Asn, Gln (Uncharged Polar).
• A. Aspartic Acid (Asp)
◦ Functions (Contributor to):
1. Pyrimidine ring synthesis.
2. Purine ring synthesis.
3. Urea synthesis (provides the second nitrogen atom).
• B. Glutamic Acid (Glu)
◦ Functions:
1. N-Acetylglutamate (NAG) Synthesis: Acetyl-CoA + Glutamate -> NAG. (NAG is an allosteric
activator of CPS-I in the urea cycle).
 2. Glutathione Synthesis: Glutathione = γ-Glutamyl-Cysteinyl-Glycine.
3. GABA Synthesis: Glutamate --(Decarboxylation)--> GABA (γ-Aminobutyric acid, an inhibitory
neurotransmitter).
• C. Asparagine (Asn)
◦ No major specific functions highlighted in the lecture beyond being incorporated into proteins.
• D. Glutamine (Gln)
◦ Functions:
1. Nitrogen donor for Purine synthesis (N3 and N9 atoms).
2. Nitrogen donor for Pyrimidine synthesis (N3 atom).
3. Major transporter of ammonia (amino groups) in the blood, especially from peripheral tissues
and the brain to the liver and kidneys. Non-toxic form of ammonia transport.
4. Source of ammonia in the kidney: Glutamine --(Glutaminase)--> Glutamate + NH3. This NH3
is excreted (as NH4+) helping in acid-base balance (renal regulation).
V. Synthesis & Catabolism Overview (Acidic AA & Amides)

• Synthesis:
◦ Aspartate: Oxaloacetate <-- (Transamination, e.g., via AST) --> Aspartate.
◦ Glutamate: α-Ketoglutarate <-- (Reductive Amidation via Glutamate Dehydrogenase, uses NH3 +
NADPH) --> Glutamate. (Reverse of oxidative deamination).
◦ Asparagine: Aspartate + Glutamine (N donor) + ATP --(Asparagine Synthetase)--> Asparagine +
Glutamate.
◦ Glutamine: Glutamate + Free NH3 + ATP --(Glutamine Synthetase)--> Glutamine. (Important for
ammonia detoxification/scavenging).
• Catabolism:
◦ Asparagine --(Asparaginase)--> Aspartate (+ NH3) --> Oxaloacetate (enters TCA cycle).
◦ Glutamine --(Glutaminase)--> Glutamate (+ NH3) --> α-Ketoglutarate (enters TCA cycle).
VI. Clinical Correlation: Canavan Disease

• Case Presentation: Infant with normal birth, followed by developmental delay, nystagmus, poor head
control (head lag), generalized hypotonia, progressive macrocephaly, and MRI showing severe
leukodystrophy (white matter degradation).
• Disease: Canavan Disease.
• Enzyme Defect: Deficiency of Aspartoacylase.
• Metabolic Consequence: Accumulation of N-Acetylaspartic Acid (NAA) in blood, CSF, and urine,
because Aspartoacylase normally breaks NAA down into Aspartate and Acetate.
• Key Features: Progressive macrocephaly, developmental delay, hypotonia, head lag, leukodystrophy,
distorted mitochondria.
VII. Key Points & Answers to Initial Questions

• Lysine & Fatty Acid Transport: Lysine is required to synthesize Carnitine, which transports fatty acids
into mitochondria. Lysine deficiency impairs carnitine synthesis and thus fatty acid transport.
• Histone Charge: Histones are rich in basic amino acids (Lysine, Arginine), giving them a net positive
charge to bind negatively charged DNA.
 • Sildenafil: Inhibits cGMP phosphodiesterase, increasing cGMP levels, which enhances NO-mediated
vasodilation, useful in impotence.
• FIGLU Test: FIGLU (an intermediate of Histidine metabolism) requires Folic Acid (THF) for its further
metabolism. In folate deficiency, FIGLU accumulates and is excreted in urine.
• Canavan Disease: Caused by Aspartoacylase deficiency, leading to NAA accumulation and
characteristic neurological symptoms including macrocephaly and leukodystrophy.
• Oxaloacetate Precursors: Aspartate and Asparagine (4-carbon AAs) yield Oxaloacetate.
• Nitrogen Donor for Asn Synthesis: Glutamine.
• Nitrogen Donor for Gln Synthesis: Free Ammonium ion (NH4+/NH3).
• Formimino Group Donor: Histidine (via FIGLU).
• Urocanate: Intermediate of Histidine metabolism.
• Ornithine Source: Arginine (via Arginase in the urea cycle).

Summary: Amino Acid Metabolism Leftover Topics

1. Entry of Amino Acids into the TCA Cycle (Anaplerotic Reactions)

• Definition: Reactions that replenish TCA cycle intermediates ("filling up reactions").

• Amino acids provide carbon skeletons that can enter the cycle at various points.
• Entry Point: Oxaloacetate (OAA)
◦ Via Pyruvate:
▪ Pyruvate is converted to OAA by Pyruvate Carboxylase.
▪ Amino Acids forming Pyruvate:
▪ Hydroxyproline, Serine, Threonine (Contain -OH group)
▪ Cysteine (Contains -SH group, thioalcohol)
▪ (Mnemonic: OH/SH group related AAs enter via Pyruvate)
◦ Via Alanine -> Pyruvate:
▪ Tryptophan can form Alanine.
▪ Alanine is converted to Pyruvate via transamination.
▪ Pyruvate is then converted to OAA (Pyruvate Carboxylase).
◦ Directly:
▪ Asparagine -> Aspartate (Enzyme: Asparaginase)
▪ Aspartate -> OAA (Via transamination)
• Entry Point: Alpha-Ketoglutarate (α-KG)
◦ Amino Acids form Glutamate first.
◦ Glutamate is converted to α-KG.
◦ Amino Acids entering via Glutamate:
▪ Histidine
▪ Proline
▪ Glutamine (converted to Glutamate by Glutaminase)
▪ Arginine
▪ (Mnemonic: All end in "-ine": Histidine, Proline, Glutamine, Arginine)*
 • Entry Point: Succinyl-CoA
◦ These amino acids are catabolized to Propionyl-CoA, which is then converted to Succinyl-CoA.
◦ Amino Acids:
▪ Methionine
▪ Threonine
▪ Isoleucine
▪ Valine
▪ (Mnemonic: MeT V I - think MTV + Isoleucine)
• Entry Point: Fumarate
◦ Amino Acids:
▪ Phenylalanine
▪ Tyrosine
▪ (Their catabolism yields both Fumarate and Acetoacetate)

2. Related Chemical Compounds & Names

• Sarcosine: N-methylglycine
• Betaine: Tri-methylglycine (Used in Homocystinuria treatment; methyl donor)
• Choline: Tri-methylethanolamine
• Ethanolamine: Formed from Serine via decarboxylation (Coenzyme: PLP)
• Ergothioneine: Derived from Histidine (Specific structure not detailed in this segment)
• Beta-mercaptoethanolamine: Formed from Cysteine via decarboxylation (Coenzyme: PLP)
• GABA (Gamma-Aminobutyric Acid): Formed from Glutamate via decarboxylation (Coenzyme: PLP)
• Carnosine: Beta-alanyl-histidine
• Anserine: Methylated Carnosine (Likely intended instead of "serine is the methylated carnosine")
• Homocarnosine: GABA + Histidine (Note: Contains GABA, not beta-alanine)

3. Urine/Body Odors in Amino Acid Metabolic Disorders

Disorder Characteristic Urine/Body Odor

Glutaric Acidemia Type 2 Sweaty feet

Isovaleric Aciduria Sweaty feet

Hawkinsinuria Swimming pool

3-Hydroxy-3-methylglutaric aciduria Cat urine

Maple Syrup Urine Disease (MSUD) Maple syrup / Caramel / Burnt sugar

Hypermethioninemia Boiled cabbage

Multiple Carboxylase Deficiency Tomcat urine

Oasthouse Syndrome Boiled cabbage / Hops-like

Phenylketonuria (PKU) Mousy / Musty

 Disorder Characteristic Urine/Body Odor

Trimethylaminuria Rotten fish (Fish Odor Syndrome)

Tyrosinemia (Type 1) Boiled cabbage

4. Fish Odor Syndrome (Trimethylaminuria)

• Cause: Deficiency of the enzyme Trimethylamine Oxidase.

• Enzyme Type: Flavin-dependent monooxygenase (Requires Riboflavin - Vitamin B2).
• Metabolic Defect: Inability to oxidize/metabolize Trimethylamine.
• Result: Accumulation of Trimethylamine.
• Clinical Feature: Characteristic odor of rotten fish in breath, sweat, and urine.
• Treatment:
◦ Dietary restriction of Choline (a precursor, being trimethyl-ethanolamine).
◦ Avoid choline-rich foods like eggs, nuts, certain green leafy vegetables.

5. Sample Question Review

• Question: Amino acids that form oxaloacetate?

• Answer: Asparagine and Aspartate (form OAA directly).

Key Points to Remember:

• Anaplerosis: Understand that amino acid catabolism replenishes TCA cycle intermediates.
• Entry Points: Know which amino acids enter at OAA, α-KG, Succinyl-CoA, and Fumarate. Memorize
the key groups (e.g., using mnemonics like MeT VI, OH/SH groups, "-ine" endings).
• Enzymes: Note key enzymes like Pyruvate Carboxylase, Asparaginase, Glutaminase, and the PLP-
dependent decarboxylases.
• Urine Odors: Associate specific odors (Sweaty feet, Maple syrup, Mousy, Rotten fish, Boiled cabbage,
Cat urine) with their respective disorders – this is frequently tested.
• Fish Odor Syndrome: Remember the deficient enzyme (Trimethylamine Oxidase), its cofactor
requirement (Flavin/Riboflavin), the accumulating substance (Trimethylamine), and the dietary
management (Choline restriction).
• Chemical Names: Be familiar with terms like Sarcosine, Betaine, Carnosine, and Homocarnosine.
Okay students, here is a summary of our discussion on the Chemistry of Lipids:
I. Definition of Lipids

• A heterogeneous group of compounds.

• Characterized by physical property: insoluble in water but soluble in non-polar solvents (e.g.,
chloroform, ether).
• Lipids are related more physically than chemically (no single general formula like carbohydrates
CnH2On or amino acids).
II. Classification of Lipids (Bloor's Classification)

1. Simple Lipids:
◦ Esters of fatty acids with an alcohol (usually glycerol).
◦ Examples: Fats, Oils, Waxes.
◦ Neutral Fat (Triacylglycerol - TAG): Glycerol + 3 Fatty Acid residues. Note: Glycerol and Fatty
Acids alone are derived lipids.
2. Compound Lipids:
◦ Esters of fatty acids and alcohol + additional groups.
◦ Examples: Phospholipids, Glycolipids, Lipoproteins.
3. Derived Lipids:
◦ Substances derived from simple or compound lipids by hydrolysis.
◦ Examples: Fatty acids, Glycerol, Steroid hormones, Fat-soluble vitamins.
III. Fatty Acids (FA)

• General Formula: R-COOH

◦ R = Hydrophobic hydrocarbon chain (e.g., CH3(CH2)n).
• Classification based on Chain Length:
◦ Short Chain FA (SCFA): C2-C6 carbons.
◦ Medium Chain FA (MCFA): C8-C14 carbons.
◦ Long Chain FA (LCFA): > C16 carbons.
◦ Very Long Chain FA (VLCFA): > C20 or C22 carbons (subset of LCFA).
• Classification based on Double Bonds:
◦ Saturated FA (SFA): No double bonds.
◦ Unsaturated FA (UFA): Contain double bonds.
▪ Monounsaturated FA (MUFA): One double bond.
▪ Polyunsaturated FA (PUFA): More than one double bond.
IV. Examples and Sources of Fatty Acids

• Saturated Fatty Acids (SFA):

◦ SCFAs: Acetic acid (C2, vinegar), Propionic acid (C3), Butyric acid (C4), Valeric acid (C5), Capric
acid (C10). Source: Butter.
◦ MCFAs: Lauric acid (C12), Myristic acid (C14). Rich source: Coconut oil.
◦ LCFAs: Palmitic acid (C16), Stearic acid (C18). Source: Animal fat.
• Unsaturated Fatty Acids (UFA):
◦ MUFAs: Palmitoleic acid, Oleic acid, Elaidic acid. Richest source: Mustard oil / Rapeseed oil.
◦ PUFAs:
▪ Linoleic Acid (C18, 2 DB): Richest source Safflower oil.
▪ Gamma-Linolenic Acid (GLA) (C18, 3 DB): Source Oil of evening primrose.
▪ Alpha-Linolenic Acid (ALA) (C18, 3 DB): Richest source Flaxseed oil.
▪ Arachidonic Acid (AA) (C20, 4 DB): Source Animal fat.
▪ Timnodonic Acid / EPA (Eicosapentaenoic Acid) (C20, 5 DB): Sources Fish oils, algal oils,
breast milk.
 ▪ Cervonic Acid / DHA (Docosahexaenoic Acid) (C22, 6 DB): Sources Fish oils, algal oils, breast
milk.
◦ Overall PUFA Richness: Safflower oil > Sunflower oil > Coconut oil (least PUFA).
V. Essential and Semi-Essential Fatty Acids

• Essential Fatty Acids (EFAs): Cannot be synthesized in the body (lack required enzymes). Must be
obtained from diet.
◦ Linoleic Acid (LA)
◦ Alpha-Linolenic Acid (ALA)
◦ Mnemonic: Essential to Love Always (LA, ALA)
• Semi-Essential Fatty Acids: Can be synthesized from EFAs.
◦ Arachidonic Acid (AA) (synthesized from Linoleic Acid)
◦ Gamma-Linolenic Acid (GLA) (synthesized from Linoleic Acid)
VI. Omega Classification of Unsaturated Fatty Acids

• Based on the position of the first double bond counting from the terminal methyl (CH3) group (the
omega end).
• Delta numbering counts from the carboxyl (COOH) group.
• Omega-3 Fatty Acids: First double bond at the 3rd carbon from the omega end.
◦ Examples: Mnemonic ATC
▪ Alpha-Linolenic Acid (ALA)
▪ Timnodonic Acid (EPA)
▪ Cervonic Acid (DHA)
• Omega-6 Fatty Acids: First double bond at the 6th carbon from the omega end.
◦ Examples: Mnemonic GLA
▪ Gamma-Linolenic Acid (GLA)
▪ Linoleic Acid (LA)
▪ Arachidonic Acid (AA)
VII. Omega-3 vs. Omega-6: Health Implications

• Omega-6 Pathway: Linoleic Acid -> Arachidonic Acid -> Eicosanoids (Prostaglandins, Thromboxanes,
Leukotrienes). These can mediate inflammation, platelet aggregation, increasing cardiovascular risk
and risk of degenerative disorders.
• Omega-3 Pathway: Generally considered anti-inflammatory. Compete with Omega-6 pathway.
◦ Significance of Omega-3s: Decrease cardiovascular risk, platelet aggregation, inflammation;
important for infant brain/retina development (DHA); may decrease mental illness (ADHD,
depression) and chronic degenerative disorders (Rheumatoid Arthritis, Alzheimer's).
• Conclusion: Omega-6 FAs can be more harmful in excess compared to Omega-3 FAs due to their pro-
inflammatory potential. A balanced dietary ratio is important.
VIII. Docosahexaenoic Acid (DHA)

• An Omega-3 fatty acid (C22, 6 DB).

• Sources: Breast milk, algal oils, fish oils.
• Uses: Crucial for infant/fetal brain development and retina development.
• Low DHA associated with Retinitis Pigmentosa.
 • Transplacental transport is possible, making supplementation during pregnancy potentially beneficial.
IX. Cis vs. Trans Fatty Acids

• Isomers of unsaturated fatty acids based on spatial arrangement around the double bond.
• Cis Form: Hydrocarbon chains on the same side of the double bond. Creates a bend (~120°). The
predominant form in nature; increases plasma membrane fluidity.
• Trans Form: Hydrocarbon chains on opposite sides of the double bond. More linear/extended structure.
• Sources of Trans Fats:
◦ Partially hydrogenated vegetable oils (solidified fats): Margarine, Dalda, Vanaspati (richest
source), cake butter. Used in bakery items, fast foods. Advantage for industry: Improves shelf life.
◦ Deep Frying (e.g., French fries) at high temperatures.
◦ Reheating vegetable oils repeatedly.
• Health Risks of Trans Fats:
◦ Maximum recommended daily intake: 2-7 grams.
◦ Cause Essential Fatty Acid Deficiency.
◦ Decrease membrane fluidity.
◦ Negatively impact lipid profile: Increase TAG, Increase LDL, Decrease HDL -> Increases
Cardiovascular Risk.
◦ Promote inflammatory response.
◦ Cause Insulin Resistance.
X. Revisiting Initial Questions

• Omega-6 vs. Omega-3: Omega-6 (e.g., from sunflower/safflower oil) can be more harmful due to pro-
inflammatory eicosanoid production pathway.
• French Fries: Harmful due to deep frying process which generates trans fatty acids.
• Coconut Oil vs. Sunflower Oil:
◦ Sunflower Oil: Rich in PUFA (Linoleic acid, Omega-6), potential source of inflammatory mediators,
susceptible to becoming trans fat upon heating.
◦ Coconut Oil: Rich in Saturated MCFA (Lauric, Myristic). Less likely to form trans fats (as it's
saturated). MCFAs can be absorbed directly into the portal vein. Controversy remains, but less prone
to harmful trans conversion.
◦ Conclusion: Minimal intake of any oil is best for individuals at high cardiovascular risk.

Key Points to Remember:

• Lipids are defined by solubility, not a single chemical structure.

• Know the Bloor's classification: Simple, Compound, Derived.
• Triacylglycerol (TAG) is a simple lipid; Glycerol and Fatty Acids alone are derived.
• Fatty acids are classified by chain length and saturation.
• Essential FAs (LA, ALA) must be from diet.
• Omega-3 (ATC mnemonic) vs. Omega-6 (GLA mnemonic) have different health impacts; balance is
key. Omega-6 excess can be pro-inflammatory.
• DHA is crucial for brain/eye development and can cross the placenta.
• Trans fats (from hydrogenation, deep frying, reheating) are harmful: increase CV risk, inflammation,
insulin resistance. Vanaspati is a major source.
 • Cis fats are natural and important for membrane fluidity.
• Heating unsaturated oils (like sunflower) can generate trans fats; saturated fats (like coconut oil) are less
susceptible.

Summary: Phospholipids, Glycolipids &

Sphingolipidosis
I. Introduction

• Key Questions Posed:

◦ Why severe neurological deficit in Krabbe's disease but not in Gaucher's disease (both
sphingolipidoses)?
◦ Is there a relationship between cardiolipin and syphilis? (Answered implicitly later - Cardiolipin is
antigenic).
II. Phospholipids

• Definition: Compound lipids containing lipids + a phosphate group.

• Structure (General): Consist of 4 parts:
1. Fatty Acid (typically 2)
2. Alcohol (Glycerol or Sphingosine)
3. Phosphoric Acid
4. Base (Nitrogenous or Non-nitrogenous)
5. Example Structure (Glycerol based): Glycerol backbone, 2 Fatty Acid residues (R1, R2), Phosphate
group, Base (X).
• Classification:
1. Glycerophospholipids: Glycerol backbone.
2. Sphingophospholipids: Sphingosine backbone.
III. Glycerophospholipids

• Sub-classification:
◦ Nitrogen-containing: Lecithin, Cephalin, Phosphatidylserine.
◦ Non-nitrogen containing: Phosphatidylglycerol, Diphosphatidylglycerol (Cardiolipin).
• Phosphatidic Acid:
◦ Structure: Glycerol + 2 Fatty Acids + Phosphate (NO Base).
◦ Significance: Simplest glycerophospholipid; precursor for others.
• Lecithin (Phosphatidylcholine):
◦ Structure: Phosphatidic acid + Choline (nitrogenous base).
◦ Significance:
▪ Most abundant phospholipid in cell membranes.
▪ Major component of lung surfactant (e.g., Dipalmitoylphosphatidylcholine).
▪ Important for L/S (Lecithin/Sphingomyelin) ratio in assessing fetal lung maturity (L should
increase).
▪ Storehouse of choline.
• Cephalin (Phosphatidylethanolamine):
◦ Structure: Phosphatidic acid + Ethanolamine (nitrogenous base).
 ◦ Significance: Important in blood coagulation pathways.
• Cardiolipin (Diphosphatidylglycerol):
◦ Structure: 2 Phosphatidic acids linked by a Glycerol molecule.
◦ Significance:
▪ First isolated from cardiac muscle.
▪ Located in the inner mitochondrial membrane.
▪ Only antigenic phospholipid. (This antigenicity is the basis for some syphilis tests like VDRL/
RPR, though not explicitly stated in the lecture).
▪ Defects associated with mitochondrial dysfunction: Barth syndrome (cardioskeletal myopathy),
aging, hypothyroidism, heart failure.
• Phosphatidylserine:
◦ Structure: Phosphatidic acid + Serine (nitrogenous base).
◦ Significance: Important in apoptosis (programmed cell death).
• Phosphatidylinositol:
◦ Structure: Phosphatidic acid + Inositol (non-nitrogenous base).
◦ Significance: Important in cell signaling; acts as a second messenger in hormone pathways.
IV. Sphingophospholipids

• Sphingosine:
◦ Backbone alcohol for this class.
◦ An amino alcohol.
◦ Derived from the amino acid Serine.
◦ Structure includes a fatty acid chain inherently.
• Ceramide:
◦ Structure: Sphingosine + an additional Fatty Acid linked to the amino group.
• Sphingomyelin:
◦ Structure: Ceramide + Phosphate + Choline.
◦ Significance:
▪ Only phospholipid with Sphingosine backbone.
▪ Found in myelin sheath.
▪ Found in outer leaflet of plasma membrane.
▪ Component of lipid rafts (specialized membrane regions).
V. Glycolipids (Glycosphingolipids)

• Definition: Compound lipids containing carbohydrate; NO phosphate.

• Common Feature with Sphingophospholipids: Contain Sphingosine.
• Structure (General): Sphingosine + Fatty Acid (= Ceramide) + Carbohydrate.
• Classification:
1. Cerebrosides: Ceramide + Monosaccharide.
▪ Glucocerebroside: Ceramide + Glucose (Found in non-neural tissues).
▪ Galactocerebroside: Ceramide + Galactose (Found in neural tissues).
2. Globosides: Ceramide + Oligosaccharide.
▪ Example: Lactosylceramide (Ceramide + Galactose + Glucose).
 3. Gangliosides: Ceramide + Oligosaccharide containing N-acetylneuraminic acid (NANA) (a type of
sialic acid).
▪ Naming: GM1, GM2, GM3 etc. (G=Ganglioside, M=Monosialo, Number=chromatographic
separation).
▪ Simplest: GM3.
▪ GM1: Acts as a receptor for Cholera toxin.
VI. Sphingolipidosis

• Definition: Group of inherited Lysosomal Storage Disorders (LSDs).

• Cause: Deficiency of specific lysosomal hydrolase enzymes needed to degrade sphingolipids.
• Result: Intra-lysosomal accumulation of the specific sphingolipid substrate.
• Specific Disorders:
1. GM1 Gangliosidosis:
▪ Enzyme Defect: β-Galactosidase.
▪ Accumulates: GM1 Ganglioside.
▪ Features: Typical facies (frontal bossing, long philtrum, depressed nasal bridge, low-set ears),
corneal opacity (blindness), macular cherry-red spot (~50%), angiokeratoma,
hepatosplenomegaly, mental retardation.
2. GM2 Gangliosidosis:
▪ Enzyme Defect: β-Hexosaminidase.
▪ Accumulates: GM2 Ganglioside.
▪ Types:
▪ Tay-Sachs Disease: Defect in α-subunit (Hexosaminidase A affected). Features: Cherry-red
spot, hyperacusis (increased startle reflex), neurodegeneration, macrocephaly. No
hepatosplenomegaly.
▪ Sandhoff's Disease: Defect in β-subunit (Hexosaminidase A and B affected). Features: Tay-
Sachs features + hepatosplenomegaly, cardiac abnormalities, bony deformities.
3. Krabbe's Disease (Globoid Cell Leukodystrophy):
▪ Enzyme Defect: β-Galactosidase (Galactocerebrosidase).
▪ Accumulates: Galactocerebroside (mainly neural).
▪ Features: Severe neurological deficit, developmental delay/regression, opisthotonus, Globoid
cell inclusions (multinucleated macrophages in white matter), No hepatosplenomegaly. Cherry-
red spot may be present but variable.
4. Gaucher's Disease:
▪ Enzyme Defect: β-Glucosidase (Glucocerebrosidase).
▪ Accumulates: Glucocerebroside (mainly non-neural/reticuloendothelial system).
▪ Features: Most common LSD. Hepatosplenomegaly, pancytopenia (anemia, easy bruising), bone
pain/pathological fractures, Erlenmeyer flask deformity (X-ray), Gaucher cells (macrophages
with "crumpled tissue paper" appearance in bone marrow). No mental retardation (usually Type
1). No cherry-red spot (usually Type 1; pseudo-CRS reported in Type 2).
▪ Treatment: Enzyme Replacement Therapy (ERT - imiglucerase, velaglucerase, taliglucerase),
Substrate Reduction Therapy (Miglustat - inhibits glucosylceramide synthase), Bone Marrow
Transplant.
 5. Niemann-Pick Disease (Type A/B implicitly discussed):
▪ Enzyme Defect: Sphingomyelinase.
▪ Accumulates: Sphingomyelin.
▪ Features: Cherry-red spot, Zebra body inclusions (electron microscopy finding, not shown but
mentioned). Hepatosplenomegaly is also characteristic of Type A/B.
6. Farber's Disease:
▪ Enzyme Defect: Acid Ceramidase.
▪ Accumulates: Ceramide.
▪ Features: Painful, swollen joints (resembles rheumatoid arthritis), subcutaneous nodules.
7. Fabry's Disease:
▪ Inheritance: X-linked recessive.
▪ Enzyme Defect: α-Galactosidase A.
▪ Accumulates: Globotriaosylceramide (Gb3 or Ceramide Trihexoside).
▪ Features: Angiokeratomas (skin eruptions), corneal/lenticular opacities, Fabry crises (severe
pain in extremities), hypohidrosis (decreased sweating), Maltese cross appearance in urine
sediment (lipid inclusions).
▪ Treatment: ERT (Agalsidase beta - Fabrazyme; Agalsidase alpha - Replagal).
• Wolman's Disease (Cholesterol Ester Storage Disease - CESD):
◦ Note: A Lysosomal Storage Disorder, but NOT a sphingolipidosis.
◦ Enzyme Defect: Acid Lipase.
◦ Accumulates: Cholesterol Esters, Triglycerides.
◦ Features: Failure to thrive, vomiting, watery green diarrhea, hepatosplenomegaly, Adrenal gland
calcification (pathognomonic).
VII. Comparative Features of Sphingolipidoses

• Krabbe vs. Gaucher:

◦ Krabbe: Defect β-Galactosidase -> Galactocerebroside (neural) accumulates -> Severe Neurological
Deficit, No Hepatosplenomegaly.
◦ Gaucher: Defect β-Glucosidase -> Glucocerebroside (non-neural) accumulates -> No Neurological
Deficit (usually), Hepatosplenomegaly Present.
• General Rules & Exceptions: (Mnemonics suggested for key exceptions)
◦ Inheritance: All Autosomal Recessive (AR) EXCEPT Fabry (X-linked).
▪ Mnemonic: Fabry is Fabulously X-ceptional (X-linked).
◦ Mental Retardation: Present in most EXCEPT Gaucher (Type 1) and Fabry.
▪ Mnemonic: You'd be mentally Fine & Good without MR in Fabry & Gaucher.
◦ Cherry-Red Spot: Present in many (Tay-Sachs, Sandhoff, Niemann-Pick, GM1) EXCEPT Gaucher
(Type 1) and Fabry. Present but variable in Krabbe.
◦ Corneal Clouding: Seen in Fabry, GM1 Gangliosidosis (also seen in Mucopolysaccharidoses).
◦ Hepatosplenomegaly: Present in many (Gaucher, Sandhoff, GM1, Niemann-Pick, Wolman)
EXCEPT Krabbe, Tay-Sachs, Farber (usually), Fabry (less prominent).
◦ Inclusion Bodies:
▪ Globoid Cells: Krabbe's Disease.
▪ Zebra Bodies: Niemann-Pick Disease.
 ▪ Gaucher Cells: Gaucher's Disease ("Crumpled tissue paper").
◦ Maltese Cross (Urine): Fabry Disease.
VIII. Summary of Enzyme Deficiencies

Disease Deficient Enzyme Accumulating Substance

(Main)

GM1 Gangliosidosis β-Galactosidase GM1 Ganglioside

Tay-Sachs Disease (GM2) β-Hexosaminidase A GM2 Ganglioside

Sandhoff's Disease (GM2) β-Hexosaminidase A & GM2 Ganglioside (+

B Globoside)

Krabbe's Disease β-Galactocerebrosidase Galactocerebroside

(Psychosine)

Gaucher's Disease β-Glucocerebrosidase Glucocerebroside

Niemann-Pick Disease (A/B) Sphingomyelinase Sphingomyelin

Farber's Disease Acid Ceramidase Ceramide

Fabry's Disease α-Galactosidase A Globotriaosylceramide (Gb3)

Metachromatic Leukodystrophy Arylsulfatase A Sulfatides

Wolman's Disease (CESD) Acid Lipase Cholesterol Esters, TGs

Note: Metachromatic Leukodystrophy was mentioned only in

the final table.

IX. Key Points to Remember

• Phospholipids and Glycolipids are complex lipids crucial for membranes and signaling.
• Sphingolipidosis = Lysosomal storage disease due to defective sphingolipid breakdown.
• Know the specific enzyme defect and accumulated substrate for each major sphingolipidosis.
• Key Clinical Differentiators:
◦ Neurological Deficit: Prominent in Krabbe, Tay-Sachs, Sandhoff, GM1. Minimal/Absent in Gaucher
(Type 1), Fabry.
◦ Hepatosplenomegaly: Prominent in Gaucher, Niemann-Pick, Sandhoff, GM1. Absent/Minimal in
Krabbe, Tay-Sachs, Fabry.
◦ Cherry-Red Spot: Tay-Sachs, Sandhoff, Niemann-Pick, GM1. Absent in Gaucher (Type 1), Fabry.
◦ Unique Features: Globoid cells (Krabbe), Gaucher cells (Gaucher), Zebra bodies (Niemann-Pick),
Angiokeratoma/Maltese Cross (Fabry), Adrenal Calcification (Wolman).
◦ Inheritance: Fabry is X-linked; others are AR.
• Cardiolipin is the only antigenic phospholipid and is located in the inner mitochondrial membrane.
• GM1 ganglioside is a receptor for cholera toxin.
Fatty Acid Oxidation Summary
I. Concept and Importance

• Context: Fatty Acid Oxidation (FAOx) is crucial during fasting states, especially after glycogen stores
are depleted.
• Fasting Stages & FAOx Roles:
◦ Early Fasting (4-16 hrs): Glycogenolysis provides glucose.
◦ Fasting (16-48 hrs): Gluconeogenesis is the primary glucose source. FAOx is essential here for:
1. Providing ATP: Gluconeogenesis requires energy, which is supplied by β-oxidation.
2. Providing Acetyl-CoA: Acetyl-CoA, a product of β-oxidation, is an allosteric activator of
Pyruvate Carboxylase, a key gluconeogenic enzyme (Note: Acetyl-CoA is not a substrate for
gluconeogenesis).
◦ Prolonged Fasting (>48 hrs): Non-carbohydrate substrates deplete. FAOx provides Acetyl-CoA
primarily for Ketone Body Synthesis to fuel vital organs like the brain.

II. Types of Fatty Acid Oxidation

1. Beta (β)-Oxidation:
◦ Most common type.
◦ Primarily acts on saturated fatty acids (e.g., Palmitic Acid, C16).
◦ Modified versions exist for: Very Long Chain Fatty Acids (VLCFA), Unsaturated Fatty Acids, Odd-
Chain Fatty Acids.
2. Minor Pathways:
◦ Alpha (α)-Oxidation
◦ Omega (ω)-Oxidation

III. Beta (β)-Oxidation (Focus on Palmitic Acid)

• Definition: Successive cleavage of 2-carbon units (as Acetyl-CoA) from the carboxyl end of a fatty acid,
releasing energy.
• Cleavage Site: Between the α-carbon and β-carbon.
• Why "Beta"? The β-carbon undergoes oxidation during the process (CH₂ group eventually becomes a
COOH group for the next cycle).
• Location:
◦ Tissues: Liver, Muscle, Adipose Tissue.
◦ Organelle: Mitochondria.

A. Preparatory Steps:

1. Activation:
◦ Location: Cytoplasm (Enzyme located on the Outer Mitochondrial Membrane (OMM)).
◦ Reaction: Fatty Acid (RCOOH) + ATP + CoA-SH → Acyl-CoA (RCO-SCoA) + AMP + PPi
◦ Enzyme: Acyl-CoA Synthetase (Thiokinase).
◦ Characteristics:
▪ Requires energy (Uses 2 high-energy phosphate bonds, equivalent to 2 ATP).
▪ Only energy-consuming step in the entire FAOx pathway.
 ▪ Enzyme belongs to the Ligase class.
2. Transport into Mitochondria (Carnitine Shuttle):
◦ Requirement: Necessary for Acyl-CoA with > 14 carbons. Shorter chain fatty acids can diffuse
directly.
◦ Key Molecule: Carnitine.
◦ Mechanism:
▪ CPT1 (Carnitine Palmitoyltransferase I / Carnitine Acyltransferase I - CAT1): Located on
OMM. Transfers acyl group from Acyl-CoA to Carnitine → Acylcarnitine + CoA. This is the
rate-limiting step and the major regulatory point.
▪ Translocase (Carnitine-Acylcarnitine Translocase): Located on Inner Mitochondrial
Membrane (IMM). Transports Acylcarnitine into the mitochondrial matrix in exchange for free
Carnitine moving out.
▪ CPT2 (Carnitine Palmitoyltransferase II / Carnitine Acyltransferase II - CAT2): Located on
IMM (matrix side). Transfers acyl group back from Acylcarnitine to mitochondrial CoA → Acyl-
CoA (inside matrix) + Carnitine.
◦ Carnitine Synthesis: Requires amino acids Lysine and Methionine (as SAM), and Vitamin C.

B. Steps of β-Oxidation Cycle (Inside Mitochondrial Matrix):

• Starts with Acyl-CoA inside the matrix. Each cycle involves 4 steps and removes one Acetyl-CoA unit.
• Mnemonic: Order of enzyme types: DeHydrogenase, Hydratase, DeHydrogenase, Thiolase (DH-HT)
1. Oxidation (Dehydrogenation):
▪ Enzyme: Acyl-CoA Dehydrogenase.
▪ Reaction: Acyl-CoA → Enoyl-CoA (trans double bond between α and β carbons).
▪ Coenzyme: FAD → FADH₂ (Generates 1.5 ATP via ETC).
2. Hydration:
▪ Enzyme: Enoyl-CoA Hydratase.
▪ Reaction: Adds H₂O across the double bond → Hydroxyacyl-CoA (OH group on β-carbon).
3. Oxidation (Dehydrogenation):
▪ Enzyme: Hydroxyacyl-CoA Dehydrogenase.
▪ Reaction: Hydroxyacyl-CoA → Ketoacyl-CoA (Keto group C=O on β-carbon).
▪ Coenzyme: NAD⁺ → NADH + H⁺ (Generates 2.5 ATP via ETC).
4. Thiolysis (Cleavage):
▪ Enzyme: Thiolase (β-Ketothiolase).
▪ Reaction: Ketoacyl-CoA + CoA-SH → Acetyl-CoA (2C unit) + Acyl-CoA (shortened by 2
carbons).
▪ The shortened Acyl-CoA re-enters the cycle.

C. Energetics (Example: Palmitic Acid - C16):

• Number of Cycles: (C/2) - 1 = (16/2) - 1 = 7 cycles.

• Number of Acetyl-CoA: C/2 = 16/2 = 8 Acetyl-CoA.
• ATP from Cycles: 7 cycles × (1 FADH₂ + 1 NADH) = 7 × (1.5 ATP + 2.5 ATP) = 7 × 4 ATP = 28 ATP.
• ATP from Acetyl-CoA: 8 Acetyl-CoA × 10 ATP/Acetyl-CoA (via TCA cycle & ETC) = 80 ATP.
• Gross ATP: 28 + 80 = 108 ATP.
 • Activation Cost: -2 ATP.
• Net ATP Yield (Palmitic Acid): 108 - 2 = 106 ATP (using modern values).
• Old Calculation Yield (Palmitic Acid): 129 ATP (using FADH₂=2, NADH=3, Acetyl-CoA=12).
• Stearic Acid (C18) Yield (Modern): 120 ATP (as stated in lecture).

D. Regulation of β-Oxidation:

• Rate-Limiting Enzyme: CPT1 (The "Gateway").

• Regulation by Malonyl-CoA:
◦ Well-fed State: ↑ Insulin → Activates Acetyl-CoA Carboxylase → ↑ Malonyl-CoA → Inhibits
CPT1 → ↓ FAOx. (Body is storing fat, not breaking it down).
◦ Fasting State: ↓ Insulin / ↑ Glucagon → Inactivates Acetyl-CoA Carboxylase → ↓ Malonyl-CoA →
CPT1 is active → ↑ FAOx. (Body needs energy from fat stores).

IV. Oxidation of Other Fatty Acids

1. Very Long Chain Fatty Acids (VLCFA; >C20/C22):

◦ Location: Starts in Peroxisomes, completed in Mitochondria.
◦ Process: Modified β-oxidation in peroxisomes shortens the chain down to Octanoyl-CoA (C8).
Products also include Acetyl-CoA and H₂O₂ (Hydrogen Peroxide).
◦ Octanoyl-CoA then enters mitochondria for standard β-oxidation.
◦ Note: The first step in peroxisomal β-oxidation uses an Acyl-CoA Oxidase that transfers electrons
directly to O₂, forming H₂O₂, instead of FADH₂.
2. Unsaturated Fatty Acids:
◦ Location: Mitochondria.
◦ Process: Standard β-oxidation occurs until a double bond is encountered.
◦ Modifications: Requires additional enzymes (e.g., Isomerase, Reductase - implied) to handle the
double bonds (position and configuration).
◦ Energetics: Bypasses the FAD-dependent Acyl-CoA Dehydrogenase step for existing double bonds
→ Yields 1.5 ATP less per double bond compared to a saturated FA of the same length.
3. Odd-Chain Fatty Acids:
◦ Location: Mitochondria.
◦ Process: Standard β-oxidation occurs, yielding Acetyl-CoA units until the final 3 carbons remain.
◦ Final Product: Propionyl-CoA (3C).
◦ Fate of Propionyl-CoA: It is carboxylated and converted to Succinyl-CoA, which enters the TCA
cycle. Propionyl-CoA is glucogenic (can be used to synthesize glucose). This is an exception to the
rule that fat cannot be converted to glucose.

V. Minor Oxidative Pathways

1. Alpha (α)-Oxidation:
◦ Location: Endoplasmic Reticulum (ER) and Peroxisomes.
◦ Substrate: Branched-chain fatty acids, especially those with a methyl group at the β-carbon (hinders
β-oxidation). Example: Phytanic Acid.
◦ Source of Phytanic Acid: Dairy products, green leafy vegetables.
 ◦ Process: Removes one carbon at a time from the carboxyl end.
2. Omega (ω)-Oxidation:
◦ Location: Smooth Endoplasmic Reticulum (Microsomes).
◦ Process: Oxidation occurs at the omega (ω) carbon (the terminal methyl group). CH₃ → CH₂OH →
COOH.
◦ Product: Dicarboxylic Acids (COOH groups at both ends).
◦ Energy: No ATP is generated directly by this pathway. It's a minor pathway, more active when β-
oxidation is impaired.

VI. Clinical Correlations / Disorders

1. FAOx Disorders & Fasting Hypoglycemia: Defects in FAOx → Reduced ATP and Acetyl-CoA →
Impaired gluconeogenesis → Hypoglycemia during fasting.
2. FAOx Inhibiting Drugs as Hypoglycemic Agents: Drugs inhibiting FAOx (Sulfonylureas mentioned,
though primary mechanism differs) → Reduce ATP supply for gluconeogenesis → Lower blood glucose.
3. Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency:
◦ Defect: Deficient enzyme for β-oxidation of C6-C12 fatty acids.
◦ Features:
▪ Fasting Hypoglycemia (impaired gluconeogenesis).
▪ Hypoketosis (low ketone bodies, as Acetyl-CoA precursor is reduced).
▪ Dicarboxylic Aciduria (due to increased reliance on ω-oxidation).
▪ Vomiting, lethargy, seizures, coma.
▪ Possible link to Sudden Infant Death Syndrome (SIDS).
◦ Treatment: Avoid fasting (frequent meals), high-carbohydrate, low-fat diet.
4. Jamaican Vomiting Sickness:
◦ Cause: Ingestion of unripe Ackee fruit containing the toxin Hypoglycin.
◦ Mechanism: Hypoglycin inhibits Acyl-CoA Dehydrogenases.
◦ Features: Severe hypoglycemia, vomiting, convulsions, coma, death. Low ketone bodies.
5. Refsum's Disease:
◦ Defect: Deficient α-oxidation enzyme (Phytanoyl-CoA Hydroxylase or Oxidase).
◦ Accumulation: Phytanic Acid.
◦ Features: Retinitis pigmentosa, peripheral neuropathy, ichthyosis (dry, scaly skin), cardiac
arrhythmias.
◦ Treatment: Dietary restriction of phytanic acid (avoid dairy, green leafy vegetables).
6. Zellweger Syndrome (Cerebrohepatorenal Syndrome):
◦ Defect: Peroxisomal biogenesis disorder (faulty Peroxisomal Targeting Signal - PTS). Enzymes
destined for peroxisomes cannot enter.
◦ Result: "Empty" peroxisomes (Peroxisomal Ghosts).
◦ Features related to FAOx: Impaired VLCFA oxidation and α-oxidation. Accumulation of VLCFAs
and Phytanic Acid.
◦ Other Clinical Features: Distinctive facial features (high forehead, epicanthal folds, hypertelorism -
resembling Down Syndrome), hypotonia, seizures, liver and kidney problems.
VII. Summary of Locations
Pathway Primary Location(s) Key Substrate(s) / Features

β-Oxidation (Saturated) Mitochondria Palmitic Acid (C16), etc.

β-Oxidation (Unsaturated) Mitochondria Unsaturated FAs, needs extra enzymes

β-Oxidation (Odd-Chain) Mitochondria Odd-Chain FAs → Propionyl-CoA (glucogenic)

VLCFA Oxidation Peroxisomes (start), Mitochondria (end) FAs > C20/C22

α-Oxidation Peroxisomes, ER Branched FAs (Phytanic Acid)

ω-Oxidation Smooth ER (Microsomes) Any FA (minor), makes dicarboxylic acids

Activation (Acyl-CoA Synth) Cytoplasm (Enzyme on OMM) Fatty Acid

Carnitine Shuttle OMM / Intermembrane Space / IMM Long-Chain Acyl-CoA

Key Points to Remember:

• β-Oxidation is the primary energy source from fats, occurring in mitochondria.

• FAOx is crucial during fasting to provide ATP and Acetyl-CoA (activator) for gluconeogenesis.
• The Carnitine shuttle (CPT1, Translocase, CPT2) is required for long-chain fatty acid entry into
mitochondria. CPT1 is the rate-limiting, regulated step, inhibited by Malonyl-CoA (high in well-fed
state).
• Each β-oxidation cycle yields 1 FADH₂, 1 NADH, and 1 Acetyl-CoA.
• Net ATP from Palmitic Acid (C16) is 106 ATP.
• Odd-chain fatty acids yield Propionyl-CoA, which is glucogenic.
• Peroxisomes handle VLCFA oxidation (initial steps) and α-oxidation (for branched FAs like phytanic
acid).
• ω-Oxidation occurs in the ER and produces dicarboxylic acids, especially when β-oxidation is blocked.
• MCAD Deficiency causes hypoketotic hypoglycemia during fasting.
• Jamaican Vomiting Sickness is due to Hypoglycin toxin inhibiting Acyl-CoA dehydrogenases.
• Refsum's Disease is a defect in α-oxidation leading to phytanic acid accumulation.
• Zellweger Syndrome is a peroxisomal biogenesis disorder affecting VLCFA and α-oxidation.
Okay students, here is a detailed summary of the Ketone Body Synthesis lecture for your revision.

Ketone Body Synthesis Summary

I. Introduction & Rationale

• Why study Ketone Bodies? To understand conditions like ketosis during starvation and Diabetic
Ketoacidosis (DKA), including the characteristic fruity breath smell.
• Why are Ketone Bodies Synthesized?
◦ During prolonged fasting/starvation (>48 hours), glycogen stores are depleted, and gluconeogenesis is
active but cannot fully meet energy demands, especially of the brain.
 ◦ Fatty acid oxidation increases, producing large amounts of Acetyl-CoA.
◦ Ketone bodies are produced as an alternative fuel source, particularly for vital organs like the brain.
◦ The brain prefers glucose but can derive approximately 20% of its energy from ketone bodies when
glucose is scarce.

II. Site of Synthesis

• Exclusive Site: Liver Mitochondria.

◦ Mnemonic Idea: Ketone Bodies are Lovely Molecules (Ketone Bodies = Liver Mitochondria).

III. Pathway of Ketone Body Synthesis

• Starting Substrate: Acetoacetyl-CoA (a 4-carbon intermediate from the final stages of beta-oxidation of
fatty acids).
• Step 1:
◦ Acetoacetyl-CoA (4C) + Acetyl-CoA (2C) → HMG-CoA (6C) (Hydroxymethylglutaryl-CoA)
◦ Enzyme: HMG-CoA Synthase (Mitochondrial isoform)
◦ This is the Rate-Limiting Enzyme of ketogenesis.
• Step 2:
◦ HMG-CoA (6C) → Acetoacetate (4C) + Acetyl-CoA (2C)
◦ Enzyme: HMG-CoA Lyase
• Primary Ketone Body: Acetoacetate (the first one formed).
• Formation of Secondary Ketone Bodies:
1. Acetone: Formed by spontaneous decarboxylation (no enzyme needed) of acetoacetate.
2. Beta-hydroxybutyrate: Formed by the reduction of acetoacetate.
▪ Enzyme: Beta-hydroxybutyrate Dehydrogenase
▪ Requires: NADH (which gets oxidized to NAD+)
• The Three Ketone Bodies: Acetoacetate, Acetone, Beta-hydroxybutyrate.
◦ Mnemonic: AAB (Acetoacetate, Acetone, Beta-hydroxybutyrate)

IV. HMG-CoA Synthase Isozymes

• Ketone Body Synthesis: Uses Mitochondrial HMG-CoA Synthase.

• Cholesterol Synthesis: Uses Cytosolic HMG-CoA Synthase.
◦ Mnemonic: Cholesterol Synthesis uses Cytosolic HMG-CoA Synthase (C4C).

V. Ketone Body Utilization (Ketolysis)

• Site: Extrahepatic Tissues (e.g., brain, skeletal muscle, heart muscle, kidney cortex). Utilization occurs
in the mitochondria of these tissues.
• Key Enzyme for Utilization: Thiophorase (Succinyl-CoA: Acetoacetate CoA Transferase).
◦ This enzyme activates acetoacetate back to acetoacetyl-CoA for energy production.
• Organs UNABLE to Utilize Ketone Bodies:
1. Liver: Lacks the enzyme Thiophorase. (Liver makes them but cannot use them).
2. Red Blood Cells (RBCs): Lack mitochondria.
 • Acetone Fate:
◦ It is volatile and neutral (does not contribute to acidosis).
◦ Excreted primarily via the lungs, causing the fruity smell in breath during ketosis/DKA.
◦ A small amount can be metabolized, but most is excreted.

VI. Ketosis in Specific Conditions

• A. Starvation:
1. Low blood glucose, depleted glycogen.
2. Increased gluconeogenesis depletes Oxaloacetate (OAA) levels in the liver mitochondria.
3. Increased lipolysis → Increased fatty acid oxidation → High levels of Acetyl-CoA.
4. Since OAA is low, Acetyl-CoA cannot efficiently enter the TCA cycle.
5. Excess Acetyl-CoA is diverted to ketone body synthesis in the liver.
• B. Uncontrolled Diabetes Mellitus (leading to DKA):
1. Insulin deficiency or resistance: Glucose cannot enter peripheral cells (muscle, adipose) effectively
(impaired GLUT4 function).
2. Cells perceive "starvation" despite high blood glucose.
3. Gluconeogenesis is stimulated → Depletes OAA.
4. Lack of Insulin Inhibition on Hormone Sensitive Lipase (HSL): HSL activity increases
significantly.
5. Increased lipolysis → Increased fatty acids → Increased beta-oxidation → Massive production of
Acetyl-CoA.
6. Low OAA prevents Acetyl-CoA entry into the TCA cycle.
7. Excess Acetyl-CoA is shunted into ketone body synthesis, leading to ketoacidosis (as acetoacetate
and beta-hydroxybutyrate are acidic).

VII. Detection of Ketone Bodies (Urine Tests)

• Rothera's Test:
◦ Detects: Acetoacetate and Acetone.
◦ Result: Purple ring.
• Gerhard's Test:
◦ Detects: Acetoacetate.
• Ketostix (Dipsticks):
◦ Principle based on Rothera's reaction.
◦ Detects: Primarily Acetoacetate, less sensitive to Acetone.
• Limitation: Standard urine tests (Rothera's, Ketostix) DO NOT detect Beta-hydroxybutyrate.
• Beta-hydroxybutyrate Measurement: Requires specific enzymatic assays (usually performed on
blood).

VIII. Predominance of Ketone Bodies

• Normal Physiological State (minimal ketogenesis):

◦ Beta-hydroxybutyrate : Acetoacetate ratio is roughly 1:1. Neither is strongly predominant.
 • Ketotic States (Starvation, DKA):
◦ The ratio shifts significantly due to high NADH levels from fatty acid oxidation favoring the
reduction of acetoacetate.
◦ Beta-hydroxybutyrate : Acetoacetate ratio becomes ~6:1 or higher.
◦ Beta-hydroxybutyrate is the predominant ketone body in severe ketosis.

Key Points to Remember:

• Site: Liver Mitochondria ONLY.

• Rate-Limiting Enzyme: Mitochondrial HMG-CoA Synthase.
• Primary Ketone Body: Acetoacetate.
• Secondary Ketone Bodies: Acetone (neutral, volatile, fruity smell) & Beta-hydroxybutyrate (acidic).
• Utilization Enzyme: Thiophorase (Absent in Liver).
• Organs that CANNOT use Ketones: Liver & RBCs.
• Stimulus: Low Insulin/Glucagon ratio (Starvation, DKA), leading to increased fatty acid oxidation and
OAA depletion.
• Predominant in Ketosis: Beta-hydroxybutyrate (but not detected by standard urine tests).
• Fruity Breath: Due to Acetone excretion via lungs.
• DKA Cause: Uninhibited HSL activity + OAA depletion leads to massive Acetyl-CoA diversion to
ketone synthesis.

Fatty Acid Synthesis Summary

I. Introduction & Concept

• Context: Synthesis of fatty acids leads to the formation of triacylglycerol (TAG), which is stored in
adipose tissue. Excess storage contributes to obesity.
• Timing: Occurs predominantly in the well-fed state.
• Hormonal Control: Primarily stimulated by Insulin.
II. Discovery

• Also known as De novo fatty acid synthesis.

• Pathway discovered by Fyodor Lynen (also referred to as "Lynen's Spiral").
III. Location (Site)

• Organs: Liver, Kidney, Brain, Lungs, Lactating Mammary Gland.

• Cellular Compartment: Cytosol (an "extra-mitochondrial" system).
• Compartmentalization: Fatty acid synthesis (cytosol) is physically separated from Beta-oxidation
(mitochondria), preventing degradation of newly synthesized fatty acids.
IV. Starting Substrate & Transport

• Starting Material: Acetyl-CoA.

• Source in Fed State: Primarily from Pyruvate Dehydrogenase (PDH) acting on pyruvate (derived from
glucose).
◦ Note: Acetyl-CoA from beta-oxidation (fasting state) is not used for synthesis.
 • Acetyl-CoA Origin: PDH produces Acetyl-CoA inside the mitochondria.
• Transport to Cytosol: Since synthesis is cytosolic, mitochondrial Acetyl-CoA must be transported:
1. Citrate Formation: Mitochondrial Acetyl-CoA combines with Oxaloacetate (OAA) to form Citrate
(TCA cycle intermediate).
2. Citrate Transport: Citrate moves from mitochondria to cytosol via the Tricarboxylic Acid
Transporter.
3. Cytosolic Cleavage: In the cytosol, ATP Citrate Lyase cleaves Citrate back into Acetyl-CoA (now
available for synthesis) and OAA. This step requires ATP.
V. Key Enzymes & Steps

• Two main enzyme systems are involved:

1. Acetyl-CoA Carboxylase (ACC)
2. Fatty Acid Synthase (FAS) Complex
• 1. Acetyl-CoA Carboxylase (ACC):
◦ Reaction: Acetyl-CoA (2C) + HCO3- + ATP → Malonyl-CoA (3C) + ADP + Pi
◦ Function: Carboxylation of Acetyl-CoA.
◦ Cofactors: Requires Biotin (Vitamin B7) and ATP. Uses HCO3- as the carbon dioxide donor.
◦ Enzyme Class: Ligase.
◦ Significance: This is the RATE-LIMITING ENZYME of fatty acid synthesis. Malonyl-CoA acts as
the donor of 2-carbon units for the growing fatty acid chain (despite being a 3C molecule, one carbon
is lost as CO2 during condensation).
• 2. Fatty Acid Synthase (FAS) Complex:
◦ Structure: A large Multifunctional Enzyme (single polypeptide chain with multiple catalytic sites).
Exists as an X-shaped Homodimer.
◦ Domains/Units (per monomer): Contains 6 distinct enzyme activities organized into 3 domains:
▪ Condensing Unit:
▪ Malonyl/Acetyl Transacylase (MAT): Loads Acetyl-CoA (initially) and Malonyl-CoA onto the
complex.
▪ Ketoacyl Synthase (KS): Condenses the activated acyl group with Malonyl-CoA, releasing
CO2 and forming a β-ketoacyl group (adds 2 carbons).
▪ Reduction Unit: (Mnemonic: RDR -> Reduce, Dehydrate, Reduce)
▪ Ketoacyl Reductase (KR): Reduces the keto group to a hydroxyl group. Requires NADPH.
▪ Dehydratase (DH): Removes water (dehydration) creating a double bond (enoyl group).
▪ Enoyl Reductase (ER): Reduces the double bond to a single bond (saturated acyl group).
Requires NADPH.
▪ Releasing Unit:
▪ Thioesterase (TE) / Deacylase: Cleaves the completed fatty acid chain (typically Palmitate)
from the FAS complex.
◦ Reaction Cycle:
1. Loading of Acetyl-CoA (first cycle only) and Malonyl-CoA (all cycles) via MAT.
2. Condensation via KS (forms β-ketoacyl group, releases CO2).
3. Reduction via KR (uses NADPH).
4. Dehydration via DH.
 5. Reduction via ER (uses NADPH).
6. The elongated saturated acyl group is transferred, and the cycle repeats (steps 2-5) with a new
Malonyl-CoA, adding 2 carbons per cycle.
7. Release of the final fatty acid (usually Palmitate, 16C) via TE.
◦ Most Common Product: Palmitic Acid (C16:0).
VI. Cofactor Requirements (Overall)

• NADPH: For the two reduction steps catalyzed by Ketoacyl Reductase (KR) and Enoyl Reductase (ER)
within the FAS complex.
◦ Sources of NADPH: HMP Shunt (Oxidative Phase), Malic Enzyme, Cytosolic Isocitrate
Dehydrogenase.
• ATP: Required by Acetyl-CoA Carboxylase (ACC) and ATP Citrate Lyase.
• Biotin (Vitamin B7): Required by ACC.
• HCO3- (Bicarbonate): Carbon source for ACC.
• Mn2+ (Manganese): Cofactor for the FAS complex.
VII. Regulation

• Key Regulatory Enzyme: Acetyl-CoA Carboxylase (ACC) (the rate-limiting step).

• Principle: Synthesis is favored in the well-fed state (high energy, high insulin) and inhibited during
fasting/stress (low energy, high glucagon/epinephrine).
• Short-Term Regulation (Allosteric):
◦ Activator: Citrate. Allosterically activates ACC by promoting its polymerization from an inactive
dimer to an active polymer form. (High citrate indicates ample substrate from mitochondria).
Mnemonic: Citrate Cytosol Carboxylase Activation.
◦ Inhibitor: Long-chain fatty acyl-CoA (the end product). Inhibits ACC directly (product inhibition)
and also inhibits the Tricarboxylic Acid Transporter, reducing citrate export from mitochondria.
• Long-Term Regulation (Hormonal / Covalent Modification of ACC):
◦ Insulin (Well-fed): Promotes DEphosphorylation of ACC, making it ACTIVE.
◦ Glucagon & Epinephrine (Fasting/Stress): Promote PHOSPHORYLATION of ACC, making it
INACTIVE. Mnemonic: Phosphorylation Puts the Pause on Synthesis.

VIII. Key Points to Remember:

• Fatty acid synthesis occurs in the cytosol during the well-fed state, stimulated by insulin.
• The primary source of Acetyl-CoA is pyruvate via PDH, transported out of mitochondria as citrate.
• Acetyl-CoA Carboxylase (ACC) is the rate-limiting enzyme, requires biotin, ATP, and HCO3-, and
produces Malonyl-CoA.
• Fatty Acid Synthase (FAS) is a multifunctional enzyme complex that performs a cycle of
condensation, reduction, dehydration, and reduction, adding 2 carbons per cycle from Malonyl-CoA.
• NADPH is the essential reductant, primarily from the HMP shunt.
• Citrate allosterically activates ACC; long-chain fatty acyl-CoA inhibits ACC and citrate transport.
• Insulin promotes dephosphorylation (activation) of ACC; Glucagon/Epinephrine promote
phosphorylation (inactivation) of ACC.
• The most common end product is Palmitic Acid (C16:0).
Cholesterol & Bile Acids Summary
I. Introduction & Significance of Cholesterol

• Metabolic Fuel? No, cholesterol cannot be used as a metabolic fuel or to derive energy.
• Excretion: Primarily excreted via conversion to bile acids.
• Nature: An exclusively animal sterol (not found in plants).
• Functions:
◦ Synthesis of Vitamin D.
◦ Synthesis of Steroid Hormones (e.g., corticosteroids, sex hormones).
• Conservation: The body tightly conserves cholesterol.
◦ Bile acids (carrying cholesterol) undergo extensive enterohepatic circulation (~98-99% reabsorbed
from the intestine back to the liver).
◦ This conservation mechanism, evolved during times of starvation, can lead to harmful accumulation
in modern sedentary, well-fed states.
• Regulation: Cholesterol metabolism is a tightly regulated pathway due to the difficulty of excretion
and potential harm from accumulation. Dietary cholesterol intake inhibits the body's own cholesterol
synthesis.

II. Cholesterol Chemistry

• Carbon Count: 27 Carbon atoms.

• Structure: Contains a specific ring structure called the Cyclopentanoperhydrophenanthrene ring.
• Property: Amphipathic (has both hydrophilic and hydrophobic parts).

III. Cholesterol Synthesis (De Novo)

• Location: Occurs in virtually all nucleated cells. Particularly active in:

◦ Liver
◦ Adrenal Cortex
◦ Testis
◦ Ovaries
◦ Intestine
• Cellular Compartments: Cytoplasm and Smooth Endoplasmic Reticulum (SER).
• Starting Material: Acetyl-CoA (Note: Unlike ketone body synthesis which often starts from fat
breakdown in fasting, cholesterol synthesis occurs in the well-fed state).
• Key Steps:
1. Formation of HMG-CoA:
▪ 2 Acetyl-CoA → Acetoacetyl-CoA (Enzyme: Thiolase)
▪ Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA (Hydroxymethylglutaryl-CoA) (Enzyme: HMG-
CoA Synthase - Cytosolic)
2. Formation of Mevalonate:
▪ HMG-CoA → Mevalonate (Enzyme: HMG-CoA Reductase)
▪ This is the Rate-Limiting Step.
▪ Requires NADPH.
 ▪ Occurs in the Endoplasmic Reticulum.
▪ Tightly regulated enzyme.
3. Formation of Isoprenoid Units:
▪ Mevalonate → Isoprenoid unit (5-Carbon, Isopentenyl unit).
4. Condensations:
▪ 2 x 5C units → 10C unit (Geranyl pyrophosphate)
▪ 10C unit + 5C unit → 15C unit (Farnesyl pyrophosphate)
▪ 2 x 15C units → 30C unit (Squalene)
▪ Mnemonic Idea: Acetate --> HMG --> Mevalonate --> Isoprenoid --> Geranyl --> Farnesyl -->
Squalene
5. Cyclization & Final Steps:
▪ Squalene cyclizes → Lanosterol (30C, the first steroid compound formed).
▪ Lanosterol → Cholesterol (27C) through a series of reactions.

IV. Fates of Cholesterol

• No Energy Production: Cholesterol is not broken down for energy.

• Excretion (Unabsorbed): Unabsorbed dietary cholesterol is excreted in feces (e.g., as Coprostanol).
• Major Conversions (Absorbed/Synthesized):
1. Bile Acid Synthesis: ~50% of excess cholesterol is converted to bile acids (primary route for
elimination).
2. Vitamin D Synthesis: Used as needed.
3. Steroid Hormone Synthesis: Used for corticosteroids and sex hormones.

V. Regulation of Cholesterol Synthesis

• Rate-Limiting Enzyme: HMG-CoA Reductase.

• Mechanisms:
1. Feedback Regulation (Genetic Level):
▪ High dietary cholesterol inhibits the synthesis of enzymes involved in cholesterol production
(including HMG-CoA Reductase).
▪ Mediated via SREBP (Sterol Regulatory Element Binding Protein), which suppresses gene
expression for these enzymes.
2. Feedback Inhibition (Enzyme Level):
▪ High levels of cholesterol (the end-product) directly inhibit the activity of the HMG-CoA
Reductase enzyme.
3. Hormonal Regulation:
▪ Stimulate HMG-CoA Reductase Activity: Insulin, Thyroxine (promote synthesis in well-fed
state).
▪ Inhibit HMG-CoA Reductase Activity: Glucagon, Glucocorticoids (inhibit synthesis during
fasting/stress).

VI. Bile Acid Synthesis

• Starting Material: Cholesterol.

• Location: Primary synthesis in the Liver.
 • Key Steps:
1. Hydroxylation:
▪ Cholesterol → 7-hydroxycholesterol (Enzyme: 7-alpha-hydroxylase)
▪ This is the Rate-Limiting Step for bile acid synthesis.
▪ Requires NADPH and Vitamin C.
2. Formation of Primary Bile Acids:
▪ 7-hydroxycholesterol undergoes further reactions (requiring O2, NADPH; releasing Propionyl-
CoA) → Primary Bile Acids.
▪ Synthesized in the Liver.
▪ Examples: Cholic acid (most abundant), Chenodeoxycholic acid.
▪ (Mnemonic: Primary acids made Primarily in the Liver: Cholic, Chenodeoxycholic)
3. Conjugation:
▪ Primary bile acids are conjugated (linked) in the liver, mainly with Glycine (predominant) or
Taurine.
▪ Examples: Glycocholic acid, Taurocholic acid.
▪ Purpose: Increases water solubility and amphipathic nature, facilitating secretion into bile.
4. Secretion & Intestinal Modification:
▪ Conjugated primary bile acids are secreted via bile into the Intestine.
▪ In the intestine, gut bacteria perform deconjugation and dehydroxylation.
5. Formation of Secondary Bile Acids:
▪ Modification of primary bile acids in the Intestine forms Secondary Bile Acids.
▪ Examples: Deoxycholic acid (from cholic acid), Lithocholic acid (from chenodeoxycholic acid).
▪ (Mnemonic: Secondary acids made Secondarily in the Intestine: Deoxycholic, Lithocholic)

VII. Enterohepatic Circulation of Bile Acids

• Process: Most secondary (and some primary) bile acids are reabsorbed from the intestine, travel via the
portal vein back to the liver, and are re-secreted into bile.
• Efficiency: Very high, ~98-99% of bile acids are recycled.
• Significance: Conserves the body's cholesterol pool, making net cholesterol excretion difficult.
• Exception: Lithocholic acid undergoes the least enterohepatic circulation (more is excreted).

VIII. Bile Acids vs. Bile Salts

• Bile: Alkaline medium.

• Bile Acids: In the alkaline pH of bile, the acidic bile acids become ionized (negatively charged).
• Bile Salts: These ionized bile acids associate with cations like Sodium (Na+) or Potassium (K+).
• Relationship: Bile salt is the form in which bile acids exist in the alkaline environment of bile (e.g.,
Sodium glycocholate). They are functionally the same molecule in different states.

IX. Clinical Correlation: Bile Acid Sequestrants

• Mechanism: These drugs bind to bile acids within the intestine.

• Effect: Prevents the reabsorption of bile acids (interrupts enterohepatic circulation).
 • Outcome: Bound bile acids (containing cholesterol derivatives) are excreted in the feces. This loss forces
the liver to convert more cholesterol into new bile acids, thereby lowering blood cholesterol levels.
• Therapeutic Use: Used as hypocholesterolemic drugs (to lower cholesterol).

Key Points to Remember:

1. Cholesterol is NOT fuel: It's a structural component and precursor (Vit D, Steroid Hormones, Bile
Acids).
2. Excretion is Difficult: Primarily via bile acids, but most are recycled (Enterohepatic Circulation).
3. Synthesis Regulation is Crucial: HMG-CoA Reductase is the rate-limiting enzyme, regulated by
feedback (cholesterol, SREBP) and hormones (Insulin/Thyroxine vs. Glucagon/Glucocorticoids).
4. Bile Acid Synthesis: Cholesterol is the precursor. 7-alpha-hydroxylase is the rate-limiting step (needs Vit
C, NADPH).
5. Primary vs. Secondary Bile Acids: Primary (Cholic, Chenodeoxycholic) made in Liver; Secondary
(Deoxycholic, Lithocholic) formed in Intestine by bacteria.
6. Bile Salts: Ionized form of bile acids complexed with Na+/K+ in alkaline bile.
7. Enterohepatic Circulation: Efficient recycling (~98-99%) of bile acids conserves cholesterol.
Lithocholic acid is least recycled.
8. Bile Acid Sequestrants: Lower cholesterol by blocking bile acid reabsorption, forcing excretion and
increased cholesterol use for bile acid synthesis.
Okay students, here is a detailed summary of the Lipoproteins session for your revision.

Lipoproteins Summary
1. Introduction & Definition

• Relevance: Clinically important and applied topic, relevant for exams and practice.
• Core Questions:
◦ Why complex lipids with proteins? -> For transport in blood.
◦ Why is LDL "bad" and HDL "good" despite both being lipoproteins? -> Due to their different roles in
cholesterol transport.
• Definition: Lipoproteins are compound lipids formed by the complexing of lipids with proteins
(apolipoproteins or apoproteins).
• Concept: Lipids are hydrophobic, but needed by cells. Complexing with proteins allows transport in the
aqueous plasma.

2. Lipoprotein Structure

• Core (Inner Layer): Hydrophobic lipids.

◦ Triacylglycerol (TAG)
◦ Cholesterol Esters (CE) (Lecture mentions Cholesterol here, but CE is the hydrophobic form typically
in the core; LDL section confirms high CE content)
• Shell (Surface/Second Layer): Amphipathic components.
◦ Phospholipids (PL)
◦ Free Cholesterol (FC)
 • Outer Layer: Proteins (Apolipoproteins).
◦ Can be peripheral or integral.

3. Major Classes of Lipoproteins

(Listed generally from largest/least dense to smallest/most dense)

1. Chylomicrons (CM)
2. Very Low-Density Lipoprotein (VLDL)
3. Intermediate-Density Lipoprotein (IDL)
4. Low-Density Lipoprotein (LDL)
5. High-Density Lipoprotein (HDL)

4. Characteristics & Functions of Major Lipoproteins

a) Chylomicrons (CM)

• Origin: Intestine.
• Function: Transport exogenous (dietary) TAG primarily to peripheral organs (and liver).
• Size: Maximum size.
• Density: Least dense.
• Lipid Content: Maximum lipid content (primarily TAG).
• Apolipoproteins:
◦ ApoB48 (Unique to CM)
◦ ApoC2
◦ ApoE

b) Very Low-Density Lipoprotein (VLDL)

• Origin: Liver.
• Function: Transport endogenous (liver-synthesized) TAG to peripheral organs.
• Apolipoproteins:
◦ ApoB100
◦ ApoC2
◦ ApoE

c) Low-Density Lipoprotein (LDL) - "Bad Cholesterol"

• Origin: Formed from VLDL via IDL (Lipoprotein Cascade Pathway: VLDL → IDL → LDL).
• Function: Carry cholesterol to extrahepatic tissues and liver. Delivery to peripheral tissues contributes
to its "dangerous" reputation.
• Lipid Content: Maximum cholesterol and cholesterol ester content.
• Apolipoprotein: Only ApoB100.

d) High-Density Lipoprotein (HDL) - "Good Cholesterol"

• Origin: Liver and Intestine.

• Function: Reverse Cholesterol Transport (RCT) - picks up cholesterol from periphery and transports it
back to the liver.
 • Density: Highest density.
• Protein Content: Highest protein content.
• Apolipoprotein Content: Maximum overall apolipoprotein content.
• Lipid Content: Least lipid content.
• Phospholipid Content: Maximum phospholipid content.
• Apolipoproteins:
◦ ApoA1 (Most significant)
◦ ApoC (various)
◦ ApoD
◦ ApoE (various)
• Key Enzymes Associated with HDL:
◦ LCAT (Lecithin Cholesterol Acyltransferase):
▪ Action: Lecithin + Cholesterol → Lysolecithin + Cholesterol Ester (CE).
▪ Significance: Converts amphipathic Cholesterol into hydrophobic CE, allowing it to move into
the HDL core. This is essential for cholesterol uptake capacity. Activated by ApoA1.
▪ Mnemonic: Lets Cholesterol Acquire Tails (Fatty Acid tail added -> Ester)
◦ CETP (Cholesterol Ester Transfer Protein):
▪ Action: Transfers CE from HDL to other lipoproteins (IDL, LDL) in exchange for TAG.

5. Specialized Lipoproteins
a) Lipoprotein(a) / Lp(a)

• Structure: Similar to LDL, but contains Apo(a) linked to ApoB100 via a disulfide bond.
• Peculiarity: Apo(a) is structurally similar to plasminogen (a plasminogen analog).
• Mechanism: Competes with plasminogen for tissue plasminogen activator (tPA) -> Reduces plasmin
formation -> Inhibits clot lysis (fibrinolysis).
• Clinical Significance: Risk factor for thrombosis.

b) Lipoprotein X / LpX

• Formation: Accumulates in cholestasis (blockage of bile flow).

• Mechanism: Impaired bile acid excretion -> Cholesterol accumulates in the liver -> Cholesterol +
Phospholipids form abnormal lipoprotein LpX.
• Clinical Significance: Marker/Indicator of cholestasis.

6. Electrophoretic Mobility
(Separation based on charge and size; movement towards anode (+))

• Origin (Cathode/-): Chylomicrons (Largest, don't move much)

• Beta (β) Region: LDL
• Pre-Beta (Pre-β) Region: VLDL
• Broad-Beta Region: IDL (migrates between VLDL and LDL)
• Alpha (α) Region (Fastest): HDL (Smallest, highest protein/charge ratio)
 • Mnemonic (Fastest to Slowest): Healthy Volkswagen Is Less Costly (HDL > VLDL > IDL > LDL >
CM) - Note: VLDL moves faster than LDL. Or remember the Greek letters: α (HDL) -> Pre-β (VLDL) ->
Broad-β (IDL) -> β (LDL).

7. Lipoprotein Metabolism
a) Chylomicron Metabolism (Exogenous Pathway)

1. Formation: Intestine forms Nascent CM (ApoB48).

2. Maturation: Acquires ApoC2 & ApoE from HDL in plasma -> Mature CM.
3. Lipolysis: In peripheral capillaries, Lipoprotein Lipase (LPL), activated by ApoC2, hydrolyzes TAG ->
Fatty Acids (FA) + Glycerol. FA taken up by tissues (adipose, muscle).
4. Remnant Formation: TAG-depleted CM becomes CM Remnant.
5. Uptake: CM Remnants taken up by the Liver via receptor-mediated endocytosis. Ligand for receptor is
ApoE.

b) VLDL/IDL/LDL Metabolism (Endogenous Pathway)

1. Formation: Liver forms Nascent VLDL (ApoB100).

2. Maturation: Acquires ApoC2 & ApoE from HDL -> Mature VLDL.
3. Lipolysis: In peripheral capillaries, LPL (activated by ApoC2) hydrolyzes TAG -> FA uptake by tissues.
4. IDL Formation: VLDL remnant = IDL (still has B100, E, C2 temporarily).
5. IDL Fates:
◦ ~50% taken up by Liver (Ligand: ApoE).
◦ ~50% converted to LDL:
▪ Further TAG removal by Hepatic Lipase (HL).
▪ ApoE and ApoC2 returned to HDL.
▪ Result: LDL (rich in Cholesterol/CE, only ApoB100).
6. LDL Fates:
◦ ~70% taken up by Liver via LDL receptor (Ligand: ApoB100).
◦ ~30% taken up by Extrahepatic Tissues via LDL receptor (Ligand: ApoB100) - Source of
peripheral cholesterol deposition.
◦ (Pathological): LDL can become Oxidized LDL (under oxidative stress) -> Taken up by
Macrophages (via scavenger receptors) -> Foam cell formation -> Atherosclerosis risk.

c) HDL Metabolism (Reverse Cholesterol Transport)

1. Formation: Liver & Intestine secrete Nascent Discoidal HDL (PL, FC, ApoA1).
2. Cholesterol Esterification: LCAT (activated by ApoA1) esterifies FC to CE -> CE moves to core ->
HDL becomes Spherical HDL3.
3. Cholesterol Pickup: HDL3 accepts free cholesterol from peripheral cells via transporters (ABCA1,
ABCG1, SRB1).
4. Maturation: Continued LCAT action increases CE core -> HDL3 matures into larger HDL2.
5. Cholesterol Delivery to Liver: HDL2 delivers CE/C to the liver via SRB1 (Scavenger Receptor B1) -
This is selective uptake, not endocytosis of the whole particle.
6. Hepatic Processing: Liver converts cholesterol to bile acids for excretion.
 7. HDL Remodeling: Hepatic Lipase can hydrolyze TAG/PL in HDL2, regenerating HDL3 (HDL Cycle).
HDL components (Apos) are recycled.

8. Apolipoprotein Functions (Summary)

• ApoA1: Primary HDL protein; Activator of LCAT.

• ApoA2: Inhibits LPL.
• ApoA5: Promotes LPL action.
• ApoB100: Synthesized in Liver; Structural for VLDL/IDL/LDL; Ligand for LDL receptor.
• ApoB48: Synthesized in Intestine; Structural for CM; Essential for CM assembly. (Truncated version of
ApoB100 due to mRNA editing).
• ApoC1: Inhibits CETP.
• ApoC2: Activator of Lipoprotein Lipase (LPL).
• ApoC3: Inhibitor of LPL.
• ApoE: Ligand for receptor-mediated uptake of remnants (CM remnants, IDL) by the liver. Arginine-
rich.
• ApoE4 Allele: Associated with increased risk of late-onset Alzheimer's disease.
• ApoD: Associated with human degenerative disorders.
• Apo(a): Component of Lp(a); Plasminogen analog; Inhibits fibrinolysis.

Key Points to Remember

• Lipoproteins are essential for transporting hydrophobic lipids (TAG, Cholesterol) in blood.
• Chylomicrons (CM): Transport DIETARY (exogenous) TAG from intestine. Unique Apo: B48.
• VLDL: Transport LIVER-SYNTHESIZED (endogenous) TAG. Apo: B100.
• LDL: "Bad"; Delivers cholesterol TO peripheral tissues. Main Apo: B100. High levels risk
atherosclerosis. Formed from VLDL.
• HDL: "Good"; Performs Reverse Cholesterol Transport (cholesterol FROM periphery TO liver). Main
Apo: A1. Activates LCAT.
• LPL (Lipoprotein Lipase): Breaks down TAG in CM and VLDL. Activated by ApoC2.
• LCAT: Esterifies cholesterol within HDL. Activated by ApoA1.
• Receptor Ligands: ApoE for remnant uptake; ApoB100 for LDL uptake.
• Lp(a): LDL-like particle, inhibits clot lysis, thrombosis risk.
• LpX: Marker for cholestasis.
• Electrophoresis Order (Fastest to Slowest): HDL (α) > VLDL (Pre-β) > IDL (Broad-β) > LDL (β) >
CM (Origin).
• Clinical Relevance: Understanding lipoprotein metabolism is key to managing dyslipidemias and
cardiovascular risk. HDL removes cholesterol, while LDL deposits it peripherally (especially when
oxidized).

Dyslipidemia Summary (Biochemical Aspects)

I. Introduction

• Understanding lipoprotein metabolism is key to understanding dyslipidemias.

 • Focus: Biochemical basis of primary hyperlipoproteinemias and hypolipoproteinemias.
• Goal: Correlate clinical features and lab findings with underlying defects, avoiding rote memorization.
• Key Questions Addressed:
◦ Why milky plasma in Type 1 vs. clear in Type 2? (Due to high Triglycerides vs. Cholesterol)
◦ Why pancreatitis/abdominal pain in Type 1 vs. CAD/chest pain in Type 2? (High TAG vs. High
Cholesterol effects)
II. Classification of Dyslipidemias

• Dyslipidemia: Includes Hyperlipoproteinemias and Hypolipoproteinemias.

• Hyperlipoproteinemias:
◦ Primary (Genetic/Hereditary) - Focus of this lecture
◦ Secondary (Due to other diseases/drugs) - Not covered in detail
• Modern Classification (Combined Harrison & Fredrickson):
◦ Primary Hyperlipoproteinemias with Hypertriglyceridemia (HTG):
▪ Fredrickson Type 1
▪ Fredrickson Type 4
▪ Fredrickson Type 5
◦ Primary Hyperlipoproteinemias with Hypercholesterolemia (HC):
▪ Fredrickson Type 2
◦ Primary Hyperlipoproteinemias with Both HTG & HC:
▪ Fredrickson Type 3
III. Primary Hyperlipoproteinemias
A. Type 1 Hyperlipoproteinemia (Familial Chylomicronemia Syndrome)

• Clinical Vignette: Young patient, recurrent abdominal pain, blood like "cream of tomato soup", creamy
layer on refrigerated sample, milky white (lactescent) plasma, eruptive xanthomas (grouped), lipemia
retinalis.
• Biochemical Defect:
◦ Deficiency of Lipoprotein Lipase (LPL) OR
◦ Deficiency of ApoCII (LPL activator).
• Pathophysiology: Impaired breakdown of mature chylomicrons and VLDL.
• Elevated Lipoprotein: Chylomicrons >>> VLDL.
• Elevated Lipid: Triglycerides (TAG). Cholesterol is normal.
• Clinical Features:
◦ Recurrent Pancreatitis -> Acute abdominal pain.
◦ Lactescent Plasma.
◦ Eruptive Xanthomas (grouped clusters).
◦ Lipemia Retinalis.
◦ Hepatosplenomegaly.
◦ NO increased risk of Coronary Artery Disease (CAD) due to normal cholesterol.
• Mnemonic: Type 1 = 1 Lipase (LPL) problem. -> High TAG -> Tummy ache (pancreatitis), Abnormal
plasma (milky), Grouped xanthomas (eruptive).
B. Type 2 Hyperlipoproteinemia (Primarily Hypercholesterolemia)

• Clinical Vignette: Corneal arcus, Tendon Xanthomas (discrete, esp. Achilles tendon).
• Elevated Lipid: Cholesterol. TAG is normal. Plasma is clear.
• Major Clinical Association: Premature Coronary Artery Disease (CAD) -> Chest pain.
• Subtypes:
1. Familial Hypercholesterolemia (FH) / ADH Type 1:
▪ Most common primary hyperlipoproteinemia.
▪ Defect: LDL Receptor (LDLR) mutation (Autosomal Dominant).
▪ Elevated Lipoprotein: LDL.
▪ Features: Premature CAD, Tendon xanthomas (sometimes called Tuberous xanthomas), Corneal
arcus.
2. Familial Defective ApoB100 (FDB) / ADH Type 2:
▪ Defect: Mutation in ApoB100 (ligand for LDLR).
▪ Result: Impaired LDL binding to LDLR -> High LDL/Cholesterol.
3. ADH Type 3:
▪ Defect: Gain-of-function mutation in PCSK9.
▪ Result: Increased LDLR degradation -> Fewer LDLRs -> High LDL/Cholesterol.
4. Autosomal Recessive Hypercholesterolemia (ARH):
▪ Defect: Mutation in LDLR Adaptor Protein (LDLRAP1).
▪ Result: Impaired LDL internalization -> High LDL/Cholesterol.
5. Sitosterolemia (Phytosterolemia):
▪ Defect: Mutation in ABCG5 or ABCG8 transporters.
▪ Result: Impaired excretion of plant sterols -> Plant sterol accumulation -> Cellular feedback ↓
LDLR synthesis -> High LDL/Cholesterol (and high plant sterols).
• Mnemonic: Type 2 = 2 C's (Cholesterol & CAD). Problems in LDL clearance (LDLR, ApoB, PCSK9,
LDLRAP1, ABCG5/8).
C. Type 3 Hyperlipoproteinemia (Familial Dysbetalipoproteinemia / Remnant Removal Disease /
Broad-Beta Disease)

• Clinical Vignette: Tuberoeruptive xanthomas ("bunch of grapes" appearance), Palmar xanthomas (lipid
streaks in palmar creases).
• Biochemical Defect: Mutation in ApoE (often ApoE2 homozygosity, which binds poorly to receptors).
• Pathophysiology: Impaired hepatic uptake of chylomicron remnants and VLDL remnants (IDL).
• Elevated Lipoprotein: Chylomicron Remnants and VLDL Remnants (IDL). (IDL = Broad-Beta band
on electrophoresis).
• Elevated Lipid: Both Triglycerides (TAG) and Cholesterol (moderate elevation).
• Clinical Features:
◦ Tuberoeruptive Xanthomas.
◦ Palmar Xanthomas (pathognomonic when present).
◦ Slightly Increased risk of CAD (due to elevated cholesterol). Peripheral vascular disease is also
common.
• Mnemonic: Type 3 = 3 letters (Apo E). Affects Remnants. Both TAG & Cholesterol up.
IV. Recent Treatments for Hyperlipoproteinemias

• Type 1 (LPL Defect):

◦ Alipogene tiparvovec (Glybera): Gene therapy (IM injection) delivering a gain-of-function LPL
gene variant.
• Type 2 (Homozygous FH):
◦ Lomitapide: Inhibits Microsomal Triglyceride Transfer Protein (MTP) -> Reduces VLDL and
subsequently LDL assembly/secretion.
◦ Antisense Oligonucleotide Therapy: (e.g., Mipomersen targeting ApoB mRNA) -> Reduces ApoB
synthesis -> Reduces VLDL/LDL production.
V. Hypolipoproteinemias
A. Tangier Disease

• Clinical Picture: Enlarged orange/yellowish tonsils, peripheral neuropathy (e.g., mononeuritis

multiplex), hepatosplenomegaly.
• Biochemical Defect: Mutation in ABCA1 transporter.
• Pathophysiology: ABCA1 normally transfers cholesterol from peripheral cells to nascent HDL. Defect
impairs HDL maturation and cholesterol efflux.
• Lipid Profile: Very low HDL and ApoA1. Cholesterol ester accumulation in reticuloendothelial system.
B. Abetalipoproteinemia (ABL)

• Clinical Picture: Infants/children with severe fat malabsorption (steatorrhea, diarrhea, failure to thrive),
progressive neurological deficits (ataxia, weakness), retinitis pigmentosa, acanthocytes (spur cells) on
blood smear, bleeding tendency (Vit K deficiency).
• Biochemical Defect: Mutation in Microsomal Triglyceride Transfer Protein (MTP).
• Pathophysiology: MTP is essential for loading lipids onto ApoB and secreting ApoB-containing
lipoproteins (Chylomicrons, VLDL).
• Lipid Profile: Absence or near absence of Chylomicrons, VLDL, IDL, LDL (all ApoB lipoproteins).
HDL is normal or low-normal. Severe deficiency of fat-soluble vitamins (A, D, E, K).
• Name: Absence of Beta-(LDL) and Pre-Beta-(VLDL) lipoproteins.
C. LCAT Deficiency

• LCAT Function: Lecithin-Cholesterol Acyltransferase; esterifies free cholesterol on HDL. (Cholesterol

+ Lecithin -> Cholesterol Ester + Lysolecithin).
• Biochemical Changes: ↑ Free Cholesterol, ↑ Lecithin; ↓ Cholesterol Esters, ↓ Lysolecithin. Low HDL.
• Types:
1. Complete LCAT Deficiency (Norum Disease):
▪ Progressive corneal opacification.
▪ Hemolytic anemia.
▪ Progressive end-stage renal disease (fatal).
2. Partial LCAT Deficiency (Fish Eye Disease):
▪ Corneal opacification ("fish eye" appearance).
▪ Benign condition, no progressive renal disease.
VI. Summary of Key Defects
 Condition Fredrickson Defective Protein/Gene Main Lipoprotein(s) Main Lipid(s)
Type Affected Affected

Familial Chylomicronemia Type 1 LPL or ApoCII ↑↑ Chylomicrons, ↑ ↑↑ TAG, Normal

VLDL Chol

Familial Type 2a LDL Receptor (LDLR) ↑ LDL ↑ Cholesterol,

Hypercholesterolemia Normal TAG

Familial Defective Type 2a ApoB100 ↑ LDL ↑ Cholesterol,

ApoB100 Normal TAG

ADH Type 3 Type 2a PCSK9 (Gain of function) ↑ LDL ↑ Cholesterol,

Normal TAG

ARH Type 2a LDLRAP1 ↑ LDL ↑ Cholesterol,

Normal TAG

Sitosterolemia Type 2a ABCG5 or ABCG8 ↑ LDL, ↑ Plant Sterols ↑ Cholesterol

Familial Type 3 ApoE (often E2/E2) ↑ Chylomicron ↑ TAG, ↑

Dysbetalipoproteinemia Remnants, ↑ IDL Cholesterol
(Remnants)

Tangier Disease (Hypo) ABCA1 ↓↓ HDL Low HDL-C

Abetalipoproteinemia (Hypo) MTP Absent Chylomicrons, Very low TAG &

VLDL, IDL, LDL Chol

LCAT Deficiency (Hypo) LCAT (Lecithin- ↓ HDL, Abnormal LDL/ ↑ Free Chol, ↓
(Complete) Cholesterol VLDL structure Chol Esters
Acyltransferase)

LCAT Deficiency (Partial) (Hypo) LCAT (Partial activity) ↓ HDL ↑ Free Chol, ↓
Chol Esters

VII. Important Points to Remember

• Type 1 vs Type 2: High TAG -> Pancreatitis (Abd pain), Milky plasma, Eruptive xanthomas :: High
Cholesterol -> CAD (Chest pain), Clear plasma, Tendon xanthomas, Corneal arcus.
• Type 3: Affects Remnants (due to ApoE defect), causes both high TAG & Cholesterol, associated with
Palmar and Tuberoeruptive xanthomas.
• MTP Defect: Causes Abetalipoproteinemia (no ApoB lipoproteins, severe malabsorption) but MTP
inhibitors (Lomitapide) treat hypercholesterolemia by reducing VLDL/LDL production.
• ABCA1 Defect: Causes Tangier disease (very low HDL, orange tonsils).
• ABCG5/G8 Defect: Causes Sitosterolemia (plant sterol accumulation, mimics high cholesterol).
• LCAT Defect: Affects HDL maturation; complete deficiency (Norum) causes renal failure, partial (Fish
Eye) is benign corneal opacity.
• Clear Plasma vs. Lactescent Plasma: Clear plasma suggests primarily elevated cholesterol (LDL);
Lactescent/milky plasma indicates significantly elevated triglycerides (Chylomicrons, VLDL).
Summary: Lipases in Lipid Metabolism
I. Introduction to Lipases

• Primary Action: Break covalent ester bonds in lipids, specifically in Triacylglycerol (TAG).
• Reaction: TAG + 3 H₂O → Glycerol + 3 Fatty Acids
• Enzyme Class: Hydrolase (requires water).
II. Types of Lipases Discussed

1. Hormone Sensitive Lipase (HSL) - Major Focus

2. Lipoprotein Lipase (LPL) - Major Focus
3. Intestinal (Pancreatic) Lipase
4. Hepatic Lipase
5. Endothelial Lipase
III. Hormone Sensitive Lipase (HSL)

• Location: Within adipose tissue (adipocytes).

• Function: Hydrolyzes stored TAG in adipocytes, releasing energy substrates.
• Physiological State: Active during fasting.
• Mechanism: Step-wise hydrolysis of TAG:
◦ TAG → Fatty Acid (from C1) + 2,3-Diacylglycerol (DAG)
◦ 2,3-DAG → Fatty Acid (from C3) + 2-Monoacylglycerol (2-MAG)
◦ Note: HSL cannot remove the fatty acid from the second (C2) position.
◦ Net HSL Products: 2 Fatty Acids (released as Acyl-CoA) + 2-Monoacylglycerol.
◦ The final fatty acid (from C2) is removed by esterases to yield glycerol.
• Regulation:
◦ Activation State: Active when phosphorylated.
◦ Glucagon (Fasting Hormone): Activates HSL (promotes phosphorylation).
◦ Insulin (Fed State Hormone): Inhibits HSL (promotes dephosphorylation via phosphatases).
◦ Other Activators:
▪ Catecholamines (Epinephrine)
▪ Adrenocorticotropic Hormone (ACTH)
▪ Thyroid Stimulating Hormone (TSH)
▪ Glucocorticoids
▪ Thyroid Hormones
◦ Other Inhibitors:
▪ Niacin (Nicotinic Acid)
▪ Prostaglandin E1
• Diabetes Mellitus: Considered similar to a fasting state for the cell. Due to insulin deficiency/resistance,
HSL activity is increased.
IV. Lipoprotein Lipase (LPL)

• Location: Anchored to the endothelial lining of capillaries.

◦ Sites: Heart, Adipose tissue, Spleen, Renal medulla, Aorta, Diaphragm, Lactating Mammary Gland.
 • Anchoring: Attached via Heparin Sulfate (a Glycosaminoglycan/GAG).
◦ Clinical Note: Heparin injection dislodges LPL into the bloodstream, allowing measurement.
• Function: Hydrolyzes TAG within circulating Chylomicrons and VLDL.
• Physiological State: Active during the fed state.
• Regulation:
◦ Activation (Apolipoprotein): Requires Apo-C2.
◦ Insulin (Fed State Hormone): Activates LPL (increases LPL expression).
• Diabetes Mellitus: Due to decreased insulin action, LPL activity is decreased.
V. HSL vs. LPL Comparison

Feature Hormone Sensitive Lipase (HSL) Lipoprotein Lipase (LPL)

Location Inside Adipocytes Capillary Endothelium

Substrate Stored TAG (in adipose tissue) TAG in Chylomicrons & VLDL (blood)

State Fasting Fed

Main Hormone Glucagon (Activates) Insulin (Activates/ ↑Expression)

Insulin Inhibits (Dephosphorylation) Activates (↑Expression)

Glucagon Activates (Phosphorylation) Inhibited (Opposite of Insulin)

Active Form Phosphorylated (Regulation via Expression/Apo-C2)

Diabetes Activated (↓Insulin effect) Inhibited (↓Insulin effect)

VI. Other Lipases

• Hepatic Lipase:
◦ Location: Sinusoidal surface of liver cells.
◦ Function: Role in metabolism of Chylomicron remnants and conversion of HDL3 to HDL2.
• Endothelial Lipase:
◦ Location: Endothelium (distinct from LPL).
◦ Function: Role in HDL metabolism (conversion of HDL3 to HDL2 and HDL3 to pre-beta HDL).
Pre-beta HDL is key for cholesterol uptake.
• Intestinal (Pancreatic) Lipase:
◦ Source: Pancreas.
◦ Action Site: Intestine.
◦ Function: Hydrolyzes dietary TAG.
◦ Products (as stated in text): Fatty Acids + Glycerol (overall result of digestion).
VII. Mnemonics (Use Sparingly)

• HSL Activation: Think Fasting/Glucagon/Phosphorylated = Go! (Mobilize stored fat). Hormones

linked to energy need (Catecholamines, ACTH, TSH, Glucocorticoids, Thyroid) also activate HSL.
• HSL Inhibition: Think NIP HSL in the bud: Niacin, Insulin, Prostaglandin E1 inhibit.
 • LPL Activation: Think Fed/Insulin/Lipoproteins (CM, VLDL) = Load up cells! Requires Apo-C2 co-
pilot.
VIII. Key Points to Remember

• HSL acts inside adipocytes on stored fat, mainly during fasting, activated by glucagon (phosphorylated
form is active), and inhibited by insulin. Crucial for energy release from stores.
• LPL acts on the capillary walls on TAG in chylomicrons/VLDL, mainly during the fed state, activated
by insulin (expression) and Apo-C2. Crucial for clearing fat from blood after a meal and delivering it to
tissues.
• Diabetes: Mimics fasting metabolically in some ways → HSL is UP, LPL is DOWN. This contributes to
dyslipidemia.
• Hepatic and Endothelial Lipases are important for lipoprotein remnant and HDL metabolism.
• Pancreatic Lipase is essential for dietary fat digestion.
• All lipases are hydrolases breaking ester bonds in TAG.

Enzymeology: Introduction Summary

I. Importance of Enzymes

• Life processes like heartbeat, muscle contraction, and digestion require energy derived from chemical
reactions.
• Enzymes are essential catalysts for these biochemical reactions.
• Example: Sugar (sucrose) is stable on a shelf but is rapidly broken down into glucose and fructose in the
intestine due to the presence of enzymes (like sucrase).
• Enzymes make life processes possible and easier.
II. Definition

• Enzymes are specialized proteins that catalyze biochemical reactions within the body.
III. Brief History

• 1850 (Louis Pasteur): Observed fermentation (sugar → alcohol by yeast). Proposed catalysis by
"ferments" (vitalism), but couldn't isolate them.
• 1897 (Eduard Buchner): Showed that a cell-free yeast extract could catalyze fermentation, disproving
pure vitalism.
• Frederick W. Kühne: Coined the term "enzyme" (from Greek en zyme, meaning "in leaven"). This
marked the dawn of biochemistry as a science.
IV. Types of Enzymes

1. Simple Enzymes: Consist solely of amino acid residues.

2. Complex Enzymes: Require both:
◦ Apoenzyme: The protein part (amino acid residues).
◦ Chemical Component: A non-protein part essential for activity.
V. Components of Complex Enzymes

• Apoenzyme: The inactive protein component.

 • Chemical Component: Can be:
◦ Inorganic: Metal ions (e.g., Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺). Termed Cofactors.
◦ Organic/Metallo-organic: Complex organic molecules, potentially containing a metal. Termed
Coenzymes.
VI. Properties of Enzymes

• Mostly Proteins: The vast majority of enzymes are protein-based.

◦ Exception: Ribozymes - RNA molecules with catalytic activity.
• Contain approximately 16% Nitrogen by weight.
• Can be precipitated by protein precipitating agents.
• Are Heat Labile (sensitive to denaturation by heat), reflecting their protein nature.
VII. Ribozymes (Catalytic RNA)

• RNA molecules acting as catalysts.

• Examples:
◦ 28S rRNA: Component of ribosomes with peptidyltransferase activity (catalyzes peptide bond
formation).
◦ snRNA (Small Nuclear RNA): Part of the spliceosome complex, involved in RNA splicing.
◦ Group II Introns: Found in primary transcripts (hnRNA), self-splicing activity (RNA splicing).
◦ RNase P: Involved in the post-transcriptional modification of tRNA.
VIII. Coenzymes

• Definition: Heat-stable, low molecular weight organic molecules.

• Function: Often act as co-substrates or "second substrates" in the reaction.
• Derivation: Many are derived from water-soluble vitamins.
• Examples:

Vitamin (Name) Active Key Reactions Catalyzed Mnemonic Link

Form(s)

B1 (Thiamine) TPP Oxidative decarboxylation, TPP for Thiamine

Transketolase

B2 (Riboflavin) FAD, FMN Dehydrogenases, Redox reactions FAD/FMN for

Flavin

B3 (Niacin) NAD⁺, Dehydrogenases, Redox reactions NAD for Niacin

NADP⁺

B5 (Pantothenate) Coenzyme A Acyl group transfer (e.g., Acetyl- CoA for Acyl
(CoA) CoA)

B6 (Pyridoxine) PLP Transamination, Transsulfuration, PLP for Protein/

other amino acid reactions AA

B9 (Folic Acid) THF One-carbon metabolism (transfer of Folate for 1-C

formyl, methyl etc.) units

B12 (Cobalamin) Ado-B12, Me- Alkyl group & H atom transfers Cobalamin for
B12 Big groups
 Vitamin (Name) Active Key Reactions Catalyzed Mnemonic Link
Form(s)

Lipoate Lipoamide Oxidative decarboxylation (in PDH, Lipo links Redox/

α-KGDH complexes) Decarbox

Vitamin C (Ascorbate) Ascorbate Hydroxylation reactions (e.g., C for Collagen

proline/lysine in collagen)

(Note: Lipoate is often synthesized, not

strictly considered a vitamin anymore).

IX. Cofactors (Metal Ions)

• Definition: Primarily metal ions required for enzyme activity.

• Categories based on binding:
◦ Metalloenzymes: Metal ion is tightly integrated (covalently or non-covalently) into the apoenzyme
structure.
▪ Examples: Zn²⁺ in Carbonic Anhydrase, Cu²⁺ in Tyrosinase.
◦ Metal-activated Enzymes: Metal ion is loosely associated or required in the surrounding medium,
not permanently bound to the apoenzyme.
▪ Example: Ca²⁺ required for Lipase activity.
• Examples of Metal Cofactors and Associated Enzymes:
◦ Zinc (Zn²⁺):
▪ Key Examples: Carbonic Anhydrase (Gas transport), Carboxypeptidase A/B (Protein
digestion), Alcohol Dehydrogenase (Alcohol/Retinol metabolism), Cytosolic Superoxide
Dismutase (SOD) (ROS defense).
▪ Others: ALA Dehydratase, Alkaline Phosphatase, Adenosine Deaminase, Lactate Dehydrogenase.
◦ Magnesium (Mg²⁺):
▪ Key Examples: Kinases (Phosphotransferases), Phosphatases (Phosphohydrolases), Mutase,
Enolase.
▪ Note: Often complexes with ATP; required for most ATP-dependent reactions.
◦ Iron (Fe²⁺/Fe³⁺):
▪ Heme Iron: Cytochrome c Oxidase (Complex IV), Complex III (ETC), Tryptophan Pyrrolase/
Dioxygenase (Trp catabolism), Peroxidase, Catalase (ROS defense).
▪ Non-Heme Iron (Fe-S clusters): NADH Dehydrogenase (Complex I), Succinate
Dehydrogenase (Complex II) (ETC).
◦ Manganese (Mn²⁺):
▪ Key Examples: Arginase (Urea cycle), Ribonucleotide Reductase (DNA precursor synthesis),
Mitochondrial SOD (ROS defense). (Mn for Mitochondrial SOD).
▪ Others: Often substitutes for Mg²⁺ in Kinases, Phosphatases.
◦ Molybdenum (Mo):
▪ Key Example: Xanthine Oxidase (Purine degradation → Uric acid).
▪ Others: Sulfite Oxidase, Dinitrogenase.
◦ Potassium (K⁺):
▪ Examples: Pyruvate Kinase, Na⁺/K⁺-ATPase.
 ◦ Copper (Cu²⁺):
▪ Key Examples: Tyrosinase (Melanin synthesis), Cytochrome c Oxidase (Complex IV) (requires
both Cu & Fe), Lysyl Oxidase (Collagen cross-linking), Cytoplasmic SOD (requires both Cu &
Zn). (Cu for Cytoplasmic SOD & Collagen).
◦ Nickel (Ni²⁺):
▪ Example: Urease (not typically active in humans).
◦ Calcium (Ca²⁺):
▪ Examples: Lipase (Lipid digestion), Lecithinase.
X. Prosthetic Group

• Definition: A coenzyme or cofactor that is tightly bound (covalently or strongly non-covalently) to the
apoenzyme.
• It is considered an integral part of the enzyme's structure.
XI. Holoenzyme

• Definition: The complete, catalytically active enzyme complex.

• Holoenzyme = Apoenzyme (protein part) + Chemical Component (Coenzyme/Cofactor)
◦ (Mnemonic: Holo = Whole enzyme)
XII. Clinical / Applied Importance (Key Takeaways)

• Understanding enzyme components is crucial because deficiencies lead to disease.

• Vitamin Deficiencies: Impair coenzyme function → Enzyme defects → Various pathologies (e.g.,
Vitamin C → Scurvy; B vitamin issues → energy metabolism problems).
• Mineral Deficiencies: Impair cofactor function → Enzyme defects:
◦ Zn Def: Affects gas transport, digestion, alcohol/retinol metabolism (vision), immune function, ROS
defense.
◦ Cu Def: Causes hypopigmentation (Tyrosinase), energy failure (Cyt c Oxidase), collagen disorders
like Menkes disease (Lysyl Oxidase). (Menkes = Messed up copper).
◦ Fe Def: Anemia, impaired energy metabolism (ETC), reduced ROS defense.
◦ Mn Def: Problems with urea synthesis, DNA synthesis, mitochondrial ROS defense.
◦ General: Mineral roles in SODs (Cu/Zn-SOD, Mn-SOD) highlight their importance in combating
oxidative stress, linked to aging, atherosclerosis, cancer.
• Exam Focus: Shift from simple recall (e.g., "What cofactor does Enzyme X use?") to application (e.g.,
"Patient with symptoms Y likely has deficiency in which mineral affecting enzyme Z?"). Need to
understand the physiological consequence of the enzyme's function and its cofactor requirement.

Key Points to Emphasize for Revision:

• Distinguish clearly: Apoenzyme, Cofactor, Coenzyme, Prosthetic Group, Holoenzyme.

• Know the key exception: Ribozymes are RNA catalysts.
• Link major water-soluble vitamins (B complex, C) to their coenzyme forms and general reaction types.
• Associate key minerals (Zn, Mg, Fe, Mn, Cu, Mo) with their most important enzyme partners and the
metabolic/physiological consequences of deficiency.
• Remember the dual roles: Some enzymes need multiple cofactors (e.g., Cyt c Oxidase needs Fe and Cu;
Cytoplasmic SOD needs Cu and Zn).
• Focus on the clinical application and consequences of deficiencies.
Enzyme Classification Summary
I. Importance of Learning Enzyme Classification

• Accurate Naming: Helps in correctly naming enzymes in biochemical reactions without rote
memorization.
• Understanding Drug Metabolism: Essential for comprehending how drugs (e.g., via cytochromes,
which are oxidoreductases) are processed in the body.
• Understanding Free Radical Scavenging: Crucial for understanding mechanisms involving oxidation-
reduction reactions that neutralize harmful free radicals.
II. Need for Formal Classification

• To avoid confusion caused by the trivial system, where the same enzyme might have multiple names.
• The International Union of Biochemistry and Molecular Biology (IUBMB) established a systematic
classification.
• There are seven main classes (the 7th class, Translocases, was added in August 2018).
• Class numbers are fixed (e.g., Class 1 is always Oxidoreductases).
III. The Seven Classes of Enzymes

• Mnemonic (for first 6 classes): Operation Theater Has Low Intensity Light
◦ Oxidoreductases (Class 1)
◦ Transferases (Class 2)
◦ Hydrolases (Class 3)
◦ Lyases (Class 4)
◦ Isomerases (Class 5)
◦ Ligases (Class 6)
• Translocases (Class 7) - Latest addition, not in the mnemonic.
IV. Detailed Breakdown of Enzyme Classes
A. Class 1: Oxidoreductases * Function: Catalyze oxidation-reduction reactions. * Subclasses: 1.
Dehydrogenases: Transfer Hydrogen (H+, H-, H2) and electrons to an acceptor. * Acceptors: *
Nicotinamide Coenzymes (NAD+, NADP+): * NAD+ → NADH + H+ (Accepts 1 H+ & 2 e-). Many
reactions. * NADP+ → NADPH + H+ (Accepts 1 H+ & 2 e-). Fewer reactions (Examples: Glucose-6-
Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase in HMP pathway, Cytoplasmic Isocitrate
Dehydrogenase, Malic Enzyme). * Flavoproteins (FAD) (Riboflavin - Vit B2 derivative): * FAD →
FADH2 (Accepts 2 H+ & 2 e-). Few reactions (Examples: Succinate Dehydrogenase [ETC Complex II],
Acyl-CoA Dehydrogenase [Fatty Acid Oxidation], Mitochondrial Glycerol-3-Phosphate Dehydrogenase). 2.
Oxygenases: Add oxygen directly to the substrate. * Monooxygenases (Mixed Function Oxidases/
Hydroxylases): Add 1 oxygen atom. Often involves hydroxylation (adding -OH). Reaction: AH + O2 + ZH2
→ AOH + H2O + Z. * Examples: Aromatic Amino Acid Hydroxylases (Tryptophan, Tyrosine,
Phenylalanine), 7-alpha Hydroxylase, Cytochromes (P450, B5) - crucial for drug metabolism in liver/
intestine (ER & mitochondria). * Clinical Link: Barbiturates induce Cytochrome P450, which can aggravate
porphyria. * Dioxygenases: Add both oxygen atoms. Reaction: A + O2 → AO2. * Examples: Homogentisate
Oxidase (Dioxygenase), Tryptophan Pyrrolase (Dioxygenase). 3. Oxidases: Transfer Hydrogen to Oxygen as
the final acceptor. * Can produce water (H2O) or hydrogen peroxide (H2O2 - a Reactive Oxygen Species,
ROS). Reaction: AH2 + ½O2 → A + H2O or AH2 + O2 → A + H2O2. * Examples: Cytochrome C Oxidase.
* Flavoprotein Oxidases (using FAD/FMN): L-Amino Acid Oxidase, Xanthine Oxidase. 4.
Hydroperoxidases: Use H2O2 or organic peroxides as substrate; involved in free radical scavenging. Both
are Hemoproteins (contain Heme). * Peroxidases: Reduce peroxides. Electron acceptors can be Ascorbate
(Vit C), Quinones, Glutathione, Cytochrome C. Reaction: H2O2 + AH2 → 2H2O + A. * Example:
Glutathione Peroxidase (Selenium-containing enzyme, found in erythrocytes). Highlights importance of Vit
C and Glutathione in scavenging. * Catalases: Use one H2O2 molecule to reduce another. Reaction: 2H2O2
→ 2H2O + O2. Found in peroxisomes (liver, kidney, etc.). One of the fastest enzymes. * Protective Role:
Peroxisomes contain both oxidases (generate H2O2) and catalase (remove H2O2). 5. Reductases: Often
require NADPH. Involved in reductive biosynthesis (e.g., fatty acids, steroids).
B. Class 2: Transferases * Function: Transfer functional groups (e.g., methyl, amino, acyl, phosphoryl)
from one molecule (donor) to another (acceptor). * Examples: 1. Enzymes with "Trans" in the name
(Transaminase, Transketolase, Transaldolase). 2. Kinases: Transfer a phosphoryl group (-PO3^2-), typically
from ATP. * Examples: Hexokinase/Glucokinase (Glucose + ATP → G6P + ADP), Phosphofructokinase (F6P
+ ATP → F1,6BP + ADP). ATP is the phosphate donor. 3. Phosphorylases: Transfer a phosphoryl group
using inorganic phosphate (Pi). * Example: Glycogen Phosphorylase (Glycogen(n) + Pi → Glycogen(n-1)
+ Glucose-1-Phosphate).
C. Class 3: Hydrolases * Function: Catalyze cleavage of bonds (C-O, C-N, C-C, etc.) by the addition of
water (hydrolysis). Often digestive enzymes. * Examples: 1. Carbohydrate Bonds (Glycosidic): Amylase,
Sucrase, Maltase, Lactase. 2. Protein Bonds (Peptide): Peptidases, Proteases (e.g., Serine Proteases like
Trypsin, Chymotrypsin, Elastase). 3. Lipid Bonds (Ester): Lipases, Esterases. 4. Nucleic Acid Bonds
(Phosphodiester): Nucleases (Exonucleases - cleave from ends; Endonucleases - cleave within chain). 5.
Phosphatases: Remove phosphate groups using water. * Examples: Glucose-6-Phosphatase (G6P + H2O →
Glucose + Pi), Fructose-1,6-Bisphosphatase (F1,6BP + H2O → F6P + Pi). 6. Arginase (Urea Cycle).
D. Class 4: Lyases * Function: Catalyze cleavage of bonds (C-C, C-O, C-N, etc.) by mechanisms other
than hydrolysis or oxidation. Often form a double bond or ring, or add groups to a double bond. *
Examples: 1. Enzymes with "Lyase" in the name (HMG-CoA Lyase, Argininosuccinate Lyase). 2. Aldolase:
(Fructose-1,6-Bisphosphate → DHAP + Glyceraldehyde-3-Phosphate) - Cleavage. 3. Enolase: (2-
Phosphoglycerate → PEP + H2O) - Removal of H2O (atom elimination) to form a double bond. 4.
Fumarase: (Fumarate + H2O ⇌ Malate) - Addition of H2O across a double bond. (Note: classified as lyase
due to mechanism involving double bond, despite water addition). 5. Aconitase: (Citrate ⇌ Isocitrate via cis-
aconitate). 6. Simple Decarboxylases: Remove CO2 without oxidation. Require PLP (Pyridoxal Phosphate
- Vit B6). * Examples: Histidine Decarboxylase (→ Histamine), Glutamate Decarboxylase (→ GABA).
These are Lyases. 7. Contrast with Oxidative Decarboxylation: These remove CO2 with oxidation (NAD+
→ NADH) and belong to Class 1 (Oxidoreductases). They are multi-enzyme complexes requiring 5
coenzymes (TPP, Lipoate, CoA, FAD, NAD+). * Examples: Pyruvate Dehydrogenase, α-Ketoglutarate
Dehydrogenase.
E. Class 5: Isomerases * Function: Catalyze geometric or structural changes within a single molecule
(isomerization). * Examples: 1. Enzymes with "Isomerase" in the name (Phosphohexose Isomerase [G6P ⇌
F6P], Phosphotriose Isomerase [DHAP ⇌ G3P]). 2. Racemases: Interconvert stereoisomers (D ⇌ L forms).
3. Mutases: Catalyze the intramolecular shift of a chemical group (e.g., phosphate). * Examples:
Phosphoglucomutase (G6P ⇌ G1P), Phosphoglycerate Mutase (3PG ⇌ 2PG). (Distinction: Transfer within
the same molecule, not between molecules like Transferases).
F. Class 6: Ligases * Function: Join two molecules together, requiring energy input, usually from ATP
hydrolysis (ATP → ADP + Pi or ATP → AMP + PPi). Form C-C, C-S, C-O, C-N bonds. * Examples: 1.
Synthetases: (Often used when ATP is involved in synthesis). * Examples: Carbamoyl Phosphate
Synthetase, Argininosuccinate Synthetase. 2. Carboxylases: Add CO2 to a substrate. Require ATP, Biotin
(Vit B7), and CO2 (Mnemonic: ABC Ligases). * Examples: Pyruvate Carboxylase (Pyruvate [C3] + CO2
→ Oxaloacetate [C4]), Acetyl-CoA Carboxylase (Acetyl-CoA [C2] + CO2 → Malonyl-CoA [C3]),
Propionyl-CoA Carboxylase (Propionyl-CoA [C3] + CO2 → Methylmalonyl-CoA [C4]). * Clinical Link:
Raw egg white contains Avidin, which binds Biotin tightly, inhibiting these enzymes. Can lead to impaired
gluconeogenesis (via Pyruvate Carboxylase) and hypoglycemia.
G. Class 7: Translocases * Function: Catalyze the movement of ions or molecules across membranes, or
their separation within membranes. * Examples: Ion pumps (H+ pump), Ion channels (K+, Ca2+ channels),
ATP/ADP Translocase.
V. Example Reactions for Practice

• Glucose + ATP → G6P + ADP: Kinase (Glucokinase/Hexokinase) - Class 2

• G6P + H2O → Glucose + Pi: Phosphatase (Glucose-6-Phosphatase) - Class 3
• G6P ⇌ F6P: Isomerase (Phosphohexose Isomerase) - Class 5
• G6P ⇌ G1P: Mutase (Phosphoglucomutase) - Class 5
• Pyruvate + NAD+ + CoA → Acetyl-CoA + NADH + CO2: Dehydrogenase (Pyruvate Dehydrogenase
Complex) - Class 1
• Pyruvate + CO2 + ATP → Oxaloacetate + ADP + Pi: Carboxylase (Pyruvate Carboxylase) - Class 6
VI. Key Points & Clinical Correlations to Remember

• Seven Classes: Know the names and basic function of each (Oxidoreductases, Transferases, Hydrolases,
Lyases, Isomerases, Ligases, Translocases).
• Class 1 (Oxidoreductases): Diverse group including dehydrogenases (NAD+/FAD), oxidases (use O2,
can make ROS), oxygenases (add O2, Cytochromes P450/B5 for drug metabolism), peroxidases/catalase
(ROS scavenging, Heme/Selenium involved).
• Class 2 (Transferases): Kinases (use ATP to transfer Pi), Phosphorylases (use Pi to transfer Pi).
• Class 3 (Hydrolases): Break bonds using water (digestive enzymes, phosphatases).
• Class 4 (Lyases): Break bonds without water (often form double bonds); includes simple decarboxylases
(need PLP). Differentiate from Oxidative Decarboxylation (Class 1, need 5 coenzymes).
• Class 5 (Isomerases): Rearrangements within one molecule (isomerases, racemases, mutases).
• Class 6 (Ligases): Join molecules using ATP energy; includes synthetases and carboxylases (need Biotin,
ATP, CO2 - "ABC").
• Class 7 (Translocases): Membrane transport.
• Clinical Links:
◦ Drug Metabolism: Cytochrome P450 (Monooxygenase - Class 1).
◦ Free Radicals: Generation (Oxidases - Class 1) and Scavenging (Catalase, Peroxidase - Class 1).
◦ Biotin Deficiency: Raw eggs (Avidin) inhibit Biotin-dependent Carboxylases (Ligases - Class 6),
affecting gluconeogenesis -> hypoglycemia.
◦ Vitamins as Coenzymes: B1, B2, B3, B5 (Oxidative Decarboxylation), B6 (Simple Decarboxylation,
Transamination), B7 (Carboxylation), Vit C (Peroxidase reaction).

Mechanism of Action of Enzymes - Summary

I. Introduction & Core Concept

• Goal Analogy: Achieving success in life requires focused effort, similar to how enzymes work.
• Enzyme Function: Enzymes increase the rate of a reaction by providing a specific environment for it to
occur efficiently.
II. Enzyme Structure & Substrate Interaction

• Enzyme: Typically large, three-dimensional protein structures.

• Active Site:
◦ A specific, small pocket or cleft within the enzyme structure.
◦ Contains critical amino acid residues (catalytic residues) responsible for the reaction.
◦ Analogy: Like focusing on key fundamental areas when studying vast subjects.
• Substrate: The molecule(s) upon which the enzyme acts.
• Binding: The substrate must bind specifically to the active site for catalysis to occur.
III. Reaction Coordinate Diagram & Energetics

• Axes:
◦ Y-axis: Free Energy (specifically Gibbs Free Energy, denoted as G). Represents the energy content of
molecules.
◦ X-axis: Progress of the Reaction.
• Uncatalyzed Reaction:
◦ Substrate (S): Starting energy level.
◦ Transition State (TS): A high-energy, unstable intermediate state that must be reached for the
reaction to proceed. The "summit" of the energy barrier.
◦ Product (P): Final energy level.
◦ Analogy: The initial difficult phase of learning/achieving goals (like starting MBBS or PG prep)
represents the climb to the transition state.
• Activation Energy (ΔG‡):
◦ The energy difference between the substrate and the transition state (ΔG P in lecture notation,
representing S -> TS barrier).
◦ The minimum energy required for the reaction to occur.
◦ For a reversible reaction, there's also an activation energy for the reverse reaction (P -> S), denoted
ΔG S in the lecture (P -> TS barrier).
◦ (Note: Standard notation often uses ΔG‡ for activation energy).
• Standard Free Energy Change (ΔG⁰):
◦ The overall free energy difference between the products and the substrates (ΔG P - ΔG S in lecture
notation).
◦ Determines the reaction's equilibrium position.
◦ Not altered by the enzyme.
IV. How Enzymes Catalyze Reactions

• Primary Mechanism: Enzymes lower the activation energy (ΔG‡) of the reaction.
• How: They achieve this by stabilizing the transition state, making it easier to reach.
• Effect on ΔG⁰: Enzymes do not change the standard free energy change (ΔG⁰). They accelerate the rate
at which equilibrium is reached but don't change the equilibrium itself.
• Alternative Reaction Coordinate (Catalyzed): Can be drawn showing intermediate steps like Enzyme-
Substrate (ES) complex and Enzyme-Product (EP) complex, appearing as valleys in the energy profile
before/after the lowered transition state peak(s).
V. Overcoming Energy Barriers (How Enzymes Lower ΔG‡)

• Enzymes act like a "tunnel through the hill," bypassing high energy barriers.
• Barriers & Enzyme Solutions:
1. Entropy (Disorderliness): Reactants are randomly oriented in solution.
▪ Enzyme Action: Binds substrates in the precise orientation needed for reaction, reducing
entropy (increasing order). (Mnemonic: Order from Enzyme).
2. Solvation Shell: Water molecules surrounding the substrate can interfere.
▪ Enzyme Action: Causes desolvation by removing the water shell as the substrate enters the
active site. Analogy: Getting rid of negative thoughts/influences.
3. Improper Alignment: Reactive groups not positioned correctly.
▪ Enzyme Action: Ensures proper alignment of substrate(s) relative to catalytic groups in the
active site.
VI. Theories Explaining Substrate Alignment & Binding

1. Emil Fischer's Lock and Key (Template Theory):

◦ Assumes a rigid active site shape complementary to a specific substrate.
◦ Analogy: Cinderella's glass slipper fitting only her foot.
◦ Limitation: Considered too rigid; doesn't explain enzyme flexibility or transition state stabilization
well.
2. Jenck & Pauling's Transition State Stabilization Theory:
◦ Proposes the active site is most complementary to the transition state structure, not the initial
substrate.
◦ By binding and stabilizing the unstable TS, the enzyme lowers the activation energy.
◦ Example: Stickase enzyme binding the "bent stick" (transition state) better than the intact stick
(substrate).
3. Daniel Koshland's Induced Fit Theory (1958):
◦ Current widely accepted model.
◦ Substrate binding induces a conformational change in the enzyme's active site.
◦ This change optimizes the alignment of catalytic groups and improves binding interactions (multiple
weak interactions like H-bonds, van der Waals, hydrophobic).
◦ Analogy: A glove changing shape slightly to fit the hand (Donald Duck vs. Mickey Mouse vs. Human
hand).
VII. Specific Catalytic Mechanisms within the Active Site

1. Catalysis by Proximity:
◦ Enzyme brings reactants close together in the active site (within "bond-forming distance") and in the
correct orientation, increasing the effective concentration.
◦ Analogy: Needing to be close to study materials (books, videos, Qbank) to learn.
2. Acid-Base Catalysis:
◦ Active site amino acid residues act as proton donors (general acids) or proton acceptors (general
bases) during the reaction.
◦ Example: Aspartate proteases (like Pepsin) use aspartate residues in their active site for this.
3. Covalent Catalysis:
◦ Formation of a transient covalent bond between the enzyme (or a cofactor) and the substrate.
 ◦ Common in group transfer reactions.
◦ Example: Serine proteases (Chymotrypsin, Trypsin, Elastase) use a serine residue to form a
temporary covalent intermediate. Analogy: Transient positive interactions aiding progress.
4. Catalysis by Strain:
◦ Enzyme binding distorts the substrate, stretching or bending key bonds (inducing strain), weakening
them and making them easier to break.
◦ Often important in lytic reactions (Lyases, Hydrolases).
◦ Analogy: Painful effort needed to break old habits or negative bonds for growth/success.
5. Metal Ion Catalysis:
◦ Metal ions (cofactors in metalloenzymes) participate directly in catalysis.
◦ Roles include:
▪ Orienting the substrate.
▪ Stabilizing charged intermediates (electrostatic catalysis).
▪ Mediating redox reactions.
◦ Example: Zinc (Zn²⁺) in Carbonic Anhydrase. (Mnemonic: Metals Mediate / Make Alignment).
VIII. Sample Question Analysis

• Question: Which statement regarding lowering of activation energy is FALSE/LEAST TRUE?

◦ (A) Entropy lowered - TRUE (Proximity/Orientation)
◦ (B) Desolvation - TRUE (Removes water shell)
◦ (C) Conformational Change - TRUE (Induced Fit)
◦ (D) Active site always complementary to substrate - FALSE/LEAST TRUE (This is the rigid Lock &
Key model; Induced Fit and TS Stabilization are more accurate).
IX. Conclusion

• Adopt a "winner" mindset.

• Success results from consistent, small efforts ("day in and day out"), much like enzyme catalysis is a
specific, efficient process, not magic.

⭐ Important Points to Remember ⭐

1. Primary Function: Enzymes increase reaction rates by lowering activation energy (ΔG‡).
2. No Change to Equilibrium: Enzymes do not alter the overall free energy change (ΔG⁰) or the
equilibrium constant (Keq).
3. Active Site: The specific region where substrate binds and catalysis occurs.
4. Lowering ΔG‡ Mechanisms: Achieved by reducing entropy, desolvation, and ensuring proper
alignment.
5. Binding Models: Understand Lock & Key (rigid, older), Transition State Stabilization, and Induced Fit
(flexible, widely accepted).
6. Key Catalytic Strategies: Know the basics of Proximity, Acid-Base, Covalent, Strain, and Metal Ion
catalysis, and recognize examples (e.g., Serine proteases - covalent; Aspartate proteases - acid/base;
Carbonic anhydrase - metal ion).
7. Reaction Coordinate Diagram: Be able to identify Substrate, Product, Transition State, ΔG‡, and ΔG⁰,
and show how an enzyme modifies the ΔG‡ peak.
Enzyme Kinetics: Summary
1. Introduction to Enzyme Kinetics * Focuses on two main aspects: 1. Rate of Reaction: How fast an
enzyme-catalyzed reaction proceeds. 2. Factors Affecting Rate: External influences on the reaction speed. *
Considered a "hot topic" for exams due to frequent questions.
2. Rate of Reaction & Equilibrium * General Reaction: A + B ⇌ P + Q * Forward Rate (R1):
Proportional to substrate concentrations ([A][B]). * R1 = k1 * [A][B] (k1 = forward rate constant) *
Backward Rate (R2): Proportional to product concentrations ([P][Q]). * R2 = k2 * [P][Q] (k2 =
backward rate constant, Note: transcript uses k2 for backward here, but later uses k2 for product formation
from ES) * Equilibrium: A dynamic state where Forward Rate = Backward Rate (R1 = R2). * Equilibrium
Constant (Keq): Ratio of rate constants. * Keq = k1 / k2 = ([P][Q]) / ([A][B]) * Keq =
[Products] / [Substrates] at equilibrium. * Enzymes and Keq: Enzymes DO NOT alter the Keq because Keq
is a ratio of rate constants, not reaction rates. Enzymes increase both forward and backward rates equally. *
Enzymes and Energy: * Enzymes lower the activation energy. * Enzymes DO NOT alter the standard free
energy change (ΔG⁰). * ΔG⁰ and Keq Relationship: * ΔG⁰ = -RT * ln(Keq) * R = Gas constant * T
= Absolute temperature
3. Factors Affecting Enzyme-Catalyzed Reaction Rate * Four key factors: 1. Substrate Concentration ([S])
2. Temperature (T) 3. pH (H+ concentration) 4. Enzyme Concentration ([E])
4. Factor 1: Substrate Concentration ([S]) * Effect: Increasing [S] increases the reaction rate (velocity, V)
until a maximum velocity (Vmax) is reached. Beyond Vmax, increasing [S] has no effect as the enzyme is
saturated. * Graphical Representation: Plotting Velocity (V) vs. [S] gives a Hyperbolic Curve. * Kinetics
Orders: * Low [S]: First-Order Kinetics (V is directly proportional to [S]). * High [S] (Saturation): Zero-
Order Kinetics (V is independent of [S], V = Vmax). * Vmax: The maximum rate achieved when all enzyme
active sites are occupied by substrate. * Michaelis Constant (Km): * Definition: The substrate
concentration ([S]) at which the reaction velocity is half of Vmax (V = ½ Vmax). * Significance: * It is a
measure of the affinity of an enzyme for its substrate. * Inverse Relationship: Low Km = High Affinity;
High Km = Low Affinity. * Mnemonic: Low Kinda Meh value means High Attraction (Affinity). *
Independent of enzyme concentration. * Unique for a specific enzyme-substrate pair ("signature"). *
Indicates [S] required: High affinity (low Km) means less substrate is needed to reach significant velocity.
Low affinity (high Km) means more substrate is needed. * Michaelis-Menten Equation: Describes the
relationship between initial velocity (V₀ or Vi), Vmax, Km, and [S]. * V₀ = (Vmax * [S]) / (Km +
[S]) * Lineweaver-Burk Plot (Double Reciprocal Plot): * A linear transformation of the Michaelis-Menten
equation. * Plots 1/V₀ (y-axis) vs. 1/[S] (x-axis). * Equation: 1/V₀ = (Km/Vmax) * (1/[S])
+ 1/Vmax (Matches y = mx + c) * Intercepts: * Y-intercept: 1/Vmax * X-intercept: -1/Km * Slope:
Km/Vmax * Other Plots (Names Only):* * Eadie-Hofstee plot (V vs. V/[S]) - Single reciprocal * Hanes-
Woolf plot ([S]/V vs. [S]) - Single reciprocal
5. Factor 2: Temperature (T) * Effect: Increasing temperature initially increases reaction rate until an
optimum temperature is reached. Beyond this, the rate sharply declines due to denaturation of the enzyme. *
Graphical Representation: Bell-shaped curve (V vs. T). * Optimum Temperature: The temperature at
which the enzyme exhibits maximum activity (approx. 35-40°C for most human enzymes). * Maximum
Stability Temperature: Temperature range where the enzyme structure is most stable (often higher than
optimum temp, approx. 45-55°C for human enzymes). Denaturation occurs above this. * Reason for Initial
Increase: Increased kinetic energy of molecules and increased collision frequency between enzyme and
substrate. * Temperature Coefficient (Q10): For every 10°C rise in temperature (within limits), the reaction
rate approximately doubles (Q10 ≈ 2).
6. Factor 3: pH (H+ Concentration) * Effect: Enzyme activity is maximal at an optimum pH. Activity
decreases sharply on either side of the optimum. * Graphical Representation: Bell-shaped curve (V vs.
pH). * Optimum pH: The pH at which the enzyme shows maximum activity (varies widely, often 5-9 range
for human enzymes). * Reason: pH affects the ionization state (charge) of amino acid residues in the
enzyme, particularly those in the active site (catalytic residues). The correct charge state is crucial for
substrate binding and catalysis. (Related to pKa values of residues). * Examples: * Pepsin: Active in acidic
stomach pH (~1.5-2.5). * Chymotrypsin: Active in alkaline intestinal pH (~8.0), relies on Histidine (pKa ~6).
7. Factor 4: Enzyme Concentration ([E]) * Effect: The reaction rate (V) is directly proportional to the
enzyme concentration, provided the substrate is not limiting ([S] >> Km). * Graphical Representation:
Straight line (V vs. [E]).
8. Key Kinetic Constants & Concepts * Reaction Scheme: E + S ⇌ ES → E + P * Rate constants: k₁ (ES
formation), k₋₁ (ES dissociation), k₂ (Product formation, also called kcat) * Km: Michaelis Constant ([S] at
½ Vmax, indicates affinity). Units: Concentration (e.g., µM, mM). * Vmax: Maximum velocity (rate at
enzyme saturation). Units: Rate (e.g., µmol/min). * kcat (Turnover Number / Catalytic Constant): *
Measures the catalytic efficiency of a single enzyme molecule under saturating conditions. * Represents the
number of substrate molecules converted to product per enzyme molecule per unit time. * kcat =
Vmax / [Et] * [Et] = Total enzyme concentration ([Free E] + [ES Complex]). * Units: Inverse time (e.g.,
s⁻¹, min⁻¹). * kcat/Km (Specificity Constant): * Measures the overall catalytic efficiency of an enzyme;
useful for comparing different enzymes or substrates. * Takes into account both substrate binding (Km) and
catalysis rate (kcat). * Higher value indicates higher efficiency. * Units: Concentration⁻¹ Time⁻¹ (e.g., M⁻¹s⁻¹).
* Kd (Dissociation Constant): * Measures the tendency of the ES complex to dissociate back to E + S. *
Kd = k₋₁ / k₁ * Related to affinity (Low Kd ≈ High Affinity, similar to Km). Units: Concentration. *
Ka (Association Constant): * Measures the tendency of E and S to form the ES complex. * Ka = k₁ /
k₋₁ * Ka = 1 / Kd . Units: Concentration⁻¹.
9. Problem Solving Insights from Examples * Isoenzymes: Different Km values allow isoenzymes (e.g.,
Hexokinase vs. Glucokinase) to function optimally under different physiological conditions/substrate levels.
* Hexokinase (RBC): Low Km, high affinity. Works efficiently even at low blood glucose. * Glucokinase
(Liver): High Km, low affinity. Primarily active when blood glucose is high (post-meal storage). * Half-
Maximal Velocity: Occurs when 50% of the enzyme's active sites are occupied by substrate. * Calculations:
Be prepared to use M-M equation and the relationship Vmax = kcat * [Et] to find unknown
variables like Km. Pay attention to units (e.g., converting nM to µM). * Comparing Efficiency: Use the
kcat/Km ratio. The highest ratio indicates the most efficient enzyme. * Zero-Order Kinetics Problems:
If [S] >>> Km, the reaction is zero-order with respect to [S]. The rate depends only on Vmax, which is
proportional to [E]. If [E] is changed, the rate changes proportionally, and the time required to produce a
certain amount of product changes inversely proportionally. (e.g., If [E] is reduced to 1/3, rate becomes 1/3,
time becomes 3 times longer).
10. Important Points to Remember * Enzymes increase reaction rates by lowering activation energy,
without changing Keq or ΔG⁰. * Km is the [S] at ½ Vmax and is inversely related to affinity. * Vmax is the
maximum rate at enzyme saturation. * The Michaelis-Menten equation relates V₀, Vmax, Km, and [S]
(hyperbolic). * The Lineweaver-Burk plot linearizes the M-M equation (1/V vs 1/[S]). Know the intercepts
(1/Vmax, -1/Km) and slope (Km/Vmax). * kcat is the turnover number (Vmax/[Et]). * kcat/Km is the
specificity constant, used to compare enzyme efficiency. * Temperature and pH have optimum values,
beyond which activity decreases (bell-shaped curves), primarily due to denaturation (temp) or changes in
ionization state (pH). * Reaction rate is directly proportional to enzyme concentration ([E]) if substrate is
not limiting. * Understand the difference between first-order ([S] << Km) and zero-order ([S] >> Km)
kinetics with respect to substrate.
Enzyme Inhibition Summary
I. Introduction & Importance

• Biological Warfare Analogy: Ongoing conflict between organisms and humans; antibiotics are key
weapons.
• Mechanism of Antibiotics: Primarily Enzyme Inhibition.
• Significance: Enzyme inhibition has vast applications and is a frequent topic for questions.
• Cellular Control: All cellular processes require enzymes; inhibitors allow control over these processes.
• Illustrative Case Scenarios:
1. Organophosphorus Poisoning: 5-year-old ingests melathion (organophosphorus compound).
▪ Symptoms: Vomiting, severe abdominal cramps, low pulse (50/min), low BP (80/50 mmHg),
muscle fasciculations.
▪ Mechanism (explained later): Inhibition of acetylcholinesterase.
2. Methanol Poisoning (Hooch Tragedy): Multiple hospitalizations; ethanol given as an antidote.
▪ Mechanism (explained later): Competitive inhibition of alcohol dehydrogenase.
3. Carbon Monoxide Poisoning: House fire deaths without charred bodies.
▪ Mechanism (explained later): Non-competitive inhibition of cytochrome c oxidase & high
hemoglobin affinity.
II. Types of Enzyme Inhibition

• Major Categories:
◦ Reversible: Inhibition can be overcome.
◦ Irreversible: Inhibition is permanent or very long-lasting.
• Specific Types:
◦ Reversible:
▪ Competitive Inhibition
▪ Uncompetitive Inhibition
▪ Mixed Inhibition
◦ Irreversible:
▪ Suicide Inhibition
▪ Transition State Analogues
◦ Non-competitive Inhibition: Can be considered under both reversible and irreversible contexts
(discussed later).
III. Reversible Inhibition Details
A. Competitive Inhibition

• Definition: Inhibitor is a structural analogue of the substrate and competes for the active site.
• Mechanism:
◦ Normal: E + S ⇌ ES → E + P
◦ Inhibited: E + I ⇌ EI (No product formed from EI)
• Key Properties:
1. Inhibitor resembles substrate structure.
2. Inhibition can be overcome by increasing substrate concentration ([S]).
 • Kinetics:
◦ Vmax: Unchanged. (With enough substrate, the normal max rate can eventually be reached).
◦ Km: Increases (Apparent Km, Km' > Km). More substrate is needed to reach half Vmax because of
competition. (Km' = α * Km)
• Lineweaver-Burk Plot (LB Plot):
◦ Appearance: Lines intersect on the Y-axis. (Mnemonic: Competitive inhibitors 'Cross' paths at the Y-
axis, like an 'X').
◦ Y-intercept (1/Vmax): Unchanged.
◦ X-intercept (-1/Km): Moves closer to zero (magnitude decreases as Km increases).
• Examples:
◦ Melathion Poisoning: Melathion → Melaxone (active form) → Competitively inhibits
Acetylcholinesterase → Acetylcholine accumulates → Symptoms (vomiting, cramps, fasciculations,
low pulse/BP, sweating). Note: Can become irreversible at high doses.
◦ Methanol Poisoning Antidote: Methanol --(Alcohol Dehydrogenase)--> Formaldehyde (Toxic).
Ethanol competes with methanol for Alcohol Dehydrogenase's active site. Ethanol --(ADH)-->
Acetaldehyde (Less toxic). Giving excess ethanol prevents toxic formaldehyde formation.
B. Non-Competitive Inhibition

• Definition: Inhibitor binds to a site distinct from the active site (allosteric site). It does not need to be a
structural analogue. Binds to both free enzyme (E) and enzyme-substrate complex (ES).
• Mechanism:
◦ E + I ⇌ EI
◦ ES + I ⇌ ESI
◦ EI + S ⇌ ESI
◦ ESI complex forms product at a negligible/very slow rate.
• Key Properties:
1. No competition for the active site.
2. Inhibition cannot be overcome by increasing substrate concentration.
• Kinetics:
◦ Vmax: Decreases (Apparent Vmax, Vmax' < Vmax). Enzyme function is impaired regardless of
substrate binding. (Vmax' = Vmax / α)
◦ Km: Unchanged. Substrate binding affinity to the active site is not directly affected.
• Lineweaver-Burk Plot (LB Plot):
◦ Appearance: Lines intersect on the X-axis. (Mnemonic: Non-competitive Vmax is affected, shifting
the Y-intercept Vertically, lines meet on X-axis, like a 'V').
◦ Y-intercept (1/Vmax): Increases (as Vmax decreases).
◦ X-intercept (-1/Km): Unchanged.
• Examples (Often Poisons):
◦ Carbon Monoxide (CO): Inhibits Cytochrome C Oxidase.
◦ Cyanide: Inhibits Cytochrome C Oxidase.
◦ Fluoride: Inhibits Enolase.
◦ Iodoacetate: Inhibits Glyceraldehyde 3-phosphate Dehydrogenase.
• Case Link (CO): House fire → CO inhibits Cytochrome C Oxidase (non-competitively); CO also binds
Hemoglobin with higher affinity than O2, preventing oxygen transport.
C. Uncompetitive Inhibition

• Definition: Inhibitor binds only to the Enzyme-Substrate (ES) complex. It has no affinity for the free
enzyme (E).
• Mechanism:
◦ E + S ⇌ ES
◦ ES + I ⇌ ESI (Inhibitor only binds after substrate has bound)
• Key Properties:
1. Requires substrate binding first.
2. Relatively rare.
• Kinetics:
◦ Vmax: Decreases (Apparent Vmax, Vmax' < Vmax). Formation of ESI removes productive ES
complex. (Vmax' = Vmax / α)
◦ Km: Decreases (Apparent Km, Km' < Km). By binding ES, inhibitor shifts E+S⇌ES equilibrium
right (Le Chatelier's), increasing enzyme's apparent affinity for substrate. (Km' = Km / α)
• Lineweaver-Burk Plot (LB Plot):
◦ Appearance: Parallel lines. (Mnemonic: Both Vmax and Km change proportionally).
◦ Y-intercept (1/Vmax): Increases.
◦ X-intercept (-1/Km): Moves away from zero (magnitude increases as Km decreases).
• Example:
◦ Placental Alkaline Phosphatase (ALP) inhibited by Phenylalanine.
D. Mixed Inhibition

• Definition: Inhibitor binds to both the free enzyme (E) and the ES complex, typically at a site distinct
from the active site. Combines features of non-competitive and uncompetitive inhibition.
• Mechanism:
◦ E + I ⇌ EI
◦ ES + I ⇌ ESI
◦ EI + S ⇌ ESI
• Kinetics:
◦ Vmax: Decreases (Apparent Vmax, Vmax' < Vmax). (Vmax' = Vmax / α)
◦ Km: Can increase or decrease, depending on the relative affinities of the inhibitor for E vs. ES.
IV. The "Tricky Part" of Non-Competitive Inhibition (Multi-Substrate Context)

• Issue: Simple models assume one substrate, but many enzymes use multiple (A, B).
• Scenario: An inhibitor (I2) might be a structural analogue of substrate B but not substrate A.
◦ It binds to the 'B' binding site.
◦ With respect to substrate A: I2 acts as a Non-competitive inhibitor (binds elsewhere, Vmax↓, Km
unchanged when varying [A]). This inhibition is not reversed by adding excess A.
◦ With respect to substrate B: I2 acts as a Competitive inhibitor (competes for B's site). This
inhibition is reversed by adding excess B.
• Classical Non-Competitive: An inhibitor (I3) binds to a completely separate allosteric site, not
interfering with A or B binding sites directly. This is non-competitive w.r.t. both A and B (like the poison
examples).
 • Takeaway: Classification (competitive/non-competitive) and reversibility by substrate can depend on the
specific inhibitor and which substrate is being considered in multi-substrate systems.
V. Irreversible Inhibition Details
A. Suicide Inhibition (Mechanism-Based Inactivation)

• Definition: The inhibitor is initially unreactive. The enzyme's own catalytic mechanism converts the
inhibitor into a highly reactive intermediate. This reactive intermediate then binds covalently/
irreversibly to the enzyme's active site, 'killing' it.
• Examples:
◦ Allopurinol: Treats gout. Xanthine Oxidase converts Allopurinol → Alloxanthine (reactive).
Alloxanthine irreversibly inhibits Xanthine Oxidase, reducing uric acid production.
◦ Aspirin: Inhibits Cyclooxygenase.
◦ Difluoromethylornithine (DFMO): Treats African Sleeping Sickness (Trypanosomiasis). Inhibits
Ornithine Decarboxylase.
B. Transition State Analogues

• Definition: Inhibitor structurally resembles the unstable transition state of the normal substrate-enzyme
reaction, rather than the substrate itself.
• Mechanism: Enzymes bind transition states very tightly (Pauling/Jencks theory). Analogues exploit this,
binding with high affinity, often irreversibly.
• Example:
◦ Penicillin: Antibiotic. Mimics the transition state of the Transpeptidase enzyme reaction.
Transpeptidase is crucial for bacterial cell wall (proteoglycan) synthesis. Inhibition disrupts cell wall
→ bacterial death.
VI. Kinetic Parameter Summary

Inhibition Type Effect on Km Effect on Vmax LB Plot Appearance

Competitive Increases (αKm) Unchanged Intersect on Y-axis ('X' shape)

Non-competitive Unchanged Decreases (Vmax/α) Intersect on X-axis ('V' shape)

Uncompetitive Decreases (Km/α) Decreases (Vmax/α) Parallel Lines

Mixed Increases or Decreases Decreases (Vmax/α) Intersect off-axis (left of Y, above X)

(α is a factor related to inhibitor concentration and binding affinity)

VII. Key Takeaways & Important Points

• Enzyme inhibition is a fundamental mechanism in biology and pharmacology (esp. antibiotics, poisons,
drugs).
• Distinguish Reversible (Competitive, Uncompetitive, Mixed, some Non-competitive) from Irreversible
(Suicide, Transition State Analogues, some Non-competitive).
• Competitive: Inhibitor = Substrate analogue, competes for Active Site. Km ↑, Vmax unchanged.
Reversed by ↑[S]. (e.g., Ethanol for Methanol poisoning, Melathion initially).
• Non-competitive: Inhibitor binds Allosteric Site. Km unchanged, Vmax ↓. Not reversed by ↑[S]. (e.g.,
CO, Cyanide, Fluoride). Be aware of multi-substrate complexities.
 • Uncompetitive: Inhibitor binds ES complex only. Km ↓, Vmax ↓. Rare. (e.g., Phenylalanine on
Placental ALP).
• Irreversible: Covalent binding or extremely tight binding.
◦ Suicide: Enzyme activates its own inhibitor. (e.g., Allopurinol, Aspirin, DFMO).
◦ Transition State Analogue: Mimics the transition state. (e.g., Penicillin).
• Understand how Vmax and Km changes are reflected in Lineweaver-Burk plots (Y-intercept = 1/Vmax,
X-intercept = -1/Km).
Enzyme Regulation Summary
I. Introduction: Why Regulate Enzymes?

• Enzymes control nearly all cellular processes.

• Regulating enzyme activity is crucial for maintaining homeostasis and adapting to changing conditions
(e.g., fed vs. fasting states).
• Examples Illustrating Importance:
◦ Insulin/Glucagon: Regulate blood glucose via enzyme control based on dietary status.
◦ Cholera Toxin: Inhibits G protein interaction with adenylyl cyclase, causing cholera symptoms.
◦ Plague (Yersinia pestis): Disables macrophage phagocytosis.
◦ Neurodegenerative Diseases (Alzheimer's, Parkinson's): Involve issues with protein/enzyme
processing or degradation.
◦ Phenobarbital: Induces heme synthesis, potentially aggravating porphyria.
II. Major Types of Enzyme Regulation
(Mnemonic: Think "How Much?" vs "How Well?")

1. Regulation of Enzyme Quantity (How Much?)

◦ Long-term regulation (takes time, involves changes in protein amount).
◦ A. Control of Enzyme Synthesis (Induction & Repression):
▪ Acts at the gene level (regulating transcription/translation).
▪ Induction: Increases enzyme synthesis.
▪ Repression: Decreases enzyme synthesis.
▪ Examples:
▪ Cholesterol Synthesis: High dietary cholesterol represses the gene for HMG-CoA reductase
(rate-limiting enzyme).
▪ Heme Synthesis:
▪ Enzyme: ALA Synthase (Delta-aminolevulinic acid synthase).
▪ High Heme levels repress the ALA synthase gene.
▪ Low Heme levels induce the ALA synthase gene.
▪ Phenobarbital Example Explained:
1. Phenobarbital metabolism requires cytochrome P450 enzymes (heme-containing).
2. Drug use depletes cytochromes -> lowers cellular heme levels.
3. Low heme induces the ALA synthase gene.
4. Increased ALA synthase activity boosts heme synthesis pathway.
5. In porphyria (where a heme synthesis enzyme is defective), this induction leads to the
accumulation of toxic intermediates (porphyrins), aggravating the condition.
 ◦ B. Control of Enzyme Degradation:
▪ Removes enzymes/proteins when no longer needed or if defective.
▪ Targets: Short-lived proteins (e.g., cyclins), regulatory proteins, aberrant/defective proteins.
▪ Mechanism: Ubiquitin-Proteasome System
1. Target protein tagged with ubiquitin (a small protein).
2. Ubiquitinated protein is recognized and degraded by the proteasome (a large protein
complex).
▪ Clinical Relevance:
▪ Cancer: Failure to degrade cyclins can lead to uncontrolled cell cycle progression.
▪ Neurodegenerative Diseases (Alzheimer's, Parkinson's): Associated with the failure of the
proteasome system to degrade accumulating abnormal/defective proteins.
2. Regulation of Enzyme Quality (How Well?)
◦ Short-term regulation (rapid changes in activity of existing enzyme molecules).
◦ Also called regulation of intrinsic catalytic activity.
◦ A. Allosteric Regulation:
▪ Definition: Enzyme activity at the active site is modulated by a modifier (effector) binding to a
separate allosteric site ("allo" = other).
▪ Allosteric Enzymes:
▪ Often multi-subunit (possess quaternary structure).
▪ Have distinct catalytic (active) and regulatory (allosteric) sites.
▪ Modifiers:
▪ Positive Modifier (Activator): Increases enzyme activity.
▪ Negative Modifier (Inhibitor): Decreases enzyme activity.
▪ Key Point: Modifiers are NOT structural analogs of the substrate.
▪ Mechanism: Modifier binding induces a conformational change in the enzyme, affecting
substrate binding affinity or catalytic efficiency at the active site.
▪ Activators often stabilize the high-affinity R (Relaxed) state.
▪ Inhibitors often stabilize the low-affinity T (Taut) state.
▪ Kinetics:
▪ Exhibit Sigmoid (S-shaped) kinetics on a Velocity vs. [Substrate] plot (unlike Michaelis-
Menten hyperbolic curves).
▪ Reason for Sigmoid Curve: Cooperative Binding (binding of one substrate molecule to one
subunit increases the affinity/likelihood of substrate binding to other subunits - similar to
hemoglobin).
▪ Kinetic Parameters: Vmax (maximum velocity), K.5 or S.5 (substrate concentration at Vmax/
2 - indicates affinity, NOT Km).
▪ Effect of Modifiers on Kinetics:
▪ Activator: Shifts curve to the left, decreases K.5 (increases apparent affinity).
▪ Inhibitor: Shifts curve to the right, increases K.5 (decreases apparent affinity).
▪ Classification:
▪ K-series: Modifier affects K.5 (affinity), Vmax remains constant. (Kinetic pattern similar to
competitive inhibition).
 ▪ V-series: Modifier affects Vmax, K.5 remains constant. (Kinetic pattern similar to non-
competitive inhibition).
▪ Important Distinction: Allosteric regulation involves a distinct site and sigmoid kinetics,
differing fundamentally from competitive/non-competitive inhibition (which follow
Michaelis-Menten kinetics).
▪ Examples:
▪ ALA Synthase: Inhibited by Heme.
▪ Aspartate Transcarbamoylase (ATCase): Inhibited by CTP (pyrimidine), Activated by ATP
(purine).
▪ HMG-CoA Reductase: Inhibited by Cholesterol.
▪ Acetyl-CoA Carboxylase: Inhibited by Acyl-CoA, Activated by Citrate.
▪ Carbamoyl Phosphate Synthetase I (CPS I): Activated by N-acetylglutamate (NAG).
▪ General Principle: Often, the end product of a pathway allosterically inhibits an early enzyme in
that pathway (feedback inhibition).
◦ B. Covalent Modification:
▪ Definition: Enzyme activity regulated by the covalent attachment or removal of a chemical
group.
▪ Types:
▪ Irreversible:
▪ Partial Proteolysis (Zymogen Activation): Inactive precursor enzyme (zymogen) is
activated by cleavage of specific peptide bonds.
▪ Examples: Trypsinogen → Trypsin, Chymotrypsinogen → Chymotrypsin, Fibrinogen →
Fibrin (Blood Clotting). (Mnemonic: ZIP it! Zymogen Irreversible Proteolysis)
▪ Reversible:
▪ Phosphorylation/Dephosphorylation:
▪ Addition/removal of phosphate groups (PO4³⁻).
▪ Target Amino Acids: Serine (Ser), Threonine (Thr), Tyrosine (Tyr) (contain -OH
groups).
▪ Enzymes: Protein Kinases (add P, use ATP), Protein Phosphatases (remove P, use
H2O).
▪ Crucial Point: Phosphorylation can either activate or inactivate an enzyme,
depending on the specific enzyme.
▪ Example: Glycogen Phosphorylase
▪ Glucagon (fasting) → activates Protein Kinase A → Phosphorylates Glycogen
Phosphorylase b (inactive) → Glycogen Phosphorylase a (active) → Glycogen
breakdown ↑.
▪ Insulin (fed) → activates Protein Phosphatase 1 → Dephosphorylates Glycogen
Phosphorylase a → Glycogen Phosphorylase b → Glycogen breakdown ↓.
▪ Acetylation:
▪ Addition of acetyl group (-COCH3).
▪ Target: Lysine residues. Enzyme: Acetyltransferase (e.g., HAT).
▪ Example: Histone acetylation → reduces positive charge → loosens DNA binding →
euchromatin → increased gene expression (Epigenetic modification). Also occurs on
other enzymes.
 ▪ Methylation:
▪ Addition of methyl group (-CH3).
▪ Donor: S-adenosylmethionine (SAM).
▪ Example: DNA methylation (on Cytosine) → often leads to gene silencing (Epigenetic
modification).
▪ ADP-ribosylation:
▪ Addition of ADP-ribose.
▪ Donor: NAD+.
▪ Example: Cholera Toxin ADP-ribosylates a G-protein, locking it in an active state →
persistent activation of adenylyl cyclase → cholera symptoms.
▪ Ubiquitination: (Mentioned earlier for degradation, but is itself a covalent modification).
III. Compartmentation

• An additional regulatory strategy (not strictly Quantity or Quality).

• Definition: Segregation of metabolic pathways or enzymes into different cellular compartments
(organelles).
• Prevents interference between opposing pathways and allows for specialized environments.
• Examples:
◦ Fatty Acid Synthesis: Cytosol.
◦ Fatty Acid Beta-Oxidation: Mitochondria.
◦ Heme Synthesis: Starts in mitochondria, continues in cytosol, finishes in mitochondria.
IV. Linking Examples Back to Mechanisms:

• Insulin/Glucagon: Primarily Covalent Modification (Phosphorylation/Dephosphorylation).

• Cholera Toxin: Covalent Modification (ADP-ribosylation).
• Plague (Yersinia pestis): Induces host enzyme (Tyrosine Phosphatase) production/activity (Involves
aspects of Quantity/Quality control manipulation).
• Neurodegenerative Diseases: Defect in Control of Enzyme/Protein Degradation (Ubiquitin-Proteasome
System).
• Phenobarbital/Porphyria: Control of Enzyme Synthesis (Induction).
V. Key Points to Remember:

• Enzyme regulation occurs via Quantity (synthesis/degradation, long-term, gene level/proteasome) and
Quality (allosteric/covalent mod, short-term, affects existing enzyme activity).
• Allosteric regulation involves a separate binding site, cooperative binding, and sigmoid kinetics (K.5,
not Km). Modifiers are not substrate analogs.
• Covalent modification involves attaching/removing chemical groups. Phosphorylation/
dephosphorylation (kinases/phosphatases) is a key reversible example, crucial in hormone signaling
(insulin/glucagon). Zymogen activation is an irreversible example.
• Defects in regulation underlie many diseases (cancer, neurodegeneration, metabolic disorders, infectious
disease pathology).
• Compartmentation provides spatial regulation.
Clinical Enzymology Summary
I. Introduction

• Core Concept: Clinical enzymology applies biochemistry principles (enzyme study) to medical
diagnostics.
• Main Topics Covered:
1. Isoenzymes
2. Clinical Enzyme Profiles (Organ-specific: Heart, Liver, etc.)

II. Isoenzymes (Isozymes)

• Definition: Physically distinct forms of the same enzyme that catalyze the same reaction.
◦ Analogy: Different physical forms of actor Allu Arjun, but all perform the same function (acting).
• Basis for Physical Distinction:
◦ Different Genes: e.g., Salivary Amylase vs. Pancreatic Amylase.
◦ Different Subunits: e.g., Lactate Dehydrogenase (LDH) with H and M subunits (H4, H3M1, etc.).
◦ Electrophoretic Mobility: Different migration speeds during electrophoresis (e.g., LDH1 is fastest,
LDH5 is slowest).
◦ Heat Stability: Different responses to heat (e.g., Placental Alkaline Phosphatase is heat stable, liver
ALP is heat labile).
◦ Km / Substrate Specificity: Different affinities for substrate (e.g., Glucokinase (liver) has lower
affinity/higher Km for glucose than other Hexokinases).
◦ Cofactor Requirement: Different coenzymes needed (e.g., Cytoplasmic Isocitrate Dehydrogenase
uses NADP+, Mitochondrial uses NAD+).
◦ Tissue Localization: Predominant presence in specific tissues (e.g., LDH1 in heart, LDH2 in RBCs).

III. Functional vs. Non-Functional Enzymes in Blood

• Functional Enzymes:
◦ Definition: Have a known physiological function within the bloodstream.
◦ Examples: Lipoprotein Lipase (LPL), Coagulation/Clotting Factors.
◦ Location: Often act in blood or at the blood-vessel interface (e.g., LPL on capillary endothelium).
• Non-Functional Enzymes:
◦ Definition: Do not have a known physiological function in the blood. Their presence usually
indicates normal cell turnover ("wear and tear") or organ injury.
◦ Clinical Significance: Used as Diagnostic Biomarkers.
◦ Utility:
1. Location of Disease: Elevated levels point towards injury in the organ where the enzyme
normally resides (e.g., cardiac enzymes in Myocardial Infarction).
2. Nature & Severity of Disease:
▪ Cytoplasmic Enzymes: Increase with reversible inflammation or injury causing increased
cell membrane permeability. Suggests milder/reversible damage.
▪ Mitochondrial Enzymes: Increase significantly only with necrosis or irreversible cell injury
involving mitochondrial membrane damage. Suggests more severe damage.
IV. Specific Isoenzyme Examples & Clinical Profiles
A. Lactate Dehydrogenase (LDH)

• Reaction: Pyruvate <=> Lactate

• Location: Cytoplasmic
• Structure: Tetramer (4 subunits), H (Heart) and M (Muscle) types.
• Isoforms (5 + 2 others):

Isoform Subunits Tissue Origin Electrophoretic Normal Clinical

Mobility Serum % Note

LDH-1 H4 Heart, RBC, Fastest ~14-26% ↑↑ in MI

Kidney ("Flipped
Pattern")

LDH-2 H3M1 RBC, Kidney, Fast ~29-39% Highest in

Heart normal
serum

LDH-3 H2M2 Spleen, Lungs, Intermediate ~20-26% Non-

Lymphs, specific
Leukocytes

LDH-4 HM3 Liver, Skeletal Slow ~8-16% ↑ in Liver/

Muscle Muscle
injury

LDH-5 M4 Liver, Skeletal Slowest ~6-16% ↑ in Liver/

Muscle Muscle
injury

* Note: LDH-4 predominates over

LDH-5 in skeletal muscle.

* Other LDH Forms:

* LDH-X / LDH-C: Post-pubertal

testes.

* LDH-6: Seen in severely ill

patients (associated with poor
prognosis).

* Clinical Point: Flipped LDH

Pattern: In Myocardial Infarction
(MI), LDH-1 > LDH-2 (Normally
LDH-2 > LDH-1). Less used now.

B. Creatine Kinase (CK / CPK)

• Reaction: Creatine + ATP <=> Creatine Phosphate + ADP

• Location: Primarily Cytoplasmic (except CK-MT)
• Structure: Dimer (2 subunits), M (Muscle) and B (Brain) types.
• Isoforms (3 + 1 other):
 Isoform Subunits Tissue Electrophoretic Normal Clinical Note
Origin Mobility Serum %

CK-1 BB Brain, Fastest Trace ↑ in brain injury,

Lung (<1%) some cancers

CK-2 MB Heart Intermediate <6% Key cardiac

marker for MI

CK-3 MM Skeletal Slowest ~94-100% Highest in

Muscle normal serum; ↑
in muscle disease/
injury

* Mnemonic: Think Brain (BB)

-> Heart (MB) -> Muscle (MM)
for 1, 2, 3.

* Other CK Forms:

* CK-MT: Mitochondrial form.

Release indicates severe cell
injury involving mitochondria.

C. Alkaline Phosphatase (ALP)

• Reaction: Hydrolyzes phosphate esters at alkaline pH.

• Location: Cell membranes primarily.
• Isoforms (Multiple & Complex):

Isoform Source / Heat Clinical Significance

Name Location Stability

Alpha-1 ALP Biliary Labile Cholestasis marker (esp. Extrahepatic obstruction - marked
Canaliculi elevation)
Epith.

Alpha-2 Heat Hepatocytes Labile Parenchymal Liver Disease (e.g., Hepatitis - mild/moderate
Labile elevation)

Alpha-2 Heat Placenta Most Stable Pregnancy; Inhibited by Phenylalanine

Stable

Pre-beta ALP Osteoblasts Labile Bone Formation marker (Paget's disease, Rickets/
Osteomalacia, Hyperparathyroidism). Normal in Multiple
Myeloma. (Mnemonic: Pre-Beta for Bone Blast)

Gamma ALP Intestinal Labile Intestinal disease; Also ↑ in liver disease (↓ clearance by
Mucosa sinusoidal cells)

Leukocyte Leukocytes Variable Hematologic disorders (e.g., CML vs Leukemoid reaction)

ALP (LAP)

Regan Tumor (Germ Stable (like Carcinoplacental; Derepressed in malignancies (Liver, Lung,
Isoenzyme cell origin) Placental) etc.)

Tumor Stable Similar to Regan; Associated with malignancy.

 Isoform Source / Heat Clinical Significance
Name Location Stability
Nagao /
Kasahara

D. Cardiac Biomarkers (for Myocardial Infarction - MI)

• Key Markers: CK-MB, Cardiac Troponins (cTnI, cTnT).

• Less Used Now: LDH (flipped pattern), AST.
• Timing Profile:

Marker Rises Peaks Duration Specificity Notes

Myoglobin ~1-3 6-9 hrs ~24 hrs Low Earliest marker, but non-
hrs specific (muscle injury)

CK-MB 4-8 hrs ~24 hrs 2-3 days Moderate Good early marker, useful
for re-infarction detection
after normalization

cTnI/cTnT 4-6 hrs ~24 hrs 7-10+ High Most specific & sensitive;
days Prolonged elevation good
for late diagnosis

LDH ~12-24 3-6 ~10-14 Low Historically used (Flipped

hrs days days Pattern: LDH1 > LDH2)

AST ~8-12 ~36-48 ~4-5 days Low Non-specific (liver,

hrs hrs muscle)

* High-Sensitivity Troponin
Assays: Allow earlier detection at
very low levels (<1 ng/L).

* Brain Natriuretic Peptide (BNP /

pro-BNP): Marker of heart failure /
volume overload, not primarily
ischemia/MI.

E. Liver Disease Markers

• 1. Markers of Hepatocellular Injury (Transaminases):

◦ ALT (Alanine Aminotransferase):
▪ Location: Cytoplasmic only.
▪ Specificity: More specific for liver injury.
◦ AST (Aspartate Aminotransferase):
▪ Location: Cytoplasmic + Mitochondrial.
▪ Specificity: Less specific (found also in heart, skeletal muscle, kidney, brain, RBCs).
◦ AST/ALT Ratio (AAR):
▪ Ratio < 1 (ALT > AST): Typical in most liver injuries (Viral Hepatitis, NAFLD, Toxic Hepatitis,
Acetaminophen toxicity).
 ▪ Ratio > 1 (AST > ALT):
▪ Alcoholic Liver Disease: Mitochondrial damage releases more AST. Ratio often > 2.
▪ Cirrhosis / Liver Neoplasia: Reduced clearance of AST by damaged sinusoidal cells.
◦ Degree of Elevation:
▪ Marked (>>10x ULN): Ischemic/Toxic injury, Acute Viral Hepatitis.
▪ Moderate: Chronic Hepatitis.
▪ Mild: Cirrhosis, Alcoholic Liver Disease (though ratio might be high).
◦ Isolated Mild ALT elevation: Suspect Fatty Liver.
• 2. Markers of Cholestasis (Impaired Bile Flow):
◦ ALP (Alkaline Phosphatase):
▪ Source: Biliary canalicular membrane (Alpha-1 isoform).
▪ Note: Marked elevation suggests extrahepatic obstruction. Non-specific (bone, placenta
source).
◦ GGT (Gamma-Glutamyl Transpeptidase):
▪ Source: Biliary epithelial cells (membrane + microsomes/SER).
▪ Note: Sensitive to cholestasis (intra & extrahepatic), but less specific than 5'NT. Induced by
alcohol and drugs (phenytoin, phenobarbital) - good marker for alcohol abuse.
◦ 5'-Nucleotidase (5'NT):
▪ Source: Biliary canalicular membrane.
▪ Note: Most specific marker for cholestasis (not elevated in bone disease or pregnancy).
• Liver Disease Pattern Summary:
◦ Hepatocellular: Markedly ↑ ALT & AST (ALT often > AST, unless alcohol/cirrhosis). ALP/GGT
mildly ↑ or normal.
◦ Cholestatic: Markedly ↑ ALP & GGT, ↑ 5'NT. ALT/AST mildly ↑ or normal.
◦ Mixed: Elevations in both hepatocellular and cholestatic markers.
F. Prostate Disease Markers

• Acid Phosphatase (ACP):

◦ Prostatic form is Tartrate-Labile.
◦ Less specific; also elevated in bone resorption states (osteoclasts have tartrate-resistant ACP). Largely
replaced by PSA.
• Prostate-Specific Antigen (PSA):
◦ Also called Kallikrein-related peptidase 3 (KLK3).
◦ More specific marker for prostate tissue (cancer, BPH, prostatitis).
◦ Cutoff: Generally < 4 ng/mL (age-dependent).
G. Pancreatic Disease Markers (Acute Pancreatitis)

• Serum Amylase:
◦ Rises quickly, falls quickly (short half-life).
◦ Non-specific: Elevated in salivary gland disease, bowel obstruction/infarction, ectopic pregnancy,
renal failure, macroamylasemia.
• Serum Lipase:
◦ Rises slightly later than amylase, stays elevated longer.
 ◦ More specific for pancreatitis than amylase. Can be >5000 times elevated.
H. Bone Disease Markers

• Markers of Bone Formation (Osteoblast activity):

◦ ALP (Pre-beta / Bone-specific Isoform)
◦ Osteocalcin
◦ Pro-peptides of Type I Collagen (PINP)
• Markers of Bone Resorption (Osteoclast activity):
◦ N-telopeptide (NTX) of Type I Collagen (Urine/Serum)
◦ C-telopeptide (CTX) of Type I Collagen (Urine/Serum)
◦ Urine Free Deoxypyridinoline (DPD)
◦ Tartrate-Resistant Acid Phosphatase (TRAP) - Not mentioned but relevant
I. Acute Kidney Injury (AKI) Markers (Novel)

• Examples: (Focus on unique names)

◦ Kidney Injury Molecule-1 (KIM-1)
◦ Neutrophil Gelatinase-Associated Lipocalin (NGAL)
◦ Interleukin-18 (IL-18)
◦ Cystatin C (Also a marker of GFR)
◦ Liver Fatty Acid Binding Protein (L-FABP)
◦ Osteopontin (Note: Osteocalcin is bone formation marker)
◦ Exosomal Fetuin-A
◦ Others mentioned: Alanine aminopeptidase, GST, GGT, Beta-2 microglobulin, Alpha-1
microglobulin, RBP, NHE3.
J. Marker Enzymes for Cell Organelles

• Plasma Membrane: 5'-Nucleotidase, Adenylyl Cyclase, Na+/K+ ATPase

• Endoplasmic Reticulum (ER): Glucose-6-Phosphatase
• Golgi Apparatus: Galactosyltransferase
• Mitochondria: ATP Synthase, Glutamate Dehydrogenase (not mentioned but common)
• Lysosomes: Acid Phosphatase, Cathepsins

V. Key Takeaways & Important Points

1. Isoenzymes: Different forms, same function. Clinically useful for identifying the source of elevated total
enzyme activity (e.g., LDH-1 vs LDH-5).
2. Non-Functional Enzymes: Key diagnostic tools. Cytoplasmic release suggests reversible injury;
mitochondrial release suggests necrosis/severe injury.
3. Cardiac Markers: Troponins (T or I) are the preferred markers for MI due to high specificity and
sensitivity, and longer detection window. CK-MB is useful for early detection and re-infarction.
4. Liver Markers:
◦ ALT is more liver-specific than AST.
◦ AST/ALT ratio > 1 (or >2) suggests alcoholic liver disease, cirrhosis, or neoplasia.
◦ 5'-Nucleotidase is the most specific marker for cholestasis.
 ◦ GGT is sensitive for cholestasis and highly induced by alcohol.
5. Pancreatic Markers: Lipase is more specific for pancreatitis than Amylase.
6. Bone Markers: Differentiate between formation (ALP, Osteocalcin) and resorption (Telopeptides, DPD).
ALP is normal in Multiple Myeloma.
7. Prostate: PSA is the primary marker, replacing ACP.
8. AKI: Novel markers like KIM-1 and NGAL offer earlier detection potential.
(Remember to consider the clinical context alongside enzyme levels for accurate diagnosis.)
Okay students, here is a summary of the introductory session on vitamins, focusing specifically on Vitamin
A, formatted for easy revision.

Vitamins: Introduction & General Concepts

1. Definition & Importance: * Vital: Essential for health and frequently tested in exams. * Organic
Compounds: Required in small amounts from food for body growth and maintenance. * Self-Explanatory
Topic: Can often be learned through reading; less conceptual complexity.
2. Essentiality & Synthesis: * Essential: Generally means they cannot be synthesized by the body and must
be obtained from the diet. * Exception - Endogenous Synthesis: Some vitamins can be synthesized. * By
the Body: Using the body's own metabolic pathways. * Niacin (B3): From the amino acid Tryptophan. *
Vitamin D: From 7-dehydrocholesterol in the skin upon exposure to UVB sunlight (280-315 nm). * In the
Body: Synthesized by gut bacteria (intestinal flora). * Vitamin K * Pantothenic Acid (B5) * Biotin
3. Classification: * Fat-Soluble Vitamins: * Mnemonic: ADEK (Vitamins A, D, E, K) * Water-Soluble
Vitamins: * Vitamin C * B-Complex Vitamins: * Energy-Releasing: B1 (Thiamine), B2 (Riboflavin), B3
(Niacin), B5 (Pantothenic Acid), Biotin. * Hematopoietic (Blood Cell Formation): B9 (Folic Acid), B12
(Cobalamin). * Others: B6 (Pyridoxine).
4. Fat-Soluble vs. Water-Soluble Vitamins: Key Differences

Feature Fat-Soluble (ADEK) Water-Soluble (B-Complex, C)

Absorption Require Chylomicrons (and fat) Do not need chylomicrons; direct absorption

Excretion Not readily excreted in urine Excreted in urine

Storage Stored in the body (mainly liver/ Minimal storage (except B12)
fat)

Toxicity Toxicity possible (due to storage) Generally no toxicity

Toxicity Exceptions N/A Niacin (B3) and Pyridoxine (B6) can be toxic in
excess

Primary Function Varied functions Primarily act as Coenzymes

Coenzyme Role Vitamin K acts as a coenzyme N/A

Exception

Vitamin A
1. Introduction & Types: * Deficiency illustrated by: Bitot's Spot (foamy, heaped conjunctiva) and
Follicular Hyperkeratosis (skin manifestation). * Two Main Forms: * Preformed Vitamin A (Retinoids):
Active form, mainly from animal sources. Includes Retinol, Retinal, Retinoic Acid. * Provitamin A
(Carotenoids): Precursor form, mainly from plant sources. Must be converted to active form. Example:
Beta-carotene.
2. Structure: * Basic structure: Beta-ionone ring + Isoprenoid unit. * Beta-carotene (a provitamin) is
unique: contains two Beta-ionone rings.
3. Retinoids (Preformed Vitamin A): * Compounds related to Retinol. ("Vitamin A" strictly refers to
Retinol). * Retinol: * Forms: All-trans-Retinol, 11-cis-Retinol (component of Rhodopsin). * Function:
Primarily Vision. Also Reproduction. * Retinal: * Forms derived from Retinol oxidation. Involved in the
visual cycle (as 11-cis and All-trans forms). * Retinoic Acid: * Forms: All-trans-Retinoic Acid, 9-cis-
Retinoic Acid, 13-cis-Retinoic Acid. * Function: Growth, Morphogenesis, Cellular Differentiation (acts
like a hormone).
4. Carotenoids (Provitamin A): * Mainly from plants. * Most prevalent: Beta-carotene. * Non-
Provitamin A Carotenoids (cannot be converted to Vitamin A but have other benefits): * Lutein &
Zeaxanthin: Used for macular degeneration. * Lycopene: May have role in prostate cancer prevention/
treatment.
5. Metabolism: * Absorption (Intestine): * Plant sources (Beta-carotene) -> converted to Retinal, then
Retinol (by dioxygenase enzyme). * Animal sources (Retinyl esters) -> hydrolyzed to Retinol. * Inside
intestinal cells: Retinol + Fatty Acid -> Retinyl esters. * Transport to Liver: Retinyl esters incorporated
into Chylomicrons. * Liver Uptake: Chylomicrons remnants taken up via receptors recognizing ApoE. *
Storage (Liver): Stored mainly as Retinyl palmitate in Perisinusoidal stellate cells (Ito cells). *
Transport from Liver to Target Tissues: * Retinyl esters -> Retinol. * Retinol binds to Retinol-Binding
Protein (RBP). * RBP-Retinol complex binds to Transthyretin (TTR). * Transported in blood as this Tri-
molecular complex (Retinol-RBP-TTR).
6. Functions:

• Vision (Visual Cycle):

◦ Rhodopsin (visual pigment in rods) = Opsin protein + 11-cis-Retinal (lecture stated 11-cis retinol,
but retinal is the aldehyde form bound).
◦ Light exposure -> Photoisomerization: 11-cis-Retinal -> All-trans-Retinal.
◦ This change leads to conformational changes (e.g., Metarhodopsin II formation).
◦ Metarhodopsin II activates a G-protein (Transducin).
◦ Activated Transducin activates Phosphodiesterase (PDE).
◦ PDE hydrolyzes cGMP -> 5'-GMP.
◦ Decreased cGMP causes closure of Na+ channels.
◦ Leads to Hyperpolarization of the rod cell membrane.
◦ Hyperpolarization generates the nerve impulse sent to the brain.
◦ Wald's Visual Cycle: Regeneration of 11-cis-Retinal from All-trans-Retinal (requires isomerase
enzymes).
• Regulation of Gene Expression:
◦ Retinoic Acid acts like a steroid hormone.
◦ Binds to nuclear receptors:
▪ RAR (Retinoic Acid Receptor) - Ligand: All-trans-Retinoic Acid.
▪ RXR (Retinoid X Receptor) - Ligand: 9-cis-Retinoic Acid.
◦ Receptor-ligand complex binds to DNA response elements, regulating gene transcription.
 ◦ RXR often forms dimers with other receptors (like the Vitamin D receptor) for function. Important
link between Vitamin A and D action.
• Epithelial Cell Health: Maintenance of normal epithelium in skin and mucous membranes.
• Reproduction: Essential for normal reproductive processes (associated with Retinol).
• Antioxidant: Especially Beta-carotene, protects against oxidative damage.
• Photoprotective Properties: Protects skin from light-induced damage.
7. Deficiency (Hypovitaminosis A): * Most common vitamin deficiency worldwide. * Leading cause of
preventable blindness in children. * Ocular Manifestations (Xerophthalmia - Dry Eye): * Earliest
symptom: Impaired adaptation to low light / Loss of sensitivity to green light. * Night Blindness
(Nyctalopia): Difficulty seeing in dim light. * Conjunctival Xerosis: Dryness of the conjunctiva. * Bitot's
Spots: Triangular, foamy, white/grey patches on the conjunctiva (keratinized epithelial debris), usually
temporal side. * Corneal Xerosis: Dryness of the cornea. * Corneal Ulceration. * Keratomalacia:
Softening, necrosis, and potential perforation of the cornea -> leads to blindness. * Skin Manifestations: *
Follicular Hyperkeratosis (Phrynoderma or "Toad Skin"): Rough, goosebump-like lesions, often on
elbows, knees, buttocks, due to keratin plugging of hair follicles/adnexal glands. Also called Papular
Dermatosis. * Mucosal Manifestations: * Squamous Metaplasia: Normal mucus-secreting epithelium is
replaced by stratified squamous epithelium (e.g., in respiratory tract, urinary tract), leading to increased
infection risk. * Other: * Impaired immunity. * Loss of taste sensation.
8. Therapeutic Uses: * Beta-carotene: Used in conditions with photosensitivity like Cutaneous Porphyria.
* All-trans-Retinoic Acid (ATRA / Tretinoin): Differentiation therapy for Acute Promyelocytic Leukemia
(APL). Also used topically for acne and photoaging. * 13-cis-Retinoic Acid (Isotretinoin): Oral medication
for severe cystic acne. Also used investigationally for childhood neuroblastoma and sometimes psoriasis.
9. Toxicity (Hypervitaminosis A): * Occurs with excessive intake of preformed Vitamin A (Retinoids), not
usually from Carotenoids. * Risk Group Example: Arctic explorers consuming polar bear liver (very high in
Vit A). * Cellular Damage Target: Lysosomes. * Acute Toxicity: (Single large dose) * Nausea, vomiting,
headache, dizziness. * Pseudotumor Cerebri: Increased intracranial pressure mimicking a brain tumor. *
Exfoliative Dermatitis: Peeling skin. * Hepatomegaly, Hyperlipidemia. * Chronic Toxicity: (Long-term
excessive intake) * Nausea, anorexia, weight loss, listlessness. * Dry skin, hair loss (alopecia). *
Hepatomegaly, potential cirrhosis. * Bony Exostoses: Abnormal bone growths. * Increased Risk of
Fractures: Retinoic acid promotes osteoclast activity, leading to bone demineralization. * Teratogenicity: *
Highly teratogenic (causes birth defects), especially Isotretinoin. Absolutely contraindicated in pregnancy
or women planning pregnancy without effective contraception.
10. Required Daily Allowances (RDA): (Approximate values) * Children: ~400 µg/day * Adult Men/
Women: ~600 µg/day * Pregnancy: ~800 µg/day * Lactation: ~950 µg/day (Note: Often measured in Retinol
Activity Equivalents - RAE)
11. Sources: * Richest Source Overall: Halibut liver oil. * Animal Sources (Retinoids): Cod liver oil,
Liver (beef, chicken), Eggs, Dairy products (milk, cheese, butter). * Plant Sources (Carotenoids - Beta-
carotene mainly): * Richest Plant Source: Carrots. * Dark green leafy vegetables (Spinach, Kale). *
Yellow/Orange fruits (Mangoes, Papayas, Apricots). * Yellow/Orange vegetables (Sweet potatoes, Pumpkin,
Squash).
12. Assay (Assessment of Status): * Dark Adaptation Time: Functional test assessing time taken to adapt
to darkness. * Serum Vitamin A (Retinol) Levels: Measured biochemically. * Reaction used: Carr-Price
reaction (colorimetric test with antimony trichloride).

Key Points to Remember:

• Vitamins are vital organic compounds needed in small amounts.

 • Know the endogenously synthesized vitamins (Niacin, D by body; K, B5, Biotin by gut flora).
• Distinguish Fat-soluble (ADEK) from Water-soluble (B, C) based on Absorption, Storage, Excretion,
Toxicity.
• Vitamin A: Exists as Retinoids (animal, active) and Carotenoids (plant, precursor).
• Metabolism: Involves intestinal conversion/absorption, chylomicrons, liver storage (Ito cells), and
transport via RBP & Transthyretin.
• Functions: Vision (Rhodopsin cycle), Gene regulation (Retinoic acid receptors RAR/RXR), Epithelial
health, Reproduction, Antioxidant (Beta-carotene).
• Deficiency: Xerophthalmia (Night blindness, Bitot's spots, Keratomalacia) and Phrynoderma. Leading
cause of preventable blindness.
• Toxicity: Possible with excess Retinoids (not Carotenoids); Acute (Pseudotumor cerebri) & Chronic
(Liver damage, bone issues); Highly Teratogenic.
• Therapeutic Uses: ATRA for APL, Isotretinoin for severe acne.
• Richest Sources: Halibut liver oil (overall), Carrots (plant).

Okay students, here is a detailed summary of the lecture on Vitamins D, E, and K for your revision.

Fat-Soluble Vitamins: D, E, K Summary

Vitamin D (Sunshine Vitamin / Steroid Hormone-like)

• Nature: Not a true vitamin (endogenously synthesized), acts like a steroid hormone (regulates gene
expression). Chemically a group of steroids.
• Forms & Sources:
◦ Vitamin D2 (Ergocalciferol): Plant source (fungus ergot), commercially available.
◦ Vitamin D3 (Cholecalciferol): Animal sources & endogenous synthesis.
• Endogenous Synthesis & Metabolism (Requires 3 Organs: Skin -> Liver -> Kidney)
1. Skin: 7-dehydrocholesterol exposed to UV light (290-315 nm) → Cholecalciferol (D3).
2. Transport: Cholecalciferol binds to D-binding protein (an alpha-1 globulin) in blood.
3. Liver: Cholecalciferol undergoes 25-hydroxylation → 25-hydroxycholecalciferol (Calcidiol). This
is the form measured to assess Vitamin D status.
4. Kidney: 25-hydroxycholecalciferol undergoes 1-alpha-hydroxylation (via 1-alpha-hydroxylase) →
1,25-dihydroxycholecalciferol (Calcitriol).
▪ Calcitriol is the biologically MOST ACTIVE form.
▪ 1-alpha-hydroxylase is the RATE-LIMITING enzyme.
▪ Stimulated by: Parathyroid Hormone (PTH) & Low serum Phosphate.
5. Inactivation: If Calcium levels are sufficient, 25-hydroxycholecalciferol undergoes 24-hydroxylation
→ 24,25-dihydroxycholecalciferol (Calcitetrol) (Inactive form).
• Functions:
◦ Primary Function: Calcium & Phosphorus Homeostasis (Goal: Increase Serum Ca++ & P)
▪ Intestine: Increases absorption of dietary Calcium & Phosphorus.
▪ Mechanism: Increases synthesis of TRPV6 (calcium channel) and Calbindin-9k (calcium-
binding protein).
▪ PTH Role: Indirectly stimulates absorption by increasing Calcitriol synthesis.
 ▪ Kidney: Increases reabsorption of Calcium & Phosphorus in Distal Convoluted Tubules (DCT).
▪ Mechanism: Increases TRPV5 (calcium channel) and Calbindin-28k.
▪ PTH Comparison: PTH also increases Ca++ reabsorption but decreases P reabsorption (causes
phosphaturia).
▪ Bone: Promotes bone resorption (releases Ca++ & P into blood) when serum Ca++ is low.
▪ Mechanism: Both Vit D & PTH stimulate Osteoblasts to express RANK Ligand (RANKL).
RANKL binds to RANK receptor on pre-osteoclasts, promoting their maturation into active
Osteoclasts, which resorb bone.
◦ Bone Mineralization: Paradoxically, when serum Ca++ is adequate, Vitamin D promotes bone
mineralization.
▪ Mechanism: Stimulates Osteoblasts to produce Osteocalcin (a calcium-binding protein), which
deposits calcium into the bone matrix.
◦ Immunomodulatory Function:
▪ Macrophages can activate Vit D (convert 25-OH to 1,25-(OH)2).
▪ In Tuberculosis: Mycobacterium tuberculosis binding to Toll-Like Receptors (TLRs) on
macrophages enhances local Calcitriol production. Calcitriol increases Cathelicidin (an
antimicrobial peptide/defensin), which kills the bacteria.
▪ Protective role suggested in COVID-19, certain Cancers (Colon, Breast - risk increases if 25-OH
Vit D < 20 ng/mL), Diabetes Mellitus, Prediabetes, and Metabolic Syndrome.
• Assay / Status Assessment:
◦ Measure serum 25-hydroxycholecalciferol (Normal range: 20-100 ng/mL).
◦ Serum Osteocalcin level can also indicate status.
• Deficiency:
◦ Rickets (Children) / Osteomalacia (Adults): Defective bone mineralization.
◦ Clinical Signs (Rickets): Skeletal deformities (e.g., Windswept deformity - valgum on one leg, valgus
on the other), Rachitic rosary (enlarged costochondral junctions).
◦ Biochemical Changes in Nutritional Rickets:
1. Vit D deficiency → ↓ Serum Ca++ & ↓ Serum P.
2. Low Ca++ → Secondary Hyperparathyroidism (↑ PTH).
3. ↑ PTH → Stimulates renal 1-alpha-hydroxylase → ↑ Calcitriol (if substrate available).
4. ↑ PTH & ↑ Calcitriol → Increase Ca++ absorption/reabsorption → Serum Ca++ may normalize.
5. ↑ PTH → Increases P excretion → Serum P remains LOW.
6. Key: Low serum Phosphate is a more consistent finding than low Calcium. Normal Calcium does
not rule out Rickets.
◦ Types of Rickets (Genetic):
▪ Vitamin D Dependent Rickets Type 1 (VDDR-I / Pseudo Vitamin D Deficiency Rickets):
Defect in renal 1-alpha-hydroxylase gene.
▪ Vitamin D Dependent Rickets Type 2 (VDDR-II / Hereditary Vitamin D Resistant Rickets):
Defect in the Vitamin D Receptor (VDR). True resistance.
▪ X-linked Hypophosphataemic Rickets (XLH): Most common hypophosphataemic rickets. X-
linked dominant. Mutation in PHEX gene (Text mentions FEX) → ↑ Fibroblast Growth Factor 23
(FGF23).
▪ Autosomal Dominant Hypophosphataemic Rickets (ADHR): Mutation in FGF23 gene itself.
 ▪ Autosomal Recessive Hypophosphataemic Rickets (ARHR): Defect in Dentin Matrix Protein
1 (DMP1) gene.
• Toxicity (Hypervitaminosis D):
◦ Dose > 4000 IU/day.
◦ Manifestations: Hypercalcemia, Hyperphosphatemia → Metastatic Calcification (calcium deposition
in soft tissues like blood vessels, kidneys) → Calcinosis, Hypertension (due to vascular contraction).
• Sources:
◦ Sunlight (main source).
◦ Richest Dietary Source: Halibut liver oil, Fish liver oils, Fish. (Text notes fish as the "only dietary
source" mentioned in Harrison). Animal sources are generally richer than plant sources.
• RDA (Required Daily Allowance):
◦ Children: 10 mcg/day (400 IU).
◦ Adults: 5 mcg/day (200 IU).
◦ Pregnancy: 10 mcg/day (400 IU).

Vitamin E (Tocopherol)

• Nature: Stereoisomers of Tocopherol. Alpha-tocopherol is the most biologically potent form. Possesses
a Chromane ring (also called Tocol ring).
• Function:
◦ Most potent naturally occurring LIPOPHILIC antioxidant.
◦ Chain-breaking antioxidant located within cell membranes.
◦ Protects Polyunsaturated Fatty Acids (PUFAs) and LDL cholesterol from lipid peroxidation
(oxidative damage).
• Synergy: Works with Selenium (a mineral), which decreases the requirement for Vitamin E.
• Deficiency: (Relatively rare, usually due to severe malabsorption)
◦ Neurological: Axonal degeneration, Peripheral neuropathy, Spinocerebellar ataxia.
◦ Hematological: Hemolytic anemia (due to increased RBC membrane fragility from oxidative stress).
◦ Ocular: Pigmented retinopathy, Ophthalmoplegia, Nystagmus.
• Therapeutic Uses:
◦ Retrolental fibroplasia (Retinopathy of Prematurity).
◦ Intermittent claudication.
◦ Bronchopulmonary dysplasia.
◦ Intraventricular hemorrhage of prematurity.
◦ May slow the aging process.
• Toxicity:
◦ Generally considered low toxicity.
◦ High doses may interfere with platelet aggregation and antagonize Vitamin K's function (increasing
bleeding risk, especially if on anticoagulants).
• RDA:
◦ Males: 10 mg/day.
◦ Females: 8 mg/day.
◦ Pregnancy: 10 mg/day.
 ◦ Lactation: 12 mg/day.

Vitamin K (Koagulation Vitamin)

• Nature: Naphthoquinone derivative.

• Forms:
◦ K1 (Phylloquinone): Dietary origin (green leafy vegetables).
◦ K2 (Menaquinone): Synthesized by intestinal bacterial flora.
◦ K3 (Menadione): Synthetic, water-soluble form.
• Function:
◦ Essential cofactor for the enzyme gamma-glutamyl carboxylase.
◦ Required for the post-translational gamma-carboxylation of glutamic acid (Glu) residues into
gamma-carboxyglutamate (Gla) residues on specific proteins.
◦ Gla residues are crucial for calcium-binding and the function of these proteins.
• Proteins Requiring Gamma-Carboxylation:
◦ Clotting Factors: Factor II (Prothrombin), Factor VII, Factor IX, Factor X. (Mnemonic
suggestion: 2 + 7 = 9, and 10)
◦ Anticoagulant Proteins: Protein C, Protein S.
◦ Bone Protein: Osteocalcin (involved in bone mineralization).
◦ Kidney/Vascular Protein: Nephrocalcin (Text name; often known as Matrix Gla Protein - MGP,
inhibits vascular calcification).
◦ Other: GAS6 (Growth Arrest Specific gene 6) protein.
• Vitamin K Cycle & Anticoagulants:
1. During carboxylation, reduced Vitamin K (Hydroquinone, KH2) is oxidized to Vitamin K Epoxide.
2. Vitamin K Epoxide Reductase enzyme reduces Vit K Epoxide back to Vit K Quinone, and then
another reductase (or VKOR) reduces it back to the active KH2 form.
3. Warfarin and Dicumarol are structural analogs of Vitamin K. They act as competitive inhibitors of
Vitamin K Epoxide Reductase.
4. Inhibition prevents regeneration of active Vit K (KH2), thus blocking gamma-carboxylation of
clotting factors → Anticoagulant effect.
• Deficiency:
◦ Impaired blood clotting → Prolonged Prothrombin Time (PT), increased bleeding time, bleeding
manifestations (easy bruising, hemorrhage).
◦ Newborns are susceptible due to:
▪ Low fat stores (Vit K is fat-soluble).
▪ Sterile gut (limited bacterial synthesis of K2).
▪ Poor placental transport of Vit K.
▪ Breast milk is a poor source of Vit K.
▪ Immature liver function (Text mentions immature lungs).
▪ Routine practice: Newborns receive a prophylactic Vit K injection.
• Toxicity:
◦ Excess natural Vit K (K1, K2) is generally non-toxic.
 ◦ Excess synthetic Vit K (Menadione, K3) can cause:
▪ Hemolysis (especially in G6PD deficient individuals).
▪ Hemolytic Jaundice.
▪ Kernicterus (bilirubin deposition in the brain) → Brain damage.

Key Points to Remember:

• Vitamin D Activation: Skin (UV) → Liver (25-OH) → Kidney (1-alpha-OH) → Active Calcitriol (1,25-
(OH)2 D).
• Rate-limiting step in Vit D activation: 1-alpha-hydroxylase in the kidney (stimulated by PTH, low P).
• Active form of Vit D: 1,25-dihydroxycholecalciferol (Calcitriol).
• Vit D Status Check: Measure 25-hydroxycholecalciferol.
• Vit D vs. PTH: Both ↑ Ca++; Vit D ↑ P, PTH ↓ P (phosphaturic).
• Rickets: Serum Ca++ can be normal due to secondary hyperparathyroidism; Serum P is typically low.
• Vitamin E: Main lipophilic antioxidant, protects cell membranes (PUFAs, LDL). Works with Selenium.
Deficiency causes hemolysis & neurological issues.
• Vitamin K: Essential for gamma-carboxylation of Glu residues.
• Vit K Dependent Factors: II, VII, IX, X, Protein C, Protein S, Osteocalcin, Nephrocalcin/MGP,
GAS6.
• Warfarin/Dicumarol: Inhibit Vitamin K Epoxide Reductase, preventing Vit K recycling and clotting
factor activation.
• Newborns: Prone to Vit K deficiency (sterile gut, poor transfer).
• Vit K Toxicity: Primarily with synthetic K3 (Menadione) → Hemolysis, Jaundice, Kernicterus.

Final thought from the lecture: "Life is not about how hard a hit you can give it is about how many you can
take and still keep moving forward." Keep studying!

Hematopoietic Vitamins Summary: Folic Acid (B9) &

Vitamin B12 (Cobalamin)
This summary covers the key aspects of Folic Acid and Vitamin B12, identified as crucial hematopoietic
vitamins frequently tested in exams.
I. Introduction

• Hematopoietic Vitamins: Primarily Folic Acid (B9) and Vitamin B12 (Cobalamin).
• Importance: High-yield topic for exams (e.g., 4-5 questions in AIIMS June 2020).
• Approach: Problem-based learning.
II. Folic Acid (Vitamin B9)

• Name Origin: From "folium" (leaves), indicating its source.

• Sources: Primarily plant origin, especially green leafy vegetables. Heat-labile (cooking destroys ~95%
in 5-10 min), making deficiency common despite dietary availability.
• Active Form: Tetrahydrofolate (THFA).
• Core Function: Acts as a carrier of one-carbon (1C) units.
◦ 1C Units Carried:
▪ Methyl (-CH3)
▪ Methylene (=CH2)
▪ Methenyl (-CH=)
▪ Formyl (-CHO)
▪ Formimino (=CH-NH)
◦ 1C Metabolism: All forms are interconvertible EXCEPT the reduction of Methylene-THFA to
Methyl-THFA, which is irreversible.
• Sources of 1C Units (Entry Points):
◦ Methylene-THFA: Primarily from Serine -> Glycine conversion (via Serine
Hydroxymethyltransferase). Also from Glycine Cleavage System (GCS). This is the most important
entry point.
◦ Formimino-THFA: From Histidine metabolism.
◦ Formyl-THFA: From Tryptophan metabolism.
• Utilization of 1C Units (Exit Points):
◦ Methyl-THFA: Donates methyl group to B12 (forming Methyl-B12), regenerating free THFA.
Methyl-B12 then donates the methyl group to Homocysteine to form Methionine (catalyzed by
Methionine Synthase). This is the key link between Folate and B12 metabolism and the main way free
THFA is regenerated.
◦ Methylene-THFA: Required for dTMP (Thymidylate) synthesis (pyrimidine synthesis for DNA).
◦ Formyl-THFA: Required for Purine synthesis (for DNA).
• Folic Acid Deficiency:
◦ Mechanism: Lack of THFA impairs 1C transfer -> Decreased DNA synthesis (due to ↓dTMP and
↓purines) -> Defective nuclear maturation.
◦ Manifestations:
1. Megaloblastic Anemia: Affects rapidly dividing cells (bone marrow).
▪ Peripheral Smear: Macrocytes, Teardrop cells, Hypersegmented neutrophils (>5 lobes),
Anisopoikilocytosis.
▪ Bone Marrow: Megaloblasts (large RBC precursors with immature nuclei).
2. Neural Tube Defects (NTDs): (e.g., Spina bifida, Anencephaly). Due to impaired DNA synthesis
during fetal development. Folate supplementation is crucial BEFORE conception and during
early pregnancy.
3. Increased Cancer Risk (e.g., Colorectal): Potentially due to impaired DNA methylation (↓SAM
from ↓methionine regeneration affecting epigenetic modifications).
◦ Biochemical Assessment:
▪ ↓ Serum Folate / ↓ Red Cell Folate (better indicator of stores).
▪ FIGLU Test: Increased urinary excretion of Formiminoglutamic acid (FIGLU) after a histidine
load (FIGLU requires THFA for metabolism).
▪ ↑ Urinary ICAR (Aminoimidazole carboxamide ribose-5-phosphate): An intermediate in purine
synthesis that accumulates.
▪ ↑ Serum Homocysteine (shared with B12 deficiency).
• Folinic Acid (Leucovorin):
◦ Chemically: 5-Formyl-THFA.
 ◦ Use: "Leucovorin Rescue" - Administered to counteract the toxicity of Methotrexate (a
Dihydrofolate Reductase inhibitor/antifolate drug) by providing a reduced folate source downstream
of the enzyme block.
III. Vitamin B12 (Cobalamin)

• Name/Chemistry: Cobalamin. Contains a Corrin ring (similar to porphyrin) with a central Cobalt (Co)
atom (~4.35% by weight).
• Sources: Exclusively animal origin. Not found in plants. Also called "Extrinsic Factor of Castle".
• Absorption (Complex Multi-step Process):
1. Mouth: B12 bound to food protein. Salivary glands secrete Haptocorrin (R-binder/Cobalophilin).
(Minor passive absorption ~1%).
2. Stomach: Acid/Pepsin release B12 from food. B12 binds Haptocorrin. Stomach Parietal Cells
secrete Intrinsic Factor (IF).
3. Duodenum: Pancreatic proteases degrade Haptocorrin, releasing B12. B12 binds Intrinsic Factor
(IF).
4. Terminal Ileum: IF-B12 complex binds to specific receptors (Cubilin) on ileal enterocytes ->
Receptor-mediated endocytosis. This is the primary site of active absorption.
5. Blood: B12 absorbed into portal blood binds to Transcobalamin II (TC-II) for delivery to tissues.
(TC-I mainly carries B12 analogs).
• Active Forms (Coenzymes):
1. Adenosylcobalamin (AdoB12): Coenzyme for Methylmalonyl-CoA Mutase. Converts
Methylmalonyl-CoA -> Succinyl-CoA (important in odd-chain fatty acid and branched-chain amino
acid metabolism).
2. Methylcobalamin (MeB12): Coenzyme for Methionine Synthase (aka Homocysteine
Methyltransferase). Receives methyl group from Methyl-THFA and transfers it to Homocysteine ->
Methionine. Links B12 and Folate cycles.
• Vitamin B12 Deficiency:
◦ Causes:
1. Nutritional: Strict Vegans (no animal products).
2. Malabsorption:
▪ Pernicious Anemia: Autoimmune gastritis -> destruction of Parietal cells or antibodies
against IF -> No IF production/function -> B12 cannot be absorbed.
▪ Gastric Issues: Gastrectomy (loss of IF production).
▪ Intestinal Issues: Ileal resection, Crohn's disease affecting the ileum, Stagnant Loop
Syndrome (bacterial overgrowth consumes B12).
▪ Fish Tapeworm (Diphyllobothrium latum): Competes with the host for dietary B12 in the
intestine.
◦ Manifestations:
1. Megaloblastic Anemia: Identical hematologically to folate deficiency. Caused by functional
folate deficiency ("Folate Trap") - Methyl-THFA accumulates as it requires B12 to be converted
back to THFA, thus trapping folate in an unusable form and impairing DNA synthesis. The
proximate cause of anemia is folate deficiency, secondary to B12 deficiency.
2. ↑ Homocysteine: Due to impaired Methionine Synthase activity (shared with folate deficiency).
3. ↑ Methylmalonic Acid (MMA) & Methylmalonic Aciduria: Due to impaired Methylmalonyl-
CoA Mutase activity. This is SPECIFIC to B12 deficiency, NOT seen in folate deficiency.
 4. Neurological Deficits: Subacute Combined Degeneration (SCD) of the spinal cord (posterior
and lateral columns).
▪ Mechanism: Accumulation of MMA and upstream Propionyl-CoA leads to abnormal fatty
acid synthesis/incorporation into neuronal lipids and myelin -> Demyelination. This is
UNIQUE to B12 deficiency.
◦ Biochemical Assessment:
▪ ↓ Serum B12.
▪ ↑ Serum Homocysteine (non-specific).
▪ ↑ Serum Methylmalonic Acid (MMA) (Highly specific).
▪ Schilling Test (Historically used to determine the cause of malabsorption, less common now).
▪ Peripheral Smear / Bone Marrow: Megaloblastic changes.
IV. Key Distinctions & Clinical Pearls

• Shared Features (Folate & B12 Deficiency): Megaloblastic Anemia, ↑ Homocysteine.

• Unique to B12 Deficiency: ↑ Methylmalonic Acid (MMA), Neurological Symptoms (SCD).
• Crucial Clinical Point: ALWAYS rule out B12 deficiency before treating suspected megaloblastic
anemia with folic acid alone.
◦ Why? Giving folate can partially correct the anemia (provides substrate for DNA synthesis) but does
NOT correct the underlying B12 deficiency.
◦ This masks the B12 deficiency while allowing the neurological damage (which depends solely on
B12 for the MMA pathway) to progress or even worsen, as the folate supplementation drives the
folate cycle, potentially using up the last remaining stores of B12 for the methionine synthase
reaction.
◦ Treatment: If B12 deficiency is present or suspected, B12 MUST be replaced (often first or
concurrently with folate if both are deficient).
V. Important Points to Remember

1. Sources: Folate = Plants (heat sensitive); B12 = Animals. Vegans are at high risk for B12 deficiency.
2. Function: Both vital for DNA synthesis via 1C metabolism.
3. Anemia: Both cause Megaloblastic Anemia (hypersegmented neutrophils, macrocytes).
4. Biochemical Markers: Both cause ↑ Homocysteine. Only B12 deficiency causes ↑ MMA.
5. Neurology: Only B12 deficiency causes neurological symptoms (SCD).
6. Treatment Caution: Never treat megaloblastic anemia with folate alone without excluding B12
deficiency to prevent irreversible neurological damage.
7. Folate Trap: B12 deficiency leads to a functional folate deficiency by trapping folate as Methyl-THFA.
8. Key Enzymes:
◦ Folate cycle relies on Dihydrofolate Reductase (inhibited by Methotrexate).
◦ B12 needed for Methionine Synthase (uses Methyl-B12) and Methylmalonyl-CoA Mutase (uses
AdoB12).
9. Absorption: B12 requires a complex process involving Haptocorrin, Intrinsic Factor (from stomach
parietal cells), and Cubilin receptors in the terminal ileum. Issues at any step cause deficiency (e.g.,
Pernicious Anemia).
Okay students, here is a summary of our lecture on energy-releasing B vitamins. Focus on understanding the
connections between the vitamins, their functions, and deficiency/toxicity signs.
Summary: Energy-Releasing B Vitamins (B1, B2, B3, B5, Biotin)
General Principle: These vitamins act as coenzymes essential for metabolic pathways that generate energy,
particularly carbohydrate, fat, and protein metabolism.
I. Vitamin B1 (Thiamine)

1. Sources:
◦ Aleurone layer of cereals (lost in polishing, retained by parboiling). Use unpolished rice, whole wheat
flour.
◦ Yeast (important when consuming fermented cereal products).
2. Active Form: Thiamine Pyrophosphate (TPP) or Thiamine Diphosphate (TDP).
3. Coenzyme Role (Key Enzymes): Remember "ATP-B"
◦ Alpha-ketoglutarate Dehydrogenase (TCA Cycle)
◦ Transketolase (HMP Shunt)
◦ Pyruvate Dehydrogenase (Glycolysis link to TCA Cycle)
◦ Branched-chain Keto Acid Dehydrogenase (BCAA Metabolism)
◦ Note: Crucial for carbohydrate metabolism.
4. Absorption: Decreased by chronic alcohol consumption.
5. Deficiency Manifestations:
◦ Wet Beriberi: Cardiovascular system involvement.
▪ High-output cardiac failure
▪ Dyspnea, Cardiomegaly
▪ Peripheral edema, Pulmonary edema
◦ Dry Beriberi: Nervous system involvement.
▪ Peripheral Nervous System (PNS): Symmetrical motor & sensory neuropathy (legs > arms), pain,
paresthesia, loss of reflexes (lower limbs), muscle cramps, muscle atrophy.
▪ Central Nervous System (CNS) - Wernicke's Encephalopathy: Triad (Confusion, Ophthalmoplegia
[nystagmus, ptosis], Ataxia [truncal]).
▪ Wernicke-Korsakoff Syndrome: Wernicke's Encephalopathy + Dementia/Memory Loss +
Confabulatory Psychosis. (Often associated with ↓ Transketolase activity).
6. Nerve Conduction: Thiamine phosphorylates and activates chloride channels in nerves.
7. Biochemical Assessment:
◦ Erythrocyte Transketolase activity (functional test).
◦ Urinary thiamine excretion.
8. Toxicity: No reported toxicity.
9. RDA: 1.0 - 1.5 mg/day.
II. Vitamin B2 (Riboflavin)

1. AKA: Yellow enzyme, Warburg enzyme.

2. Properties:
◦ Gives urine bright yellow color (when taking B complex).
◦ Redox vitamin (FAD/FADH2 in ETC).
◦ Light sensitive (Supplement during phototherapy for neonatal jaundice).
◦ Heat resistant (cooking stable).
 3. Active Forms: Flavin Mononucleotide (FMN), Flavin Adenine Dinucleotide (FAD).
4. Coenzyme Role (Key Enzymes):
◦ FMN: Complex I (NADH Dehydrogenase) of ETC, L-amino acid oxidase.
◦ FAD: Acyl-CoA Dehydrogenase (Beta-oxidation), Complex II (Succinate Dehydrogenase) of ETC,
D-amino acid oxidase, Xanthine oxidase, Pyruvate Dehydrogenase, Alpha-ketoglutarate
Dehydrogenase, BCKD.
5. Deficiency (Ariboflavinosis): Remember "The 2 C's of B2"
◦ Cheilosis (fissures at corners of mouth, angular stomatitis)
◦ Corneal vascularization (blood vessels growing into cornea)
◦ Other: Glossitis (magenta tongue, smooth due to papillary atrophy), Keratitis, Conjunctivitis,
Photophobia, Lacrimation, Seborrheic dermatitis, Normocytic normochromic anemia.
6. Biochemical Assessment:
◦ Erythrocyte Glutathione Reductase activity coefficient (measures enzyme activity before and after
adding FAD).
◦ Urinary riboflavin excretion.
7. Toxicity: No reported toxicity.
8. RDA: ~1.5 mg/day (as per lecture text).
III. Vitamin B3 (Niacin)

1. Synthesis: Can be synthesized endogenously from the amino acid Tryptophan.

2. Active Forms: Nicotinamide Adenine Dinucleotide (NAD+), Nicotinamide Adenine Dinucleotide
Phosphate (NADP+).
3. Coenzyme Role: Involved in numerous redox reactions.
◦ NADPH Generating Reactions: HMP shunt (G6PD, 6-Phosphogluconate Dehydrogenase),
Cytoplasmic Isocitrate Dehydrogenase, Malic Enzyme.
◦ NADPH Requiring Reactions (Reductive Biosynthesis): Fatty Acid Synthesis (Enoyl-CoA
Reductase, Ketoacyl Reductase), Cholesterol Synthesis (HMG-CoA Reductase), Free Radical
Defense (Glutathione Reductase), Nucleotide Synthesis (Ribonucleotide Reductase), Folate
metabolism (Folate Reductase).
◦ NAD+ Requiring Reactions: Numerous dehydrogenases in glycolysis, TCA cycle etc. (Too many to
list, understand the general role).
4. Deficiency (Pellagra): The 4 D's
◦ Dermatitis: Photosensitive, symmetrical rash on sun-exposed areas (e.g., Casal's necklace, Gauntlet of
Pellagra - lichenified, indurated, erythematous, scaly lesions).
◦ Diarrhea.
◦ Dementia / Depressive Psychosis.
◦ Death (if untreated).
5. Causes of Pellagra-like Symptoms:
◦ Hartnup Disease (defective tryptophan absorption).
◦ Carcinoid Syndrome (tryptophan diverted to serotonin synthesis).
◦ Vitamin B6 Deficiency (required for niacin synthesis from tryptophan).
◦ Maize-dominant diet (Niacin present in bound form, niacinogen).
◦ Sorghum/Jowar-dominant diet (High leucine content inhibits QPRTase, enzyme in niacin synthesis
pathway).
 6. Toxicity (Pharmacological Doses):
◦ Cutaneous Flushing (Prostaglandin-mediated; treat/prevent with aspirin or Laropiprant [PGD2
inhibitor]).
◦ Fulminant Hepatitis (monitor liver enzymes).
◦ Glucose Intolerance.
◦ Hyperuricemia (caution in gout).
◦ Gastric Irritation.
◦ Macular Edema.
7. Lipid Modifying Role: Used therapeutically to ↓ LDL, ↓ TAG, ↑ HDL.
8. RDA: Expressed as Niacin Equivalents (NE), considering dietary niacin and tryptophan conversion.
Specific RDA value not mentioned in the text but typically around 14-16 mg NE/day for adults.
IV. Vitamin B5 (Pantothenic Acid)

1. Properties: Name derived from "Pantothen" (meaning "everywhere"). Contains Beta-alanine.

2. Active Form: Component of Coenzyme A (CoA) and Acyl Carrier Protein (ACP).
3. Function:
◦ CoA: Central molecule in metabolism (Acetyl-CoA, Succinyl-CoA, HMG-CoA etc.) involved in
TCA cycle, fatty acid oxidation/synthesis, cholesterol synthesis, ketone body synthesis, etc.
◦ ACP: Essential component of the Fatty Acid Synthase complex.
4. Deficiency: Rare due to widespread presence in food.
◦ Gopalan's Burning Foot Syndrome (Nutritional Melalgia). Linked to Indian scientist Dr. C. Gopalan.
5. Toxicity: Very low toxicity. No specific syndrome mentioned.
6. RDA: Not mentioned in text, typically around 5 mg/day for adults.
V. Biotin (Vitamin B7 / Vitamin H)

1. Properties: Binding inhibited by Avidin (a protein in raw egg whites).

2. Active Form: Biocytin (biotin covalently linked to lysine in carboxylase enzymes). Carboxybiotin is the
activated intermediate.
3. Coenzyme Role (Carboxylation Reactions): Requires ATP; enzymes are ligases. Remember "ABC-P"
Carboxylases
◦ Acetyl-CoA Carboxylase (Fatty Acid Synthesis - Rate Limiting Step)
◦ B... (Think Base of Gluconeogenesis) -> Pyruvate Carboxylase (Gluconeogenesis)
◦ C... -> Propionyl-CoA Carboxylase (Metabolism of odd-chain fatty acids, certain amino acids)
4. Biotin-Independent Carboxylation Reactions:
◦ Gamma-carboxylation of glutamate (Vitamin K dependent, for clotting factors).
◦ Malic Enzyme (some forms).
◦ Carbamoyl Phosphate Synthetase (CPS) I (Urea Cycle) & II (Pyrimidine Synthesis).
◦ AIR Carboxylase (Purine Synthesis).
5. Deficiency: Rare, can be caused by excessive raw egg white consumption or genetic defects.
◦ Mental changes (depression, hallucinations).
◦ Scaly, erythematous, seborrheic rash (often around eyes, nose, mouth).
◦ Alopecia (Hair loss). Added for completeness.
 ◦ Biotinidase Deficiency: Inherited metabolic disorder preventing biotin release/recycling. Leads to
neurological problems and skin issues (Leiner's disease if severe).
6. Avidin/Streptavidin Binding: Extremely high affinity binding. Streptavidin (from Streptomyces
avidinii) binds 4 biotin molecules. This interaction is widely used in molecular biology and diagnostic
assays (e.g., ELISA).
7. Toxicity: No reported toxicity.
8. RDA: Not mentioned in text, typically around 30 mcg/day for adults.

Key Points to Remember:

• Multi-enzyme Complexes: Pyruvate Dehydrogenase, α-Ketoglutarate Dehydrogenase, and BCKD

require multiple B vitamins (TPP-B1, FAD-B2, NAD+-B3, CoA-B5, Lipoamide).
• Alcoholism: Strongly associated with Thiamine (B1) deficiency due to impaired absorption and often
poor diet.
• Clinical Clues:
◦ Beriberi (Wet/Dry), Wernicke-Korsakoff -> Think B1
◦ Cheilosis, Magenta Tongue, Corneal Vascularization -> Think B2
◦ Pellagra (4 D's, Photosensitive Dermatitis) -> Think B3
◦ Burning Feet -> Think B5
◦ Raw eggs + Rash/Neurological symptoms -> Think Biotin
• Toxicity: Significant concern mainly for Niacin (B3) at pharmacological doses (flushing, liver issues).
B1, B2, B5, Biotin generally considered non-toxic.
• Assessment: Erythrocyte enzyme activity tests are key functional assays (Transketolase for B1,
Glutathione Reductase for B2).
• Learning Approach: Focus on applying knowledge to clinical scenarios (Problem-Based Learning /
CBME).

Vitamin B6 (Pyridoxine) Summary

Basics

• Name: Vitamin B6 (also Pyridoxine)

• Structure: Contains a pyridine ring.
• Active Form: PLP (Pyridoxal Phosphate)

Key Functions & Roles (Primarily Amino Acid Metabolism)

• Required Daily Allowance (RDA): Depends on protein intake due to its major role in amino acid
metabolism. RDA is approx. 1-2 mg/day.
• Amino Acid Metabolism:
◦ Transamination: Crucial coenzyme for aminotransferases (e.g., ALT, AST).
▪ Clinical Link: Alanine:Glyoxalate Aminotransferase requires PLP to convert glyoxalate to
glycine. Deficiency leads to glyoxalate accumulation, causing oxalate stones (oxaluria).
 ◦ Decarboxylation: Required for converting amino acids to biogenic amines.
▪ Examples: Histidine → Histamine; 5-Hydroxytryptophan → 5-Hydroxytryptamine (Serotonin);
Glutamate → GABA.
◦ Transsulfuration: Involved in sulfur amino acid metabolism.
▪ Example: Cystathionine β-synthase (Homocysteine + Serine → Cystathionine) requires PLP.
Deficiency leads to homocystinuria.
◦ Tryptophan Metabolism:
▪ Kynureninase (requires PLP) is needed for the pathway converting 3-Hydroxykynurenine
towards NAD+ synthesis.
▪ Clinical Link: PLP deficiency blocks this step, shunting 3-Hydroxykynurenine to Xanthurenic
Acid, leading to xanthurenic aciduria. This impaired NAD+ synthesis can contribute to
Pellagra-like symptoms.
• Heme Synthesis:
◦ ALA Synthase (the first enzyme, Succinyl CoA + Glycine → δ-Aminolevulinate) requires PLP.
◦ Clinical Link: Deficiency impairs heme synthesis, causing microcytic hypochromic anemia.
• Carbohydrate Metabolism:
◦ Only one key enzyme: Glycogen Phosphorylase (rate-limiting enzyme of glycogenolysis in liver and
muscle).
◦ Storage: 80% of the body's PLP is stored in muscle, primarily bound to glycogen phosphorylase.

Deficiency Manifestations

• Neurological: Peripheral neuropathy, personality changes (depression, confusion), convulsions.

• Hematological: Microcytic hypochromic anemia.
• Metabolic/Skin: Pellagra-like symptoms (due to impaired NAD+ synthesis from Tryptophan).
• Urinary Analytes in Deficiency: (Mnemonic: HOX)
◦ Homocysteine (due to ↓ transsulfuration)
◦ Oxalates (due to ↓ glyoxalate transamination)
◦ Xanthurenic acid (due to ↓ kynureninase activity)

Vitamin B6 and Cancer

• Deficiency is linked to increased risk of hormone-dependent cancers (e.g., breast, endometrium,

prostate).
• Mechanism: B6 normally inhibits the binding of the hormone-receptor complex to Hormone Response
Elements (HRE) on DNA. Deficiency removes this inhibition, leading to enhanced hormone action.

Toxicity

• One of the few B vitamins with reported toxicity (along with Niacin).
• Causes sensory neuropathy.

Assay Methods

• Erythrocyte transaminase activity.

• Tryptophan load test (measures xanthurenic acid excretion after tryptophan load).
• Direct measurement of PLP in blood.
Vitamin C (Ascorbic Acid) Summary
Basics

• Name: Vitamin C (Ascorbic Acid)

• Synthesis: Synthesized from glucose via the uronic acid pathway in most animals.
• Human Essentiality: Humans and higher primates cannot synthesize Vitamin C due to the lack of the
enzyme L-gulonolactone oxidase.
• Historical Context: Deficiency (Scurvy) noted in sailors (Vasco da Gama); James Lind identified citrus
fruit (lemon) as a cure.

Key Functions & Roles (Primarily Hydroxylation)

• Hydroxylation Reactions:
◦ Copper-Containing Hydroxylases:
▪ Dopamine β-hydroxylase (Dopamine → Norepinephrine).
▪ Peptidylglycine hydroxylase (involved in peptide hormone maturation).
◦ α-Ketoglutarate-Linked Iron-Containing Hydroxylases: (Require Vit C, Fe2+, α-KG)
▪ Prolyl Hydroxylase & Lysyl Hydroxylase: Essential for hydroxylation of proline and lysine
residues in procollagen. This is critical for stable collagen formation and cross-linking.
▪ Clinical Link: Deficiency leads to unstable collagen -> impaired wound healing and fragile
blood vessels (causing bleeding manifestations).
• Iron Absorption:
◦ Acts as a reductant (ferrireductase activity).
◦ Converts dietary ferric iron (Fe3+) to ferrous iron (Fe2+) in the gut, which is the form more readily
absorbed.
◦ Clinical Link: Deficiency contributes to iron deficiency anemia by reducing iron absorption.

Deficiency: Scurvy

• Cause: Lack of dietary Vitamin C.

• Pathophysiology: Primarily due to defective collagen synthesis and impaired iron absorption.
• Manifestations (Bleeding is prominent):
◦ Gums: Swollen, spongy, bleeding gums.
◦ Skin: Petechiae (small pinpoint hemorrhages), purpura, ecchymosis (bruises), perifollicular
hemorrhages (bleeding around hair follicles).
◦ Nails: Splinter hemorrhages.
◦ Joints: Hemarthrosis (bleeding into joints), causing joint effusion and pain.
◦ Other: Poor wound healing, loose teeth.
◦ Anemia: Due to chronic blood loss AND decreased iron absorption.
• Infantile Scurvy (Barlow's Disease):
◦ Occurs typically around 6-12 months (weaning period if diet is deficient).
◦ Features include general scurvy symptoms plus:
▪ Scorbutic Rosary: Sharp angulation at the costochondral junctions (due to sternal changes),
distinct from the bead-like enlargement of Rachitic Rosary.
 ▪ Pseudo paralysis: Infant avoids moving limbs due to pain (especially from subperiosteal
hemorrhage or hemarthrosis).
▪ Pit-frog position of legs.

Key Takeaways & Important Points

• PLP is the active form of Vitamin B6.

• B6 is vital for Amino Acid metabolism (transamination, decarboxylation, transsulfuration), Heme
synthesis (ALA Synthase), and Glycogenolysis (Glycogen Phosphorylase).
• B6 Deficiency: Think Neuropathy, Microcytic Anemia, Convulsions, and specific metabolic markers
(Homocysteine, Oxalates, Xanthurenic acid). Remember the link to hormone-dependent cancers.
• B6 Toxicity: Sensory Neuropathy.
• Humans cannot synthesize Vitamin C (lack L-gulonolactone oxidase).
• Vitamin C is crucial for Hydroxylation reactions: especially Collagen synthesis (Prolyl/Lysyl
Hydroxylases) and Iron absorption (Fe3+ → Fe2+). (Mnemonic: Vit C for Collagen)
• Vitamin C Deficiency (Scurvy): Characterized by Bleeding (gums, skin, joints - due to weak collagen/
capillaries) and Anemia (bleeding + ↓ iron absorption).
• Infantile Scurvy (Barlow's Disease): Presents with scurvy symptoms + Scorbutic Rosary and Pseudo
paralysis / Pit-frog legs.
• Anemia Reasons:
◦ B6 Def: Failure of Heme Synthesis (Microcytic Hypochromic).
◦ C Def: Bleeding + Impaired Iron Absorption.
Heme Metabolism Summary
I. Introduction

• Clinical Relevance: Heme synthesis (Porphyrias) and Heme catabolism (Jaundice) are highly clinically
relevant topics.
• Lecture Scope: Focus on biochemistry of heme synthesis/catabolism. Jaundice types detailed elsewhere
(Medicine/Pediatrics). MCQ sessions will cover jaundice questions.
• Key Questions Addressed:
◦ Why no photosensitivity in Acute Intermittent Porphyria (AIP)?
◦ Why glucose treats AIP?
◦ How does lead affect heme synthesis?
◦ Why porphyria in iron toxicity?
◦ Why is porphyria associated with non-immune hydrops fetalis?
II. Heme & Porphyrin Basics

• Heme: A metalloporphyrin.
◦ Metal: Iron in the ferrous (Fe2+) state.
◦ Porphyrin: Protoporphyrin.
◦ Structure: Ferroprotoporphyrin.
• Porphyrin:
◦ Structure: 4 Pyrrole rings (5-membered rings) joined by methine bridges (=CH-).
 ◦ Types (differ by side chains):
▪ Uroporphyrin (Uro): Side chains A-P-A-P-A-P-P-A (A=Acetate, P=Propionate). Water-soluble
(excreted in urine).
▪ Coproporphyrin (Copro): Side chains M-P-M-P-M-P-P-M (M=Methyl). Intermediate solubility.
▪ Protoporphyrin (Proto): Side chains M-V-M-V-M-P-P-M (V=Vinyl). Sparingly water-soluble
(excreted in feces).
◦ Isomers: Physiologically relevant form is Type III. Type I is not usually metabolized.
• Porphyrinogens:
◦ Colorless precursors of porphyrins.
◦ Porphyrins are colored due to conjugated double bonds.
• Properties of Porphyrins:
◦ Soret Band: Sharp absorption peak at 400 nm. Diagnostic feature.
◦ Fluorescence: Emit strong red fluorescence under UV light. Basis for detection and erythrodontia.
◦ Cancer Phototherapy (Photodynamic Therapy): Cancer cells concentrate porphyrins. Argon laser
excites porphyrins -> generates free radicals -> kills cancer cells.
◦ Photosensitivity: Accumulated porphyrins on skin absorb light -> get excited -> generate free
radicals -> skin lesions.
III. Heme Synthesis

• Importance: Required for heme-containing proteins:

◦ Hemoglobin (Hb)
◦ Myoglobin (Mb)
◦ Cytochromes (e.g., Cyt C, Cyt P450)
◦ Catalase (heme enzyme - important!)
◦ Tryptophan Pyrrolase (heme enzyme)
◦ Nitric Oxide Synthase (NOS)
• Sites: Almost all tissues, principally Liver and Erythroid Precursors in bone marrow.
◦ NOT in mature RBCs (lack mitochondria).
• Location: Partly in Mitochondria, partly in Cytoplasm.
• Overall Strategy: Synthesize pyrrole ring -> Combine 4 pyrroles -> Form porphyrin ring -> Insert Fe2+.
• Steps: (Mnemonic Hint: Pathway order U -> C -> P for porphyrinogens)
1. Formation of δ-Aminolevulinate (ALA) (Mitochondria)
▪ Substrates: Succinyl CoA + Glycine
▪ Enzyme: ALA Synthase (ALAS) - Rate-limiting enzyme
▪ Cofactor: Pyridoxal Phosphate (PLP / Vitamin B6)
▪ Isoforms:
▪ ALAS1: Hepatic & other tissues. Inhibited by Heme. Induced by low heme levels & drugs
(e.g., barbiturates) that consume heme (via P450).
▪ ALAS2: Erythroid specific. Regulated by iron availability.
2. Formation of Porphobilinogen (PBG) (Cytoplasm)
▪ Substrate: 2 molecules of ALA
▪ Enzyme: ALA Dehydratase (ALAD) (also called Porphobilinogen Synthase)
 ▪ Reaction: Condensation, removes 2 H2O.
▪ Inhibited by Lead.
3. Formation of Hydroxymethylbilane (HMB) (Cytoplasm)
▪ Substrate: 4 molecules of PBG
▪ Enzyme: PBG Deaminase (also called HMB Synthase or Uroporphyrinogen I Synthase)
▪ Reaction: Linear tetrapyrrole formed, releases NH3.
4. Formation of Uroporphyrinogen III (UPG III) (Cytoplasm)
▪ Substrate: Hydroxymethylbilane (HMB)
▪ Enzyme: Uroporphyrinogen III Synthase (UROS)
▪ Reaction: Cyclizes HMB and rearranges one pyrrole ring to form the asymmetric Type III isomer.
▪ Note: Non-enzymatic cyclization of HMB forms UPG I (symmetric, dead-end pathway in
humans, relevant in CEP).
5. Formation of Coproporphyrinogen III (CPG III) (Cytoplasm)
▪ Substrate: UPG III
▪ Enzyme: Uroporphyrinogen Decarboxylase (UROD)
▪ Reaction: Decarboxylates the 4 acetate side chains (A) to methyl groups (M). Releases 4 CO2.
6. Formation of Protoporphyrinogen III (PPG III) (Mitochondria)
▪ Substrate: CPG III
▪ Enzyme: Coproporphyrinogen Oxidase (CPOX)
▪ Reaction: Oxidative decarboxylation of 2 propionate side chains (P) to vinyl groups (V).
7. Formation of Protoporphyrin III (Mitochondria)
▪ Substrate: PPG III
▪ Enzyme: Protoporphyrinogen Oxidase (PPOX)
▪ Reaction: Oxidation of methine bridges, creates conjugated double bond system (colored).
8. Formation of Heme (Mitochondria)
▪ Substrates: Protoporphyrin III + Fe2+ (Ferrous iron)
▪ Enzyme: Ferrochelatase
▪ Reaction: Inserts Fe2+ into the center of the protoporphyrin ring.
▪ Inhibited by Lead.
IV. Porphyrias: Disorders of Heme Synthesis

• Definition: Hereditary or acquired defects in heme biosynthesis enzymes, leading to accumulation of

specific intermediates.
• General Features:
◦ Accumulation Site & Symptoms:
▪ Early Pathway Defect (ALA, PBG accumulate): Primarily Neurovisceral symptoms
(abdominal pain, neurological & psychiatric issues). No Photosensitivity. Example: AIP.
▪ Late Pathway Defect (Porphyrinogens/Porphyrins accumulate): Primarily Photosensitivity.
May also have neurovisceral symptoms. Examples: PCT, CEP, EPP.
◦ Inheritance: Most are Autosomal Dominant (AD).
▪ Exceptions: (Mnemonic: Childhood Porphyrias Are Recessive, except XLP)
▪ Congenital Erythropoietic Porphyria (CEP) - AR
 ▪ ALA Dehydratase Porphyria (ADP) - AR
▪ Erythropoietic Protoporphyria (EPP) - AR
▪ X-Linked Protoporphyria (XLP) - X-Linked
• Specific Porphyrias:

Porphyria Deficient Enzyme Accumulating Key Features Inheritance Type

Intermediate(s)

Acute PBG Deaminase ALA, PBG 5 P's: Painful AD Hepatic

Intermittent (HMB Synthase) abdomen, Port wine
Porphyria (AIP) urine (can darken on
standing),
Polyneuropathy,
Psychological
disturbance,
Precipitated by drugs
(P450 inducers). No
photosensitivity.
Tachycardia
common. Most
common acute
porphyria.
Treatment: Glucose/
Hemin, Gene
therapy/RNAi.

Congenital Uroporphyrinogen UPG I, CPG I Severe AR Erythropoietic

Erythropoietic III Synthase (non-enzymatic photosensitivity
Porphyria (UROS) HMB path) (blistering), Red/
(CEP) / brown teeth
Gunther's (Erythrodontia)
Disease with red
fluorescence, Port-
wine urine,
Hemolytic anemia.
Can cause non-
immune hydrops
fetalis. Gene therapy
available.

Porphyria Uroporphyrinogen UPG III (and Most common AD Hepatic

Cutanea Tarda Decarboxylase related porphyria overall. (familial) /
(PCT) (UROD) porphyrins) Blistering Sporadic
photosensitivity on
sun-exposed areas.
Often acquired/
sporadic (80%) due
to inhibitors (Iron
overload/
Hemochromatosis,
Alcohol, Estrogens,
HIV, HCV).
Treatable
(phlebotomy,
hydroxychloroquine).

Coproporphyrinogen CPG III, ALA, Photosensitivity + AD Hepatic

Oxidase (CPOX) PBG Neurovisceral
 Porphyria Deficient Enzyme Accumulating Key Features Inheritance Type
Intermediate(s)
Hereditary symptoms (similar to
Coproporphyria AIP but often
(HCP) milder).

Variegate Protoporphyrinogen PPG III, CPG Photosensitivity + AD Hepatic

Porphyria (VP) Oxidase (PPOX) III, ALA, PBG Neuroviscentric
symptoms (like AIP/
HCP). Common in
South Africa.

Erythropoietic Ferrochelatase Protoporphyrin Non-blistering AR Erythropoietic

Protoporphyria III photosensitivity
(EPP) (swelling, pain,
erythema, eczema).
Most common
porphyria in
children. Hallmark:
↑ Free Erythrocyte
Protoporphyrin
(FEP). Treatment:
Afamelanotide.

ALA ALA Dehydratase ALA Rare. Neurovisceral AR Hepatic

Dehydratase (ALAD) symptoms. No
Porphyria photosensitivity.
(ADP) / Doss Resembles lead
Porphyria / poisoning.
Plumboporphyria

X-Linked ALAS2 (Gain-of- Protoporphyrin Similar to EPP X-Linked Erythropoietic

Protoporphyria function mutation) III (photosensitivity, ↑
(XLP) FEP), affects males.
Increased ALAS2
activity. Treatment:
Afamelanotide.

• Related Condition (Not a Porphyria):

◦ X-Linked Sideroblastic Anemia: Deficiency (loss-of-function) of ALAS2. Causes anemia due to
impaired heme synthesis in erythroid cells.
V. Key Concepts & Clinical Correlations

• Lead Poisoning: Inhibits ALA Dehydratase and Ferrochelatase. Leads to accumulation of ALA and
Protoporphyrin (seen as ↑ FEP, similar to EPP/XLP biochemically in part). Causes microcytic anemia,
neurotoxicity, GI issues. Resembles ADP clinically.
• Glucose Treatment for Acute Attacks (e.g., AIP): High glucose intake -> reduces ALAS1 induction
(mechanism likely involves PGC-1α repression). This decreases the production of ALA and PBG,
alleviating symptoms. Diabetics may have less severe porphyria attacks due to chronic hyperglycemia.
• Drug Precipitation: Drugs inducing Cytochrome P450 (e.g., barbiturates, anticonvulsants) deplete
hepatic heme pool -> derepresses/induces ALAS1 -> exacerbates porphyrias (especially acute hepatic
porphyrias like AIP, HCP, VP).
• Iron Overload (Hemochromatosis) & PCT: Excess iron can inhibit UROD, leading to or worsening
PCT.
 • Non-immune Hydrops Fetalis: Can be seen in severe, early-onset CEP due to profound anemia and
porphyrin toxicity in utero.
VI. Lab Diagnosis

• Urine: Check for ALA, PBG (AIP), Uroporphyrin (PCT, CEP), Coproporphyrin. Color changes (port-
wine, pink, darkens on standing).
• Stool: Check for Protoporphyrin, Coproporphyrin.
• Blood: Check Free Erythrocyte Protoporphyrin (FEP) (elevated in EPP, XLP, Lead poisoning).
Plasma porphyrins.
• Tests:
◦ Ehrlich's Test: Detects PBG (and Urobilinogen). Rapid screening test.
◦ Watson-Schwartz Test: Differentiates PBG (positive) from Urobilinogen (negative) after extraction.
◦ Fluorescence: Red fluorescence of porphyrins in urine, plasma, RBCs, or teeth (CEP) under UV
light.
◦ Absorption Spectroscopy: Soret band at 400 nm.
• Enzyme Assays: Measure specific enzyme activity in RBCs or fibroblasts.
• Genetic Testing: Definitive diagnosis by identifying mutations in specific genes.
VII. Heme Catabolism

• Purpose: Breakdown of heme from senescent RBCs (~120 days) or other heme proteins.
• Location: Reticuloendothelial System (RES - macrophages in spleen, liver, bone marrow), Liver,
Intestine.
• Steps:
1. Formation of Biliverdin (RES - Macrophages)
▪ Source: Heme released from Hb.
▪ Enzyme: Heme Oxygenase (Microsomal, requires NADPH, O2).
▪ Products: Biliverdin (green pigment) + Fe3+ (recycled) + Carbon Monoxide (CO) (only
endogenous source of CO).
2. Formation of Bilirubin (RES - Macrophages)
▪ Substrate: Biliverdin
▪ Enzyme: Biliverdin Reductase (Cytosolic, requires NADPH).
▪ Product: Bilirubin (yellow-orange pigment, unconjugated, water-insoluble, toxic).
3. Transport to Liver (Blood)
▪ Unconjugated bilirubin binds tightly to Albumin for transport.
4. Hepatic Uptake (Liver - Hepatocytes)
▪ Albumin-bilirubin complex reaches liver sinusoids.
▪ Bilirubin dissociates and is taken up by hepatocytes (facilitated transport).
5. Intracellular Binding (Liver - Hepatocytes)
▪ Bilirubin binds to cytosolic proteins Ligandin (Y protein / GST) and Protein Z to prevent reflux
and facilitate transport to ER.
6. Conjugation (Liver - ER)
▪ Purpose: Makes bilirubin water-soluble for excretion.
▪ Enzyme: UDP-Glucuronosyltransferase (UGT1A1).
 ▪ Reaction: Bilirubin + 2 UDP-Glucuronic Acid -> Bilirubin Diglucuronide (conjugated
bilirubin, water-soluble, non-toxic). Occurs sequentially (mono- then di-glucuronide).
▪ Deficiency causes Crigler-Najjar & Gilbert syndromes.
7. Biliary Excretion (Liver - Bile Canaliculi)
▪ Conjugated bilirubin is actively secreted into bile canaliculi.
▪ Transporter: MRP2 (Multidrug Resistance-associated Protein 2) / MOAT.
▪ Rate-limiting step in hepatic bilirubin metabolism.
▪ Deficiency causes Dubin-Johnson syndrome.
▪ (Minor pathway: Some conjugated bilirubin efflux into blood via MRP3, reuptake via OATP).
8. Intestinal Modification (Intestine - primarily ileum/colon)
▪ Conjugated bilirubin reaches the gut via bile.
▪ Bacterial enzymes (β-glucuronidases) deconjugate bilirubin.
▪ Gut bacteria reduce bilirubin to Urobilinogens (colorless).
9. Excretion Fates of Urobilinogen:
▪ Feces (~80-90%): Most urobilinogen is oxidized by gut bacteria to Stercobilinogen, then
Stercobilin (brown), excreted in feces (gives stool its characteristic color).
▪ Enterohepatic Circulation (~10-20%): Some urobilinogen is reabsorbed from the gut into the
portal blood and returns to the liver.
▪ Urine (Trace): A small fraction of reabsorbed urobilinogen bypasses the liver, enters systemic
circulation, and is filtered by the kidneys -> excreted in urine as Urobilinogen (normally present
in trace amounts). Oxidizes to Urobilin (yellow) on standing.
VIII. Key Points to Remember

• Heme is Ferroprotoporphyrin (Fe2+ + Protoporphyrin III).

• Heme synthesis involves Mitochondrial and Cytoplasmic steps.
• ALAS is the rate-limiting enzyme in heme synthesis (requires PLP). ALAS1 (hepatic, inducible by
drugs, inhibited by heme) vs ALAS2 (erythroid, iron-regulated).
• Lead inhibits ALAD and Ferrochelatase.
• Porphyrias result from enzyme defects; accumulation of ALA/PBG causes Neurovisceral symptoms,
accumulation of Porphyrin(ogen)s causes Photosensitivity.
• AIP: PBG Deaminase defect, ALA/PBG up, NO photosensitivity, acute attacks precipitated by drugs,
treat with glucose/hemin.
• PCT: UROD defect, UPG III up, most common porphyria, blistering photosensitivity, often linked to
iron, alcohol, HCV/HIV.
• CEP: UROS defect, UPG I/CPG I up, severe photosensitivity, erythrodontia, AR, hydrops fetalis
possible.
• EPP: Ferrochelatase defect, Protoporphyrin up, non-blistering photosensitivity, ↑ FEP.
• Heme Catabolism: Heme -> Biliverdin (Heme Oxygenase, produces CO) -> Unconjugated Bilirubin
(Biliverdin Reductase) -> Transported by Albumin -> Liver Uptake -> Conjugation (UGT1A1) -> Biliary
Excretion (MRP2 - rate-limiting) -> Intestinal Bacteria -> Urobilinogen -> Stercobilin (feces) /
Enterohepatic circulation / Trace Urobilinogen (urine).
• Unconjugated bilirubin is lipid-soluble, toxic, bound to albumin. Conjugated bilirubin is water-soluble,
non-toxic, excreted in bile.
Okay students, here is a summary of the key concepts from the Chemistry of Nucleotides section:
I. Introduction & Exam Focus

• New Pattern Emphasis: Concentrate on:

1. Chemistry of Nucleotides
2. Metabolism of Nucleotides
3. Molecular Biology Techniques (Very Important)
• Other Topics (Transcription, Translation): Require an overall understanding rather than deep step-by-
step memorization.
• Replication: More important than transcription and translation.
II. Nucleic Acids: The Basics

• Types: Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA).

• Composition: Polymers made of repeating units called nucleotides.
• Linkage: Nucleotides are joined by 3' to 5' phosphodiester bonds.
III. Nucleotide Structure: The Components
A nucleotide consists of three parts:

1. Nitrogenous Base: Purines or Pyrimidines.

2. Pentose Sugar: Ribose (in RNA) or Deoxyribose (in DNA).
3. Phosphate Group(s): One, two, or three phosphate groups.
IV. Nitrogenous Bases

• A. Purines:
◦ Structure: Double-ring (heterocyclic).
◦ Numbering: Ring 1 (1-6, anti-clockwise), Ring 2 (7-9, clockwise). Total 9 atoms.
◦ Nitrogen Positions: N1, N3, N7, N9 (Odd numbers except 5).
◦ Major Purines:
▪ Adenine (A): 6-amino purine.
▪ Guanine (G): 2-amino, 6-oxo purine.
◦ Minor Purines: Xanthine, Hypoxanthine, Uric Acid.
◦ Mnemonic: "Pure As Gold" (Purines are Adenine and Guanine) or "All Girls Are Pure" (Adenine,
Guanine = Purines).
• B. Pyrimidines:
◦ Structure: Single-ring (heterocyclic).
◦ Numbering: Clockwise (1-6). Total 6 atoms.
◦ Nitrogen Positions: N1, N3 (Odd numbers except 5).
◦ Major Pyrimidines:
▪ Cytosine (C): 2-oxo, 4-amino pyrimidine. (Found in both DNA & RNA).
▪ Uracil (U): 2,4-dioxo pyrimidine. (Found only in RNA).
▪ Thymine (T): 2,4-dioxo, 5-methyl pyrimidine (Essentially 5-methyluracil). (Found only in
DNA).
◦ Mnemonic: "CUT the Py" (Cytosine, Uracil, Thymine are Pyrimidines; 'Cut' also implies a single
ring compared to purines).
 ◦ Important Conversions:
▪ Deamination of Cytosine → Uracil.
▪ Deamination of Cytosine + Methylation → Thymine.
V. Pentose Sugars

• Structure: 5-carbon sugar ring.

• Types:
◦ Ribose: Has hydroxyl (-OH) groups at both 2' and 3' positions. Found in RNA.
◦ Deoxyribose: Has a hydrogen (-H) at the 2' position (oxygen is removed) and a hydroxyl (-OH)
group at the 3' position. Found in DNA.
• Carbon Numbering: Carbons in the sugar are numbered 1', 2', 3', 4', 5'.
• Prime Notation ('): Used to distinguish sugar carbons (e.g., C1') from carbons in the nitrogenous base
(e.g., C1).
VI. Nucleosides

• Composition: Nitrogenous Base + Pentose Sugar.

• Bond: β-N-glycosidic bond.
◦ Links C1' of the sugar to:
▪ N9 of a Purine.
▪ N1 of a Pyrimidine.
VII. Nucleotides

• Composition: Nucleoside (Base + Sugar) + Phosphate Group(s).

• Formation:
◦ The first phosphate group attaches to the C5' of the sugar via an ester bond. This forms a Nucleoside
Monophosphate (NMP).
◦ Subsequent phosphates (to form NDP, NTP) attach via acid anhydride bonds. These are high-energy
bonds.
• Energy Currency: Breaking acid anhydride bonds (e.g., ATP → ADP + Pi) releases energy, making
molecules like ATP the cell's energy currency.
VIII. Polynucleotides & Phosphodiester Bonds

• Formation: Nucleotides link together to form DNA or RNA strands.

• Bond: 3' to 5' phosphodiester bond.
◦ Connects the 3'-hydroxyl (-OH) group of one sugar to the 5'-phosphate group of the next nucleotide's
sugar.
◦ "Diester" because one phosphate forms ester links to two different sugars (at the 5' C of one and the 3'
C of another).
IX. Polarity of Nucleic Acids

• Directionality: Nucleic acid chains have distinct ends.

◦ 5' end: Has a free phosphate group attached to the 5' carbon of the terminal sugar.
◦ 3' end: Has a free hydroxyl group attached to the 3' carbon of the terminal sugar.
• Convention: Base sequences are always written and read from the 5' end to the 3' end (5' → 3'). This
refers to the nucleotides with the free 5'-phosphate (start) and the free 3'-hydroxyl (end).
 • Reason for Polarity: The ends have different, free (ionizable/reactive) functional groups (phosphate at
5', hydroxyl at 3'), while internal nucleotides have these positions linked in phosphodiester bonds.
X. Naming Conventions

Base Nucleoside Nucleoside Deoxy-NMP (dNMP)

Monophosphate (NMP)

Adenine (A) Adenosine Adenosine Monophosphate Deoxyadenosine MP

(AMP) (dAMP)

Guanine (G) Guanosine Guanosine Monophosphate Deoxyguanosine MP

(GMP) (dGMP)

Cytosine (C) Cytidine Cytidine Monophosphate Deoxycytidine MP

(CMP) (dCMP)

Uracil (U) Uridine Uridine Monophosphate (Not typically in DNA)

(UMP)

Thymine (T) Thymidine* Thymidine Monophosphate Deoxythymidine MP

(TMP)* (dTMP)

Hypoxanthine Inosine Inosine Monophosphate

(IMP)

Xanthine Xanthosine Xanthosine Monophosphate

(XMP)

(Note: Thymidine implies Deoxythymidine as

Thymine is primarily in DNA)

Key Points to Remember:

1. Components: Nucleotide = Base + Sugar + Phosphate. Nucleoside = Base + Sugar.

2. Bases: Purines (A, G - double ring), Pyrimidines (C, T, U - single ring).
3. Sugars: Deoxyribose (DNA - H at 2'), Ribose (RNA - OH at 2').
4. DNA vs RNA: DNA uses Thymine (T) and Deoxyribose. RNA uses Uracil (U) and Ribose. Cytosine (C),
Adenine (A), Guanine (G) are in both (with corresponding sugar).
5. Key Bonds:
◦ β-N-glycosidic bond (Base-Sugar)
◦ Ester bond (Sugar-Phosphate in NMP)
◦ Acid Anhydride bond (Phosphate-Phosphate in NDP/NTP - High Energy)
◦ 3'-5' Phosphodiester bond (Links nucleotides in a chain)
6. Polarity: Nucleic acids run 5' to 3', defined by the free 5'-phosphate and 3'-hydroxyl groups at the ends.
7. Exam Focus: Chemistry, Metabolism, and Molecular Biology Techniques are high-yield topics.
Replication is more critical than Transcription/Translation for detailed knowledge in the new pattern.

Nucleotide Metabolism Summary

I. Introduction * Nucleotide metabolism is crucial for molecular biology due to numerous applied aspects. *
Key questions addressed: * Why increased uric acid in gout? * Why neurological deficits in Lesch-Nyhan
syndrome? * Why folate deficiency affects DNA synthesis / why antifolates treat cancer? * Answers lie in
purine and pyrimidine metabolism.
II. Purine Metabolism
A. De Novo Purine Synthesis * Location: Occurs in all tissues (as DNA is required), but mainly in the
liver. Subcellular location: Cytoplasm. * Exceptions (Sites without de novo synthesis): Brain,
Erythrocytes (RBCs), Leukocytes (WBCs), Bone Marrow. These rely purely on the Salvage Pathway. *
Contributors to Purine Ring Atoms: (Numbering C4, C5, N7; N3, N9; N1; C2; C8; C6) * Glycine: C4, C5,
N7 * Glutamine (amide N): N3, N9 * Aspartate: N1 * N10-Formyl-THFA (Text mentions N5-Formyl
THFA, standard is N10-Formyl): C2 * N5, N10-Methenyl-THFA: C8 * CO2 (Respiratory): C6 *
Mnemonic Aid: Think GAG for amino acids (Glycine, Aspartate, Glutamine). Folate derivatives (THFA)
contribute carbons. * Role of Folate: Essential THFA derivatives are required. * Folate deficiency ->
Defective purine synthesis -> Defective DNA synthesis -> Megaloblastic Anemia. * Folate antagonists
(Antifolates) inhibit de novo synthesis -> Used in cancer treatment. * Key Steps: 1. Ribose 5-Phosphate +
ATP → PRPP (Phosphoribosyl Pyrophosphate) + AMP * Enzyme: PRPP Synthetase (requires ATP). *
This is a preliminary step; PRPP is used in other pathways (salvage, NAD+, pyrimidine synthesis). 2.
PRPP + Glutamine → Phosphoribosylamine + Glutamate + PPi * Enzyme: PRPP Glutamyl
Amidotransferase*. * This is the committed step and the rate-limiting step of de novo purine synthesis. It
adds the N9 atom. * *Process: The purine ring is built step-by-step upon the initial Ribose 5-Phosphate
structure. * First Purine Nucleotide Formed: Inosine Monophosphate (IMP). * IMP Conversion: * IMP
→ AMP (Adenosine Monophosphate). Requires Aspartate (amino donor). Mnemonic: A for A. * IMP →
GMP (Guanosine Monophosphate). Requires Glutamine (amino donor). Mnemonic: G for G.
B. Purine Salvage Pathway * Definition: Recycling of pre-formed purine bases and nucleosides back into
nucleotides. * Rationale: 1. Saves energy (ATP) compared to de novo synthesis. 2. Effective recycling of
compounds from nucleic acid breakdown (cell lysis). 3. Crucial for tissues lacking de novo synthesis (Brain,
RBCs, WBCs, Bone Marrow). * Pathway Overview: DNA/RNA → Nucleotides → Nucleosides → Bases
→ Uric Acid (Catabolism End Product). Salvage intercepts bases/nucleosides. * Two Main Types: 1.
Phosphoribosylation (Base → Nucleotide): * Reaction: Purine Base + PRPP → Purine Nucleotide + PPi *
Enzyme: Phosphoribosyltransferase * Examples: * Hypoxanthine → IMP; Guanine → GMP * Enzyme:
Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRTase). Deficiency causes Lesch-Nyhan
Syndrome. * Adenine → AMP * Enzyme: Adenine Phosphoribosyltransferase (APRTase). Deficiency
causes 2,8-dihydroxyadenine kidney stones. 2. Phosphorylation (Nucleoside → Nucleotide): * Reaction:
Purine Nucleoside + ATP → Purine Nucleotide + ADP * Enzyme: Kinase * Example: Adenosine → AMP *
Enzyme: Adenosine Kinase.
C. Purine Catabolism * End Product: Uric Acid. * Location: Dietary purines in the intestine; Endogenous
purines mainly in the liver (cytoplasm). * Key Steps & Clinical Correlations: 1. Adenosine → Inosine *
Enzyme: Adenosine Deaminase (ADA). * Deficiency: Severe Combined Immunodeficiency (SCID)
(Affects B and T cells). 2. Inosine → Hypoxanthine; Guanosine → Guanine (Releases Ribose-1-
Phosphate) * Enzyme: Purine Nucleoside Phosphorylase (PNP) (Text calls it Purine Nucleoside
Ribosyltransferase). Requires Pi. * Deficiency: Immunodeficiency (Affects T cells primarily; B cells
normal). 3. Hypoxanthine → Xanthine AND Xanthine → Uric Acid * Enzyme: Xanthine Oxidase.
Requires Molybdenum (Mo). Produces H2O2. * Inhibited by: Allopurinol (drug for gout/hyperuricemia). *
Deficiency: Xanthinuria (Hypouricemia, xanthine crystals in urine). 4. Guanine → Xanthine * Enzyme:
Guanine Deaminase (Guanase). Releases NH3.
D. Disorders of Purine Metabolism * Lesch-Nyhan Syndrome: * Cause: X-linked recessive; Complete
deficiency of HGPRTase. * Biochemistry: ↓ Salvage → ↑ Hypoxanthine/Guanine → ↑ PRPP & de novo
synthesis → ↑ Uric Acid (Hyperuricemia). * Clinical Features: Compulsive self-mutilation, Gout,
Dystonia, Megaloblastic Anemia (due to bone marrow's reliance on salvage), Severe Neurological Deficits
(brain relies on salvage). * Treatment: Allopurinol, high fluids, urine alkalinization. * Kelley-Seegmiller
Syndrome: * Cause: Partial deficiency of HGPRTase (>1.5-2% activity). * Features: Variable, often gout
without severe neurological features. * APRTase Deficiency: * Cause: Deficiency of Adenine
Phosphoribosyltransferase. * Biochemistry: ↑ Adenine → Oxidized to 2,8-dihydroxyadenine (insoluble). *
Features: Severe crystaluria (2,8-dihydroxyadenine crystals), kidney stones, brown/orange diaper stains.
Different presentation than HGPRTase deficiency. * Gout: * Cause: Hyperuricemia leading to deposition of
monosodium urate crystals. * Types: * Primary Gout: Due to enzyme defects (↑ PRPP Synthetase activity, ↑
PRPP Glutamyl Amidotransferase activity, HGPRTase deficiency [Lesch-Nyhan/Kelley-Seegmiller],
Glucose-6-Phosphatase deficiency [Von Gierke's]). * Secondary Gout: Due to other conditions (↑ cell
turnover [malignancy], ↓ uric acid excretion [renal failure, acidosis, drugs like thiazides]). * Aggravating
Factors: Alcohol, high purine diet (meat), fructose-rich foods/drinks. * Clinical Features: Acute inflammatory
arthritis (esp. 1st MTP joint - Podagra), Tophi (chronic gout), Uric acid nephrolithiasis. Crystals prefer
cooler peripheral joints. * Diagnosis: Synovial fluid: Negatively birefringent, needle-shaped crystals. *
Treatment: Allopurinol, high fluids, urine alkalinization, anti-inflammatories (acute), uricosuric drugs
(Probenecid), lifestyle changes. * Severe Combined Immunodeficiency (SCID): * Cause: ADA deficiency
is the second most common cause (most common is gamma chain defect). Affects B & T cells. * Treatment:
Gene therapy (pioneered by French Anderson), Enzyme replacement (PEG-ADA), bone marrow transplant. *
PNP Deficiency: * Cause: Purine Nucleoside Phosphorylase deficiency. * Features: Affects T cells; B cells
are normal.
III. Pyrimidine Metabolism
A. De Novo Pyrimidine Synthesis * Location: Liver (major site). Subcellular: Cytoplasm AND
Mitochondria. * Key Differences from Purine Synthesis: * Pyrimidine ring is synthesized first, then
Ribose-5-Phosphate (from PRPP) is added. * Involves both cytoplasm and mitochondria. * Similarities to
Urea Cycle: Uses Carbamoyl Phosphate. * Key Steps: 1. CO2 + Glutamine + ATP → Carbamoyl Phosphate
* Enzyme: Carbamoyl Phosphate Synthetase II (CPS-II) (Cytoplasmic). 2. Carbamoyl Phosphate +
Aspartate → Carbamoyl Aspartate * Enzyme: Aspartate Transcarbamoylase (ATCase). 3. →
Dihydroorotic Acid * Enzyme: Dihydroorotase. * Multifunctional Enzyme CAD: CPS-II, ATCase,
Dihydroorotase activities are on a single polypeptide CAD. 4. Dihydroorotic Acid → Orotic Acid * Enzyme:
Dihydroorotate Dehydrogenase (Mitochondrial - the only mitochondrial step). 5. Orotic Acid + PRPP →
Orotidine Monophosphate (OMP) * Enzyme: Orotate Phosphoribosyltransferase (OPRTase). 6. OMP →
Uridine Monophosphate (UMP) + CO2 * Enzyme: OMP Decarboxylase. * Multifunctional Enzyme
UMP Synthase: OPRTase and OMP Decarboxylase activities are on a single polypeptide UMP Synthase. *
First Pyrimidine Nucleotide: UMP. (OMP is carboxylated intermediate). * Formation of CTP: UMP →
UDP → UTP → CTP. Enzyme for UTP→CTP: CTP Synthase. * Formation of dTMP (Thymidine - for
DNA): 1. UDP → dUDP (deoxyUDP) * Enzyme: Ribonucleotide Reductase. 2. dUDP → dUMP. 3. dUMP
→ dTMP * Enzyme: Thymidylate Synthase. Requires N5, N10-Methylene-THFA. * Inhibited by: 5-
Fluorouracil (5-FU) (Anticancer drug). 4. N5, N10-Methylene-THFA → Dihydrofolate (DHF) during the
reaction. 5. DHF → THFA (Regeneration) * Enzyme: Dihydrofolate Reductase (DHFR). * Inhibited by:
Methotrexate (Antifolate, anticancer drug).
B. Disorders of Pyrimidine Metabolism * Hereditary Orotic Aciduria: * Cause: Autosomal recessive
defect in UMP Synthase. * Type 1: Defect in both OPRTase and OMP Decarboxylase activities (most
common). * Type 2: Defect only in OMP Decarboxylase activity. * Biochemistry: ↑ Orotic Acid excretion; ↓
UMP, CTP, TMP → Impaired DNA/RNA synthesis. * Clinical Features: Failure to thrive, Megaloblastic
Anemia (unresponsive to B12/Folate/Iron), Orotic acid crystals in urine. Normal ammonia levels. *
Treatment: Uridine supplementation. * Orotic Aciduria in Other Conditions: * Urea Cycle Defects (e.g.,
OTC Deficiency / Type II Hyperammonemia): ↑ Mitochondrial Carbamoyl Phosphate leaks to cytosol,
enters pyrimidine pathway → ↑ Orotic Acid. Associated with Hyperammonemia. * Reye's Syndrome:
Mitochondrial dysfunction can impair urea cycle → secondary orotic aciduria.
C. Pyrimidine Catabolism * End Products: * Cytosine, Uracil → Beta-Alanine (+ NH3, CO2). *
Thymine → Beta-Aminoisobutyrate (+ NH3, CO2). * Significance: These end products are water-soluble.
Their accumulation does not cause clinical problems like uric acid (explains why excess pyrimidine
catabolism is asymptomatic).
D. Pyrimidine Salvage Pathway * Exists, but less significant than purine salvage. * Mainly salvages
pyrimidine nucleosides (e.g., Uridine → UMP via kinases). * Pyrimidine bases are generally not salvaged
efficiently.
IV. Miscellaneous * Pseudouridine: An isomer of uridine found in tRNA (TΨC loop). Linkage is C5
(Uracil) to C1' (Ribose), instead of N1 to C1'.
V. Key Points to Remember * De Novo Purine Synthesis: Starts with Ribose-5-P, builds ring on it. Rate-
limiting = PRPP Glutamyl Amidotransferase. First product = IMP. Needs Glycine, Aspartate, Glutamine,
Folate, CO2. * De Novo Pyrimidine Synthesis: Builds ring first (Orotic Acid), adds Ribose-P (PRPP).
Involves CAD & UMP Synthase multifunctional enzymes. Mitochondrial step = Dihydroorotate
Dehydrogenase. First product = UMP. Needs Glutamine, Aspartate, CO2. * Salvage Pathways: Crucial for
energy saving and for tissues lacking de novo synthesis (Brain, RBCs, WBCs, Bone Marrow). HGPRTase
(purine base salvage) deficiency -> Lesch-Nyhan. APRTase (purine base salvage) deficiency -> 2,8-DHA
stones. * Catabolism End Products: Purines → Uric Acid (insoluble, causes gout/stones). Pyrimidines →
Beta-Alanine / Beta-Aminoisobutyrate (soluble, asymptomatic). * Key Enzymes & Diseases/Drugs: *
HGPRTase: Deficient in Lesch-Nyhan. * ADA: Deficient in SCID. * PNP: Deficient in T-cell
immunodeficiency. * Xanthine Oxidase: Target of Allopurinol (for Gout). Deficient in Xanthinuria. * UMP
Synthase: Deficient in Orotic Aciduria. * Thymidylate Synthase: Target of 5-Fluorouracil. * DHFR: Target
of Methotrexate. * Folate: Essential for both purine (C2, C8) and pyrimidine (dTMP synthesis) metabolism.
Deficiency affects DNA synthesis -> Megaloblastic anemia. Antifolates are anticancer drugs. * Neurological
Deficits in Lesch-Nyhan: Due to the brain's dependence on the purine salvage pathway (HGPRTase). *
Orotic Aciduria: Can be hereditary (UMP Synthase defect) or secondary to Urea Cycle Defects (esp. OTC
deficiency, check ammonia levels!).

Okay students, here is a summary of the lecture on DNA Structure and Organization for your revision.

DNA Structure: The Watson-Crick Model

1. Historical Context:
◦ Rosalind Franklin: Known as the "Dark Lady of DNA," she captured the critical X-ray diffraction
image "Photo 51," which was instrumental in determining DNA's structure.
◦ Her work was shown (reportedly by colleague Maurice Wilkins) to James Watson and Francis Crick
before she could publish.
◦ Watson and Crick published the double helix model first in 1953.
◦ Watson, Crick, and Wilkins received the Nobel Prize in 1962. Franklin had passed away in 1958 due
to ovarian cancer, likely linked to X-ray exposure.
2. Salient Features of the B-DNA Double Helix:
◦ Composition: Composed of two polydeoxyribonucleotide strands.
◦ Helical Structure: Strands twist around each other in a right-handed spiral, resembling a spiral
staircase.
▪ Handrails: Sugar-phosphate backbone.
▪ Steps: Nitrogenous bases paired in the center.
◦ Antiparallel Strands: The two strands run in opposite directions: one is 5' → 3', and the
complementary strand is 3' → 5'.
 ◦ Base Pairing (Horizontal Interaction): Bases on opposite strands pair specifically via hydrogen
bonds (H-bonds).
▪ Adenine (A) always pairs with Thymine (T) using 2 H-bonds.
▪ Guanine (G) always pairs with Cytosine (C) using 3 H-bonds.
▪ (Mnemonic: At Two, Go Crazy with 3)
◦ Chargaff's Rules:
▪ Amount of Adenine (A) = Amount of Thymine (T).
▪ Amount of Guanine (G) = Amount of Cytosine (C).
▪ Total Purines (A + G) = Total Pyrimidines (T + C).
◦ Base Stacking (Vertical Interaction):
▪ Flat, nonpolar, aromatic bases stack on top of each other inside the helix.
▪ Stabilized by Van der Waals forces.
▪ Distance between stacked bases: 0.34 nm (3.4 Å).
◦ Dimensions:
▪ Base pairs per helical turn: ~10.5 bp (often approximated as 10 bp).
▪ Pitch (height of one full turn): 3.4 nm (34 Å) (0.34 nm/bp × 10 bp/turn).
▪ Diameter of the helix: 2.0 nm.
◦ Grooves: The helical structure creates a Major Groove and a Minor Groove, which are important
sites for DNA-protein interactions.
3. Chargaff's Rule Example Calculation:
◦ If Adenine (A) = 23%, then Thymine (T) must also = 23%.
◦ Total A+T = 23% + 23% = 46%.
◦ Remaining percentage for G+C = 100% - 46% = 54%.
◦ Since Guanine (G) = Cytosine (C), Guanine (G) = 54% / 2 = 27%.

Different Forms and Types of DNA

1. Conformational Forms:
◦ A-DNA: Right-handed, 11 bp/turn, shorter/broader, found in low hydration conditions (high salt).
◦ B-DNA: Right-handed, 10.5 bp/turn, longer/thinner, the most common physiological form (high
hydration, low salt), most stable.
◦ Z-DNA: Left-handed, 12 bp/turn, elongated/thin, "zigzag" backbone, found in regions with
alternating purine-pyrimidine sequences (esp. GC repeats), often at chromosome ends. (Mnemonic:
ZigZag is Left-handed).
◦ C, D, E forms also exist (right-handed).
2. Other DNA Types:
◦ cDNA (Complementary DNA): DNA synthesized from an mRNA template using reverse
transcriptase.
◦ Chimeric DNA (Recombinant DNA): DNA molecule created by joining DNA fragments from
different sources (e.g., gene of interest + vector DNA), often using restriction enzymes and ligase.
Named after the mythological Chimera (multi-part creature).
◦ Mitochondrial DNA (mtDNA):
▪ Constitutes ~1% of total cellular DNA.
 ▪ Structure: Double-stranded, circular molecule (~16,569 bp in humans).
▪ Copies: 2-10 copies per mitochondrion.
▪ Genes: Codes for 37 genes:
▪ 2 Ribosomal RNAs (16S rRNA, 12S rRNA).
▪ 22 Transfer RNAs (tRNAs).
▪ 13 proteins involved in the Electron Transport Chain (ETC) (~19% of total ETC proteins).
▪ Unique Features:
▪ Unique Genetic Code: Differs from the nuclear code (e.g., UGA codes for Tryptophan instead
of STOP; AUA codes for Methionine instead of Isoleucine; AGA/AGG code for STOP instead
of Arginine). Uses its own 22 tRNAs for translation.
▪ High Mutation Rate: Due to lack of protective histones, no introns, less efficient DNA repair
mechanisms, and constant exposure to reactive oxygen species (free radicals) from the ETC.
▪ Matrilineal Inheritance: Inherited exclusively from the mother (via the egg cytoplasm).

DNA Denaturation and Melting Temperature

1. Denaturation (Melting):
◦ Process: Separation of the two DNA strands.
◦ Causes: Heat, extreme pH, denaturing agents (e.g., formamide).
◦ Molecular Changes:
▪ Hydrogen bonds between bases are broken.
▪ Base stacking interactions are disrupted.
▪ Phosphodiester bonds (covalent bonds within a strand) remain intact.
▪ Primary structure (nucleotide sequence) is preserved.
▪ Secondary and tertiary structures are lost.
▪ Viscosity decreases.
▪ Hyperchromicity: Absorbance of UV light at 260 nm increases significantly (by up to 40%)
upon denaturation. This property is used to monitor denaturation and explains DNA's sensitivity
to UV damage.
◦ Alkali Stability: RNA is less stable in alkali conditions than DNA due to the presence of the 2'-
hydroxyl group, which facilitates hydrolysis.
2. Melting Temperature (Tm):
◦ Definition: The temperature at which 50% of the DNA duplex has dissociated into single strands.
◦ Factors Affecting Tm:
▪ Base Composition: Higher GC content leads to a higher Tm because G-C pairs have 3 H-bonds
(stronger) compared to A-T pairs with 2 H-bonds.
▪ Salt Concentration: Increasing salt (monovalent cation) concentration increases Tm by
stabilizing the phosphate backbone repulsion. (A 10-fold increase raises Tm by ~16.6°C).
▪ Denaturing Agents: Chemicals like formamide decrease Tm by disrupting H-bonds.

DNA Supercoiling and Topoisomerases

1. Supercoiling: The coiling of the DNA double helix upon itself, introducing torsional strain.
◦ Positive Supercoiling: Over-winding the DNA (in the same direction as the right-handed helix).
 ◦ Negative Supercoiling: Under-winding the DNA (in the opposite direction of the helix), which can
help in strand separation.
2. Topoisomerases: Enzymes that regulate DNA supercoiling by introducing transient breaks ("nicks") and
resealing them.
◦ Type I Topoisomerase: Creates a single-strand break. Does not require ATP.
◦ Type II Topoisomerase: Creates a double-strand break. Requires ATP.
◦ (Mnemonic: Type I = 1 strand nick, no ATP; Type II = 2 strand nick, needs ATP)

Eukaryotic DNA Organization: Chromatin

1. Levels of Organization: Eukaryotic DNA is highly organized, unlike prokaryotic DNA.

◦ Level 1: DNA Double Helix (2 nm diameter).
◦ Level 2: 10 nm Chromatin Fiber ("Beads-on-a-string"): DNA wraps around histone proteins to
form nucleosomes.
◦ Level 3: 30 nm Chromatin Fiber: Nucleosomes coil together.
◦ Higher Levels: Folding into loops attached to a nuclear scaffold, eventually forming compact
Metaphase Chromosomes. (This compaction allows vast amounts of DNA, like "120 volumes of
Harrison," to fit into the nucleus).
2. Nucleosome Structure (Basic Unit of 10 nm Fiber):
◦ Histone Octamer Core: Composed of two copies each of four core histones: H2A, H2B, H3, H4.
◦ DNA Wrapping: Approximately 1.75 turns (around 146 base pairs) of DNA wrap around the histone
octamer in a left-handed direction.
◦ Histones:
▪ Abundant, positively charged proteins (rich in basic amino acids Lysine and Arginine).
▪ Interact ionically with the negatively charged phosphate backbone of DNA.
▪ Types: Core histones (H2A, H2B, H3, H4) and Linker Histone (H1).
◦ Linker DNA: The stretch of DNA (~35 bp) between adjacent nucleosomes. Associated with Histone
H1.
◦ Appearance: Under electron microscopy, the 10 nm fiber looks like "beads (nucleosomes) on a string
(linker DNA)".
3. 30 nm Chromatin Fiber: Formed by the coiling of about 6 nucleosomes per turn.
4. Euchromatin vs. Heterochromatin: Different states of chromatin condensation within the nucleus.
◦ Euchromatin:
▪ Less condensed, more accessible.
▪ Transcriptionally active ("permissive chromatin").
▪ Stains lightly.
▪ (Analogy: Scattered books during exam prep - active studying)
◦ Heterochromatin:
▪ Highly condensed, less accessible.
▪ Transcriptionally inactive ("repressive chromatin").
▪ Stains densely.
▪ (Analogy: Neatly packed books when distracted - no studying)
 ▪ Types:
▪ Constitutive Heterochromatin: Permanently condensed and inactive (e.g., DNA at
centromeres and telomeres).
▪ Facultative Heterochromatin: Can switch between active and inactive states; inactive in
some cells/times but potentially active in others (e.g., the Barr body - inactive X chromosome
in female mammals).

Key Points to Remember:

• DNA is a right-handed double helix with antiparallel strands (5'-3' and 3'-5').
• Base pairing is specific: A=T (2 H-bonds), G≡C (3 H-bonds).
• Chargaff's Rules: A=T, G=C, Purines = Pyrimidines.
• Key dimensions: 0.34 nm between bases, 3.4 nm per turn, 2.0 nm diameter (B-DNA).
• B-DNA is the standard physiological form; Z-DNA is left-handed.
• mtDNA is circular, maternally inherited, has a unique genetic code, and a high mutation rate.
• Denaturation separates strands, breaks H-bonds (not phosphodiester bonds), and causes
hyperchromicity (increased A260).
• Tm increases with GC content and salt concentration, decreases with denaturants.
• Nucleosomes (DNA wrapped around histone octamer) are the basic unit of chromatin ("beads-on-a-
string").
• Histones are basic (+) proteins binding to acidic (-) DNA. Core (H2A, H2B, H3, H4) and Linker (H1).
• Euchromatin is open and active; Heterochromatin is condensed and inactive.
(Final Motivational Note from Lecture: Keep moving forward, adapt, and persevere.)
I. Introduction & Definition

• Concept: DNA replication is the biological process of producing two identical replicas of DNA from one
original DNA molecule.
• Analogy: Similar to copying a digital file (PowerPoint, video) to another storage device.
• Purpose: Ensures that when a cell divides into two daughter cells, each receives an exact copy of the
parent cell's DNA, allowing the passage of genetic information from parent to progeny.
• Timing: Occurs before cell division.
• Definition: The process of copying the base sequence present in the parent strand to synthesize a new
daughter strand.
II. Salient Features of DNA Replication

1. Timing in Cell Cycle: Takes place during the S phase (Synthesis phase) of the cell cycle.
2. Template Usage: Both strands of the parent DNA molecule act as templates for the synthesis of new
daughter strands.
3. Semi-Conservative Model: Each new daughter DNA molecule consists of one original parent strand and
one newly synthesized strand. (Half of the parent DNA is conserved).
◦ Proven by: Meselson and Stahl experiments.
4. Base Pairing Rule: Complementary base pairing is strictly followed: Adenine (A) pairs with Thymine
(T), and Guanine (G) pairs with Cytosine (C).
5. Direction of Synthesis: New DNA strands are always synthesized in the 5' to 3' direction.
 6. Bi-Directional: Replication proceeds in both directions from the origin, with the replication forks
moving away from each other.
7. Semi-Discontinuous Synthesis:
◦ Leading Strand: Synthesized continuously in the 5' to 3' direction on the 3' to 5' template strand
(follows the replication fork).
◦ Lagging Strand: Synthesized discontinuously in short fragments (Okazaki fragments) in the 5' to 3'
direction on the 5' to 3' template strand (moves away from the replication fork). These fragments are
later joined together.
8. Primer Requirement: DNA synthesis cannot start from scratch; it requires a short RNA primer to
provide a free 3'-OH group for the DNA polymerase to add nucleotides.
III. Steps of DNA Replication

1. Identification of Origin of Replication (Ori):

◦ Specific DNA sequences (Ori) are recognized and bound by Origin Binding Proteins (OriBP).
◦ Nearby AT-rich regions (called DUE - DNA Unwinding Element in eukaryotes) unwind easily due
to fewer hydrogen bonds (A=T has 2 H-bonds vs G≡C has 3 H-bonds).
2. Unwinding of DNA:
◦ The enzyme Helicase unwinds the DNA double helix at the replication fork, using ATP.
◦ Single-Strand Binding Proteins (SSBs) (called RPA - Replication Protein A in humans) bind to the
separated strands to prevent them from re-annealing (coming back together).
◦ Topoisomerase relieves torsional strain ahead of the replication fork caused by unwinding.
3. Formation of Replication Fork:
◦ The unwound region forms a replication bubble.
◦ Each Y-shaped structure within the bubble where active replication occurs is a replication fork.
4. DNA Synthesis:
◦ Primase synthesizes short RNA primers complementary to the template strands.
◦ Leading Strand Synthesis:
▪ Primase adds one RNA primer.
▪ DNA Polymerase III (in prokaryotes) adds DNA nucleotides continuously in the 5' to 3'
direction, following the fork movement.
◦ Lagging Strand Synthesis (Semi-Discontinuous):
▪ Primase adds multiple RNA primers along the template as the fork opens.
▪ DNA Polymerase III (in prokaryotes) synthesizes short DNA segments (Okazaki fragments)
starting from each primer, moving 5' to 3' until it reaches the previous primer.
▪ DNA Polymerase I (in prokaryotes) removes the RNA primers (using its 5'→3' exonuclease
activity) and fills the resulting gaps with DNA nucleotides (using its 5'→3' polymerase activity).
▪ DNA Ligase seals the nicks (breaks in the sugar-phosphate backbone) between the Okazaki
fragments and the newly replaced primer sections, creating a continuous strand. Requires ATP.
IV. Key Enzymes in DNA Replication

• Topoisomerase: Relieves supercoiling/torsional strain ahead of the replication fork.

• Helicase: Unwinds the DNA double helix at the replication fork (ATP-dependent).
• Primase: Synthesizes short RNA primers. (In prokaryotes, it's a separate enzyme called DnaG; in
eukaryotes, it's part of DNA Pol α).
 • DNA Polymerase: Synthesizes new DNA strands by adding nucleotides complementary to the template
strand in the 5' to 3' direction. Requires a primer. Also has proofreading ability (3'→5' exonuclease
activity).
◦ Prokaryotes: Pol I, Pol II, Pol III.
◦ Eukaryotes: Pol α, β, γ, δ, ε.
• SSB Proteins (RPA in Eukaryotes): Stabilize single-stranded DNA, preventing re-annealing.
• DNA Ligase: Joins Okazaki fragments on the lagging strand by forming phosphodiester bonds (seals
nicks, ATP-dependent).
V. DNA Polymerases (Detailed)
A. Prokaryotic DNA Polymerases (E. coli)

• DNA Polymerase I:
◦ Functions: RNA primer removal (5'→3' exonuclease), gap filling (5'→3' polymerase), DNA repair
(major repair enzyme), Proofreading (3'→5' exonuclease).
◦ Kornberg's Enzyme: Named after Arthur Kornberg who discovered it.
◦ Klenow Fragment: Pol I lacking the 5'→3' exonuclease activity (used in labs).
• DNA Polymerase II:
◦ Functions: DNA repair (minor role), Proofreading (3'→5' exonuclease).
• DNA Polymerase III:
◦ Functions: Main replicative enzyme - synthesizes both leading strand and Okazaki fragments (5'→3'
polymerase), Proofreading (3'→5' exonuclease).
◦ Processivity: Highest processivity (adds many nucleotides before dissociating).
B. Eukaryotic DNA Polymerases

• Mnemonic: Use the athletic track analogy or simple letter association.

• DNA Polymerase α (Alpha): Contains Primase activity (synthesizes RNA primers) and initiates DNA
synthesis. (Alpha = A = At the start)
• DNA Polymerase β (Beta): Involved in Base Excision Repair (BER). (Beta = B = Base repair)
• DNA Polymerase γ (Gamma): Replicates Mitochondrial DNA. (Gamma = G = Goes to mitochondria)
• DNA Polymerase δ (Delta): Synthesizes the Lagging strand; also involved in repair and proofreading.
(Delta = D = Does lagging/Discontinuous)
• DNA Polymerase ε (Epsilon): Synthesizes the Leading strand; also involved in repair and
proofreading. (Epsilon = E = Excels/Leads/Elongates continuously)
• Proofreading: Pol γ, δ, and ε possess 3'→5' exonuclease activity for proofreading.
VI. DNA Damage, Repair Mechanisms & Associated Disorders

• DNA is constantly susceptible to damage from endogenous (e.g., replication errors, free radicals) and
exogenous sources (e.g., radiation, chemicals).

Damaging DNA Defect(s) Repair Mechanism Defect Disorder(s)

Agent

Ionizing Double-strand breaks NHEJ (Non-Homologous NHEJ Defect: SCID (Severe Combined
Radiation (X- (DSBs), Single- End Joining - error-prone, Immunodeficiency)
rays), Anti- strand breaks (SSBs), joins ends directly, major HR Defect: ATLD (Ataxia-Telangiectasia Like
cancer drugs Cross-links in humans) Disorder), Nijmegen Breakage Syndrome,
HR (Homologous Bloom's Syndrome, Werner Syndrome,
 Damaging DNA Defect(s) Repair Mechanism Defect Disorder(s)
Agent
Recombination - accurate, Rothmund-Thomson Syndrome, BRCA1/2
uses sister chromatid related cancers
template, major in yeast)

UV Light, Bulky Adducts, NER (Nucleotide Excision Xeroderma Pigmentosum (XP) (often involves
Chemical Pyrimidine Dimers Repair) helicase defect), Cockayne Syndrome,
Carcinogens (esp. Thymine Trichothiodystrophy
Dimers)

Oxygen Abasic sites (AP BER (Base Excision MUTYH-associated polyposis

Radicals, sites - base missing), Repair)
Alkylating altered bases
Agents

Replication Base Mismatches, MMR (Mismatch Repair) HNPCC (Hereditary Non-Polyposis Colon
Errors Insertions, Deletions Cancer) / Lynch Syndrome

VII. Telomeres & Telomerase

• Telomeres: Protective caps at the ends of linear eukaryotic chromosomes. Consist of repetitive DNA
sequences (typically TTAGGG repeats in humans).
• End Replication Problem: During replication of linear chromosomes, RNA primer removal at the 5' end
of the lagging strand leaves a gap that cannot be filled by DNA polymerase (needs a 3'-OH). This leads to
progressive shortening of the chromosome with each cell division (loss of 3' end of parent strand
replication).
• Telomerase:
◦ A ribonucleoprotein enzyme (RNA + Protein) that counteracts chromosome shortening.
◦ Acts as a reverse transcriptase.
◦ Contains an intrinsic RNA template complementary to the telomere repeat sequence.
◦ Adds repetitive DNA sequences to the 3' overhang of the template strand, allowing primase and DNA
polymerase to complete replication of the lagging strand end.
◦ Analogy: Like the plastic tip (aglet) on a shoelace, preventing fraying.
• Presence & Activity:
◦ Absent/Low Activity: Most somatic cells (contributes to cellular aging and the Hayflick limit).
◦ High Activity: Germline cells, stem cells, and many cancer cells (allows for continued replication/
immortality).
• Clinical Significance:
◦ Decreased Activity: Can lead to premature aging syndromes (e.g., Progeria).
◦ Increased Activity: Contributes to the uncontrolled proliferation of cancer cells (making telomerase
a potential anti-cancer drug target).
• Hayflick Limit: The finite number of times a normal human somatic cell population will divide (around
50 times) before cell division stops, largely due to telomere shortening. Cells with active telomerase (like
cancer cells) can bypass this limit.
VIII. Prokaryotic vs. Eukaryotic DNA Replication Differences
 Feature Prokaryotes (e.g., E. coli) Eukaryotes (e.g., Humans)

Origin (Ori) Single Multiple

SSB Protein SSB RPA (Replication Protein A)

Primase DnaG protein DNA Polymerase α (alpha) subunit

Main DNA Pol DNA Polymerase III DNA Pol ε (epsilon - leading), δ (delta - lagging)

Primer Removal DNA Polymerase I (5'→3' exonuclease) RNase H & FEN1 (Flap Endonuclease 1)

Okazaki Frag. Longer (~1000-2000 nt) Shorter (~100-200 nt)

Genome Usually circular Linear chromosomes (with telomeres)

Speed Faster (~1000 nt/sec) Slower (~50-100 nt/sec)

IX. Important Points to Remember

• Replication is semi-conservative, proceeds 5' to 3', and is bi-directional.

• It is semi-discontinuous due to the leading (continuous) and lagging (discontinuous - Okazaki
fragments) strands.
• Key enzymes: Helicase (unwinds), Primase (RNA primer), DNA Polymerase (synthesis &
proofreading), Ligase (seals nicks), Topoisomerase (relieves strain), SSB/RPA (stabilizes ssDNA).
• DNA Pol III (prok) and Pol δ/ε (euk) are the main replicative polymerases.
• DNA Pol I (prok) removes primers; RNase H/FEN1 (euk) remove primers.
• Proofreading (3'→5' exonuclease activity) is crucial for fidelity.
• Major repair pathways (NER, BER, MMR, NHEJ, HR) correct specific types of DNA damage; defects
cause diseases (XP, Lynch Syndrome, SCID, BRCA cancers).
• Telomerase solves the end replication problem in eukaryotes with linear chromosomes; its activity is
linked to aging and cancer.
• The Hayflick limit restricts somatic cell divisions, partly due to telomere shortening.

Transcription Summary
1. Definition & Core Concept

• Transcription: The process of synthesizing RNA from a DNA template.

• Analogy: Similar to medical transcription (e.g., "edema" to "swelling"), the language (nucleotides)
remains the same (DNA uses deoxyribonucleotides, RNA uses ribonucleotides), but the form changes.
• Key Focus Areas: Enzymes, Promoters, Salient Features, Post-transcriptional Modifications.

2. Salient Features of Transcription

• Template: Only one DNA strand serves as the template for RNA synthesis (unlike DNA replication
where both strands are templates).
◦ Template Strand: Also called Minus (-) strand or Antisense strand.
◦ Non-Template Strand: Also called Coding strand, Plus (+) strand, or Sense strand.
• Direction of Synthesis: RNA is always synthesized in the 5' → 3' direction.
 • Primer: RNA Polymerase does not require a primer to initiate synthesis (unlike DNA Polymerase).
• RNA Sequence Relationship:
◦ The newly synthesized RNA sequence is complementary to the template strand.
◦ The RNA sequence is identical to the coding (non-template) strand, with the exception that Uracil
(U) replaces Thymine (T).
◦ Example (based on principle):
▪ Template (3'→5'): ... A T G C ...
▪ RNA (5'→3'): ... U A C G ...
▪ Coding (5'→3'): ... A T G C ...

3. Enzymes for Transcription: RNA Polymerase

A. Prokaryotic RNA Polymerase

• Type: Only one type exists.

• Structure: Multi-subunit enzyme.
◦ Core Enzyme: Composed of α₂, β, β', and ω subunits. Responsible for polymerization.
◦ Sigma (σ) Factor: Binds to the core enzyme to form the Holoenzyme.
▪ Function of σ Factor: Crucial for recognizing and binding to the promoter site on DNA,
ensuring transcription starts at the correct location.
◦ Function of β Subunit: Contains the catalytic site for RNA synthesis (addition of ribonucleotides).

B. Eukaryotic RNA Polymerase

• Types: Three distinct types (I, II, III).

• Distinction: Based on sensitivity to α-amanitin (a mushroom poison).
◦ Sensitivity Mnemonic: 2 > 3 > 1 (Pol II is most sensitive, Pol III is intermediate, Pol I is least
sensitive).
• Products Synthesized:
◦ RNA Polymerase I: Synthesizes most rRNA (ribosomal RNA - the most abundant RNA).
▪ Location: Nucleolus
◦ RNA Polymerase II: Synthesizes mRNA (messenger RNA), miRNA (microRNA), snRNA (small
nuclear RNA), lncRNA (long non-coding RNA).
▪ Mnemonic (Burger Analogy): Pol I & III are the buns, Pol II synthesizes all the diverse 'fillings' in
the middle (mRNA, miRNA etc.).
◦ RNA Polymerase III: Synthesizes tRNA (transfer RNA), 5S rRNA, and some snRNAs.
▪ Mnemonic: 't' for tRNA, 't' for third (Pol III).

4. Promoters for Transcription

• Definition: Specific, conserved DNA sequences located on the coding strand (usually) that signal the
start site (+1) of transcription. The promoter region encompasses sequences on both strands.
• Location: Typically located upstream (before the +1 start site, indicated by negative numbers like -10,
-35). Downstream elements are indicated by positive numbers.
• Nomenclature: Often referred to as "boxes" or "elements".
• Transcription: Promoters themselves are generally not transcribed into RNA.
A. Prokaryotic Promoters

• Pribnow Box: Located around the -10 position (upstream). Consensus sequence often TATAAT.
• -35 Sequence: Located around the -35 position (upstream). (Text mentions TGG Box, common
consensus is TTGACA).

B. Eukaryotic Promoters

• TATA Box (Hogness Box): Located around the -25 position (upstream). Similar to Pribnow box.
Recognized by TATA-binding protein (TBP), part of TFIID.
• CAAT Box: Located around the -75 position (upstream). Influences promoter efficiency.
• GC Box: Variable location (upstream), often found in housekeeping genes. Rich in Guanine and
Cytosine.
• Promoterless Sequences: Some genes lack typical promoters and rely on:
◦ Initiator Sequence (INR): Surrounds the +1 start site.
◦ Downstream Promoter Element (DPE): Located downstream of the start site.
• Exception: Some promoters (e.g., for RNA Pol III genes, some used by Pol II with DPE) can be located
downstream (intragenic) and are transcribed.

5. Transcription Process (Cycle Overview)

1. Template Binding: RNA Polymerase (holoenzyme in prokaryotes, requires transcription factors in

eukaryotes) binds to the promoter sequence on DNA, forming a Closed Promoter Complex.
2. Promoter Opening (DNA Unwinding): The DNA strands separate around the start site, forming an
Open Promoter Complex and a transcription bubble.
3. Chain Initiation: RNA Polymerase synthesizes the first few ribonucleotides. In prokaryotes, the sigma
factor dissociates after initiation.
4. Chain Elongation: RNA Polymerase moves along the template strand (reading 3'→5'), synthesizing the
RNA transcript (5'→3') by adding complementary ribonucleotides. The transcription bubble moves with
the polymerase.
5. Chain Termination: RNA synthesis stops at a specific termination signal, and the RNA transcript is
released.

6. Termination of Transcription
A. Rho (ρ)-Dependent Termination (Prokaryotes)

• Requires the Rho (ρ) factor protein.

• Rho factor is an ATP-dependent RNA-DNA helicase.
• It binds to a specific recognition site (rut site) on the nascent RNA transcript and moves towards the 3'
end.
• When Rho catches up to the paused RNA Polymerase at the termination site, it uses its helicase activity
to unwind the RNA-DNA hybrid, releasing the RNA transcript.

B. Rho (ρ)-Independent Termination (Intrinsic Termination - Prokaryotes)

• Does not require Rho factor.

 • Relies on specific sequences in the DNA template strand which are transcribed into RNA:
◦ A GC-rich region that forms a stable hairpin loop structure in the nascent RNA through intra-strand
base pairing.
◦ This is followed immediately by a sequence of Uracils (U) at the 3' end of the RNA (transcribed from
an A-rich region in the DNA template).
• Mechanism: The hairpin formation destabilizes/pauses the RNA Polymerase, and the relatively weak
bonds between the U's in the RNA and A's in the DNA template facilitate the dissociation of the RNA
transcript.

7. Post-Transcriptional Modifications

• Prokaryotes: Most RNAs (rRNA, tRNA) undergo modification, but mRNA generally does not.
Transcription and translation are often coupled.
• Eukaryotes: All types of RNA (rRNA, tRNA, mRNA) undergo processing/modification. For mRNA,
this occurs primarily in the nucleus before export to the cytoplasm for translation.
• Primary Transcript (Eukaryotes): The initial unmodified RNA molecule is called hnRNA
(heteronuclear RNA).

A. Eukaryotic mRNA Modifications

1. 5' Capping:
◦ What: Addition of a 7-methylguanosine (7-mG) cap to the 5' end via an unusual 5'-5' triphosphate
linkage.
◦ Process:
▪ Removal of one phosphate from the 5' end of hnRNA.
▪ Addition of GMP (from GTP) by guanylyltransferase (in nucleus).
▪ Methylation of the guanine base at position N7 by methyltransferase using SAM (S-adenosyl
methionine) as the methyl donor (in cytoplasm).
◦ Functions:
▪ Protects mRNA from degradation by 5' exonucleases.
▪ Enhances mRNA stability.
▪ Required for efficient export from the nucleus.
▪ Essential for binding to the ribosome (initiation of translation) via cap-binding proteins (e.g.,
eIF4E).
2. 3' Polyadenylation (Poly-A Tailing):
◦ What: Addition of a "tail" of 40-200 adenine nucleotides (poly-A tail) to the 3' end.
◦ Process:
▪ hnRNA is cleaved downstream of a consensus sequence (AAUAAA).
▪ The enzyme Polyadenylate Polymerase (PAP) adds the adenine residues one by one, using ATP
as a precursor. This process does not require a DNA template.
◦ Functions:
▪ Protects mRNA from degradation by 3' exonucleases.
▪ Enhances mRNA stability.
▪ Aids in export from the nucleus.
 ▪ Helps in initiation of translation (PABP binds the tail and interacts with initiation factors at the
5' cap).
3. Splicing:
◦ What: Removal of non-coding intervening sequences (introns) and joining together of coding
sequences (exons).
◦ Machinery: Spliceosome.
▪ Composed of snRNAs (U1, U2, U4, U5, U6) complexed with proteins to form snRNPs (small
nuclear ribonucleoproteins, pronounced "snurps").
▪ snRNAs are catalytic (ribozymes).
◦ Mechanism:
▪ snRNPs recognize conserved sequences at the 5' splice site (donor), 3' splice site (acceptor), and
an internal branch point Adenine within the intron.
▪ Two transesterification reactions occur:
▪ The 2'-OH of the branch point A attacks the 5' splice site, cleaving it and forming a loop
structure called a lariat.
▪ The freed 3'-OH of the upstream exon attacks the 3' splice site, joining the two exons together
and releasing the intron lariat, which is then degraded.
◦ Clinical Link: Autoantibodies against snRNPs are found in Systemic Lupus Erythematosus (SLE).
◦ Self-Splicing Introns: Some introns (Group I and Group II) can catalyze their own removal without
a spliceosome. Discovered by Thomas Cech. Group II forms a lariat; Group I does not. No ATP is
needed for self-splicing itself.
◦ Exon Percentage: Exons constitute only about 1.14% of the human genome.
4. Alternate mRNA Processing (Differential Splicing):
◦ What: Different combinations of exons from the same hnRNA gene transcript can be spliced
together.
◦ Result: A single gene can produce multiple different mRNA molecules, leading to the synthesis of
different protein isoforms.
◦ Significance: Increases the coding capacity of the genome. Important for generating tissue-specific
proteins or different functional forms (e.g., membrane-bound vs. secreted immunoglobulins).

8. RNA Editing

• What: A process that changes the nucleotide sequence of an mRNA molecule after transcription and
processing, but before translation.
• Significance: An exception to the central dogma (information flow usually DNA → RNA → Protein,
without RNA sequence alteration). Occurs in approx. 0.01% of human mRNAs.
• Example: ApoB Gene
◦ Liver: ApoB gene → transcribed → ApoB mRNA → translated → ApoB100 protein (full length).
◦ Intestine: ApoB gene → transcribed → ApoB mRNA → RNA Editing Occurs: A specific Cytosine
(C) in a CAA codon is chemically modified to Uracil (U) by the enzyme Cytosine Deaminase. →
Codon changes from CAA (Glutamine) to UAA (Stop Codon). → Translation terminates prematurely
→ ApoB48 protein (truncated).

9. Miscellaneous Facts

• Histone mRNA: An exception - typically lacks both introns and a poly-A tail.
 • Homopolymer Codons:
◦ Poly-A (AAA): Codes for Lysine
◦ Poly-G (GGG): Codes for Glycine
◦ Poly-U (UUU): Codes for Phenylalanine
◦ Poly-C (CCC): Codes for Proline
• Site of Modifications: Most post-transcriptional modifications occur in the nucleus. (Exception: 5' cap
methylation occurs in the cytoplasm).
• snoRNA (small nucleolar RNA): Involved in the processing and modification of rRNA within the
nucleolus.

10. Key Points to Remember

• Transcription uses one DNA strand as a template, synthesizes RNA 5'→3', and requires no primer.
• RNA sequence mirrors the coding strand (with U for T).
• Prokaryotes: One RNA Pol, Sigma factor for promoter binding, Rho-dependent/independent
termination. mRNA not typically processed.
• Eukaryotes: Three RNA Pols (I, II, III) with distinct roles and sensitivities (α-amanitin: 2>3>1).
Extensive mRNA processing (5' cap, 3' poly-A tail, splicing).
• Promoters (Pribnow/-10, -35 in prokaryotes; TATA/-25, CAAT/-75, GC in eukaryotes) direct RNA Pol
binding upstream of the start site.
• Splicing removes introns via the spliceosome (snRNPs), forming a lariat structure. Alternate splicing
generates protein diversity.
• RNA Editing (e.g., ApoB C→U deamination) modifies mRNA sequence post-transcriptionally, creating
different proteins.
• 5' Cap & 3' Poly-A Tail: Crucial for mRNA stability, export, and translation initiation.
Okay class, let's summarize the key aspects of Translation, the third process in the Central Dogma.

Translation: The Basics

1. Definition: Translation is the process where the genetic information encoded in the language of
nucleotides (specifically mRNA) is converted into the language of amino acids (forming a protein).
◦ Analogy: Translating French (one alphabet/language) to English (another alphabet/language).
◦ Formal Definition: The process by which protein is synthesized from an mRNA template.
2. Location: Translation occurs in the ribosomes. These can be:
◦ Attached to the Rough Endoplasmic Reticulum (RER).
◦ Free ribosomes in the cytoplasm.

Prerequisites for Understanding Translation

Before diving into the steps, we need to understand three key components:

1. The Genetic Code

• Concept: The relationship between the sequence of nucleotides in mRNA (derived from DNA) and the
sequence of amino acids in a protein.
• Discoverers: Cracked by Sir Marshall Nirenberg and Har Gobind Khorana.
• Cistron: The smallest unit of genetic expression.
◦ Monocistronic: One cistron codes for one polypeptide (common in Eukaryotes).
◦ Polycistronic: One cistron codes for more than one polypeptide (common in Prokaryotes).
▪ Mnemonic: PE for PE (Prokaryotes are Polycistronic).
• Codon: A sequence of nucleotides that specifies an amino acid.
◦ Triplet Nature: Based on the need to code for ~20 amino acids using 4 nucleotide bases (A, U, G, C
in mRNA):
▪ 1 base/codon -> 4^1 = 4 possibilities (Insufficient)
▪ 2 bases/codon -> 4^2 = 16 possibilities (Insufficient)
▪ 3 bases/codon -> 4^3 = 64 possibilities (Sufficient) - Hence, codons are triplets.
◦ Total Codons: 64
◦ Stop (Terminator) Codons (3): Signal the end of translation.
▪ UAA (Ochre)
▪ UGA (Opal)
▪ UAG (Amber)
▪ Mnemonics:
▪ U Go Away (UGA), U Are Gone (UAG), U Are Away (UAA)
▪ UGA = U Got an Opal
▪ UAG = AG (like Silver/Fire) = Amber
▪ UAA = Ochre (remaining one)
◦ Coding Codons (61): Specify amino acids.
◦ Start (Initiator) Codon (1): AUG.
▪ Codes for Methionine in Eukaryotes.
▪ Codes for N-formylmethionine (fMet) in Prokaryotes.
▪ Signals the beginning of translation.
◦ Exceptions to Stop Codons:
▪ UGA can code for Selenocysteine.
▪ UAG can code for Pyrrolysine.
• Salient Features of the Genetic Code:
◦ Degenerate (Redundant): More than one codon can specify the same amino acid (e.g., UUU and
UUC both code for Phenylalanine). Degeneracy often occurs at the third base of the codon.
▪ AAs coded by only one codon: Methionine (AUG), Tryptophan (UGG).
▪ AAs coded by the most codons (6 each): Serine, Leucine, Arginine.
◦ Unambiguous: One specific codon will always code for the same amino acid.
◦ Non-overlapping: Codons are read sequentially in groups of three without sharing bases (e.g., AUG
UUU GGG is read as AUG, then UUU, then GGG).
◦ Non-punctuated (Comma-less): The code is read continuously from the start codon to the stop
codon without skipping any bases.
◦ Universal: The same codons specify the same amino acids in almost all organisms, from bacteria to
humans.
▪ Exception: Mitochondrial DNA shows some variations.
2. Ribosomes (The Translating Machinery)

• Composition: A complex of ribosomal RNA (rRNA) and proteins.

• Eukaryotic Ribosome (80S):
◦ Composed of two subunits: 60S (large) and 40S (small).
◦ Contains 4 types of rRNA: 28S, 18S, 5.8S, and 5S.
◦ Contains approximately 80 different proteins.
◦ Subunit Composition:
▪ 40S Subunit: Contains 18S rRNA + ~30-33 proteins.
▪ 60S Subunit: Contains 28S, 5.8S, 5S rRNA + ~50 proteins.

3. Transfer RNA (tRNA - The Adapter Molecule)

• Aliases: Soluble RNA (sRNA) - Do not confuse with snRNA (small nuclear RNA).
• Structure:
◦ Secondary Structure: Cloverleaf shape.
◦ Tertiary Structure: L-shape.
◦ Size: 74-95 nucleotides long.
◦ Contains unusual bases (e.g., Dihydrouracil, Pseudouridine, Ribothymidine, Hypoxanthine). tRNA is
unique among RNAs for containing Thymine (as Ribothymidine).
• Key Arms/Regions (Cloverleaf Model):
◦ Acceptor Arm: At the 3' end, always ends in CCA (unpaired). Function: Attaches to a specific
amino acid.
◦ Anticodon Arm: Contains a 3-base anticodon. Function: Base-pairs complementarily with the
corresponding mRNA codon.
◦ DHU Arm (D Arm): Contains Dihydrouracil. Function: Recognized by and binds to the specific
aminoacyl-tRNA synthetase enzyme.
◦ TΨC Arm (Pseudouridine Arm): Contains Pseudouridine and Ribothymidine. Function: Binds to
the ribosome.
• Function: Acts as an adapter molecule. It reads the codon on the mRNA via its anticodon and carries the
corresponding specific amino acid to the ribosome for protein synthesis. Ensures the correct amino acid is
added based on the mRNA sequence.
• Wobble Hypothesis (Crick):
◦ Explains how ~31 tRNAs can recognize 61 coding codons.
◦ The base pairing between the third base of the mRNA codon and the first base of the tRNA
anticodon does not always follow strict Watson-Crick rules (A-U, G-C).
◦ This "wobble" allows a single tRNA to recognize multiple codons that code for the same amino acid
(often differing only in the 3rd base).

Steps of Translation
Translation occurs in four main stages:

1. Charging of tRNA (Aminoacylation)

• Process: Attaching the correct amino acid to the 3' CCA end of its specific tRNA.
 • Enzyme: Aminoacyl-tRNA synthetase (there's a specific synthetase for each amino acid). The enzyme
recognizes the correct tRNA (via DHU arm) and the correct amino acid.
• Energy: Requires ATP. ATP is hydrolyzed to AMP + PPi (using two high-energy phosphate bonds).

2. Initiation

• Goal: Assemble the translation machinery (ribosome subunits, mRNA, initiator tRNA) at the correct start
codon.
• Identifying the Start Codon (AUG): The ribosome finds the first AUG codon after a specific
recognition sequence on the mRNA.
◦ Prokaryotes: Shine-Dalgarno sequence.
◦ Eukaryotes: Kozak consensus sequence.
• Eukaryotic Initiation (Simplified Overview):
◦ Ribosomal subunits (80S) dissociate into 40S and 60S (aided by eukaryotic Initiation Factors - eIFs
like eIF3, eIF1A).
◦ A ternary complex (GTP + eIF2 + initiator Met-tRNA) forms.
◦ Ternary complex binds to the 40S subunit -> 43S pre-initiation complex.
◦ 43S complex binds mRNA and scans for the Kozak-associated AUG -> 48S initiation complex.
◦ The 60S subunit joins -> 80S initiation complex. GTP is hydrolyzed, eIFs are released.
• Result: The ribosome is assembled on the mRNA, with the initiator Met-tRNA positioned in the P site.

3. Elongation

• Goal: Add amino acids one by one to the growing polypeptide chain according to the mRNA sequence.
Requires Elongation Factors (EFs).
• Ribosome Sites:
◦ A site (Aminoacyl): Entry site for new charged tRNAs.
◦ P site (Peptidyl): Holds the tRNA carrying the growing polypeptide chain.
◦ E site (Exit): Site where the uncharged tRNA leaves the ribosome.
• Elongation Cycle:
◦ (a) Binding of Aminoacyl-tRNA: A charged tRNA with an anticodon complementary to the mRNA
codon in the A site enters. Requires eEF1 (in eukaryotes) and GTP hydrolysis.
◦ (b) Peptide Bond Formation: The amino acid (or peptide chain) on the tRNA in the P site is
transferred and covalently linked to the amino acid on the tRNA in the A site.
▪ Catalyzed by peptidyltransferase, an enzymatic activity (ribozyme) of the 28S rRNA within the
60S subunit.
▪ No ATP/GTP is consumed in this specific step.
◦ (c) Translocation: The ribosome moves one codon down the mRNA (in the 5' to 3' direction).
Requires eEF2 (in eukaryotes) and GTP hydrolysis.
▪ The tRNA that was in the A site (now carrying the peptide chain) moves to the P site.
▪ The uncharged tRNA that was in the P site moves to the E site and exits.
▪ The A site is now vacant, ready for the next charged tRNA.
• This cycle repeats, extending the polypeptide chain.
4. Termination

• Signal: A stop codon (UAA, UAG, UGA) enters the A site.

• Process: No tRNA recognizes stop codons. Instead, Releasing Factors (RFs) bind to the A site.
• Requirements: RFs, GTP hydrolysis, and peptidyltransferase activity (helps release the polypeptide).
• Result:
◦ The completed polypeptide chain is cleaved from the tRNA in the P site and released.
◦ The ribosome subunits, mRNA, tRNA, and RFs dissociate.

Energetics of Translation
• Per Peptide Bond Formed (during elongation cycle):
◦ Charging (Aminoacylation): 1 ATP -> AMP + PPi (2 high-energy bonds)
◦ Binding (tRNA to A site): 1 GTP -> GDP + Pi (1 high-energy bond)
◦ Translocation: 1 GTP -> GDP + Pi (1 high-energy bond)
◦ Total = 4 high-energy phosphate bonds per amino acid added (or peptide bond formed).
• Actual Peptide Bond Formation Step: Requires 0 ATP/GTP.
• Initiation & Termination: Also require GTP, but are not counted in the "per peptide bond" cost as they
occur only once per polypeptide synthesis.

Classification of RNA
RNAs can be broadly classified based on whether they code for protein:

1. Protein Coding RNA:

◦ mRNA (messenger RNA): The only type that carries the code for protein synthesis.
2. Non-Protein Coding RNA (ncRNA): Perform various structural, catalytic, and regulatory roles.
◦ Large ncRNAs:
▪ rRNA (ribosomal RNA): Major components of ribosomes (e.g., 28S, 18S).
▪ lncRNA (long non-coding RNA): >200 nucleotides, involved in gene regulation.
▪ circRNA (circular RNA): Role in gene expression regulation.
◦ Small ncRNAs:
▪ rRNA (ribosomal RNA): Smaller components (5.8S, 5S).
▪ tRNA (transfer RNA): Adapter molecule in translation.
▪ snRNA (small nuclear RNA): Component of spliceosomes (involved in mRNA splicing).
▪ miRNA (microRNA): Regulate gene expression post-transcriptionally.
▪ siRNA (small interfering RNA): Similar to miRNA, often exogenous origin, used in RNA
interference.

Details on Specific ncRNAs

miRNA (microRNA) & siRNA (small interfering RNA)

• Shared Features:
◦ Small (~21-22 nucleotides), single-stranded, non-coding RNAs.
◦ Function: Post-transcriptional gene silencing via RNA interference (RNAi).
 ◦ Mechanism:
▪ Processed by enzymes like Dicer.
▪ Loaded into the RISC (RNA-Induced Silencing Complex) containing Argonaut proteins.
▪ Guide RISC to target mRNAs based on sequence complementarity (often in the 3' UTR).
▪ Outcome:
▪ Perfect complementarity: mRNA degradation/cleavage.
▪ Imperfect complementarity: Translational repression (mRNA not translated).
▪ Net effect: Reduced protein production from the target gene (gene silencing).
• Key Difference:
◦ miRNA: Endogenous origin (encoded by the organism's own genome, processed from pri-miRNA ->
pre-miRNA -> mature miRNA by Drosha & Dicer). Involved in normal development and disease (can
be oncogenic - oncomirs - or tumor suppressive). Discovered by Craig Mello & Andrew Fire.
◦ siRNA: Typically Exogenous origin (e.g., from viral RNA, or experimentally introduced dsRNA).
Processed by Dicer. Primarily leads to mRNA cleavage due to often perfect complementarity. Used as
a research tool and potential therapeutic strategy.

lncRNA (long non-coding RNA)

• Definition: ncRNAs > 200 nucleotides (produced by RNA Pol II).

• Function: Diverse roles in gene expression regulation, often via epigenetic mechanisms.
• Mechanisms:
◦ Activate genes: By helping transcription factors bind.
◦ Repress genes: By sequestering transcription factors (acting as 'decoys') or guiding inhibitory
complexes to DNA/histones.
◦ Modify chromatin: By directing histone-modifying enzymes (methylases, acetylases) to specific
genomic locations.

Key Points to Remember:

• Translation: mRNA nucleotide sequence -> Amino acid polypeptide sequence. Occurs on ribosomes.
• Genetic Code: Triplet, Degenerate, Unambiguous, Non-overlapping, Comma-less, Universal (mostly).
◦ Start: AUG (Met/fMet)
◦ Stop: UAA, UAG, UGA
• tRNA: Adapter molecule with Anticodon (binds mRNA codon) and Acceptor Arm (binds amino acid).
Needs charging by aminoacyl-tRNA synthetase (uses ATP). Wobble allows fewer tRNAs.
• Ribosome (Eukaryotic 80S = 60S + 40S): Site of translation with A, P, E sites. Peptidyltransferase
(28S rRNA activity) forms peptide bonds.
• Translation Steps: Initiation (finding AUG, assembly), Elongation (cycle of tRNA binding, peptide bond
formation, translocation), Termination (stop codon recognition by RFs).
• Energetics: 4 high-energy bonds (2 ATP equiv. for charging, 2 GTP for binding/translocation) per
peptide bond. Actual bond formation = 0 energy cost.
• RNA Types: Only mRNA codes protein. ncRNAs (rRNA, tRNA, snRNA, miRNA, siRNA, lncRNA,
circRNA) have structural, catalytic, or regulatory roles.
• RNAi: Gene silencing by miRNA (endogenous) or siRNA (exogenous) via RISC complex targeting
mRNA.
 • lncRNA: Regulate gene expression through various mechanisms including chromatin modification.

Trust yourself, trust your preparation! Good luck.

# Regulation of Gene Expression Summary

## I. Introduction & Concept

* **Regulation Concept:** The same gene can be expressed differently depending on the situation or ce
* *Example:* Insulin gene exists in liver and pancreas but is only expressed in the pancreas.
* **Gene Expression (Central Dogma):** DNA (Gene) -> Transcription -> mRNA -> Translation -> Protein.
* **Regulation Goal:** Control which genes are expressed, when, and how much.

## II. Levels of Regulation

Gene expression can be regulated at multiple stages:

1. **DNA/Gene Level:**
* Gene Amplification
* Gene Rearrangement
* Epigenetic Modifications
* Transposons
* Gene Switching
2. **Transcription Level:**
* Induction & Repression (Operon Concept)
3. **mRNA Level (Post-Transcription):**
* RNA Editing
* Alternate RNA Processing
* RNA Interference (RNAi)
*(Note: mRNA level regulations are covered in separate topics as per the lecture)*

## III. Regulation at Transcription Level: The Operon Concept

* **Types of Genes:**
* **Housekeeping (Constitutive) Genes:** Expressed constantly at a basal level (e.g., Hexokinase
* **Inducible Genes:** Expressed only in response to a specific stimulus (e.g., Glucokinase, indu
* **Operon:** An array of genes coordinately regulated, often with a related function (e.g., metaboli
* Concept by: **Francois Jacob & Jacques Monod**.
* **Lac Operon (E. coli - Lactose Metabolism):**
* **Components:**
* **`lacI` Gene:** Codes for the **Repressor protein** (Inhibitor). This gene is **constituti
* **Promoter Site (P):** Binding site for **RNA Polymerase**.
* **Operator Site (O):** Binding site for the **Repressor protein**. Binding blocks RNA polym
* **Structural Genes (Inducible):**
* **`lacZ`:** Codes for **β-galactosidase** (breaks lactose into glucose + galactose). *A
* **`lacY`:** Codes for **Permease** (allows lactose entry into the cell).
* **`lacA`:** Codes for **Thiogalactoside transacetylase** (function unknown).
* **Default State (No Lactose):** `lacI` produces active repressor -> binds Operator -> RNA Polym
* **Catabolite Repression (Glucose Effect):** E. coli prefers glucose over lactose.
* **Mediator:** **Catabolite Activator Protein (CAP)**, also called CRP.
* **Activation:** CAP is active **only when bound to cyclic AMP (cAMP)**.
* **cAMP Levels:**
* **Glucose Absent:** High cAMP -> cAMP binds CAP -> **Active CAP**.
* **Glucose Present:** Low cAMP -> cAMP doesn't bind CAP -> **Inactive CAP**.
* **CAP Function:** Active CAP is a **positive regulator** of the lac operon (enhances transcript
* *Mnemonic:* **CAP** needs a **cAMP** to put its **CAP** on (bind DNA) and **A**ctivate transcri
* **Lac Operon Scenarios:**
1. **Glucose PRESENT, Lactose ABSENT:**
* *Repressor:* Active (no lactose to inactivate it) -> binds Operator.
* *CAP:* Inactive (glucose present -> low cAMP).
* *Result:* **Lac Operon OFF** (double block: repressor bound, no positive activation by CAP)
2. **Glucose ABSENT, Lactose PRESENT:**
* *Repressor:* Inactive (lactose binds to it).
* *CAP:* Active (glucose absent -> high cAMP).
* *Result:* **Lac Operon ON** (Repressor removed, CAP actively promotes transcription).
 3. **Glucose PRESENT, Lactose PRESENT:**
* *Repressor:* Inactive (lactose binds to it).
* *CAP:* Inactive (glucose present -> low cAMP).
* *Result:* **Lac Operon OFF** (or very low expression). Although the repressor is off, the l
* **Isopropyl β-D-1-thiogalactopyranoside (IPTG):** A lactose analog that can bind and inactivate the

## IV. Regulation at DNA Level (Other Mechanisms)

* **Gene Amplification:** Increasing the copy number of a specific gene.

* *Mechanism:* More gene copies available for transcription -> increased protein production.
* *Example:* Methotrexate resistance. Methotrexate inhibits Dihydrofolate Reductase (DHFR). Prolo
* **Gene Rearrangement:** Bringing different gene segments together in various combinations to create
* *Mechanism:* DNA segments are physically rearranged. This occurs at the **DNA level**, distinct
* *Example:* Immunoglobulin (antibody) variable chain diversity. V, D, and J gene segments recomb
* **Transposons ("Jumping Genes"):** Mobile DNA sequences that can move from one location to another
* *Discoverer:* Barbara McClintock.
* *Enzyme:* Transposase.
* *Effect:* Can alter gene expression by inserting into or near genes, or by carrying genes/regul
* *Classes:*
* **Class 1 (Retrotransposons):** Move via an **RNA intermediate** (DNA -> RNA -> Reverse Tra
* **Class 2 (DNA Transposons):** Move as **DNA** directly (cut/copy and paste mechanism). Con

## V. Regulation at DNA Level: Epigenetics

* **Definition:** Reversible, heritable changes in gene expression that occur **without altering the
* *Analogy:* Playing different music (gene expression) on the same piano (DNA sequence) by pressi
* **Epigenome:** The complete set of epigenetic modifications across the genome.
* **Types of Epigenetic Modifications:**

1. **DNA Modification: DNA Methylation**

* *Site:* **Cytosine** residues, typically in **CpG islands** (C followed by G, linked by pho
* *Enzyme:* **DNA Methyltransferase (DNMT)**.
* *Methyl Donor:* **S-Adenosyl Methionine (SAM)**.
* *Effect:* Generally leads to **gene silencing / decreased expression**. Promotes **heteroch
* *Inhibitors (Drugs):* Azacitidine, Decitabine (5-aza-2'-deoxycytidine).

2. **Histone Modifications (Post-Translational Modifications):** Changes to the histone proteins a

* **Histone Acetylation:**
* *Site:* **Lysine** residues on histone tails.
* *Enzyme:* **Histone Acetyltransferase (HAT)**.
* *Effect:* Adds negative acetyl group -> neutralizes positive charge of lysine -> loosen
* *Mnemonic:* **HAT**s open up the chromatin for expression.
* **Histone Deacetylation:**
* *Site:* Removes acetyl groups from lysine.
* *Enzyme:* **Histone Deacetylase (HDAC)**.
* *Effect:* Restores positive charge -> tightens DNA-histone interaction -> **heterochrom
* *Inhibitors (Drugs):* Vorinostat, Valproic Acid.
* *Mnemonic:* **HDAC**s hide the DNA, decreasing expression.
* **Other Histone Modifications:**
* *Phosphorylation:* H3 -> dual effect; H1 -> condensation.
* *Methylation:* Dual effect (can activate or repress depending on site/degree).
* *ADP-ribosylation:* Involved in DNA repair.
* *Mono-ubiquitination:* Increase or decrease expression.
* *SUMOylation:* (Small Ubiquitin-related Modifier) -> Chromatin condensation (decreased

* **Physiological Roles of Epigenetics:**

* Normal gene regulation.
* **Genomic Imprinting:** Differential expression of a gene depending on whether it was inherited
* Aging process.
* Embryogenesis & Development (e.g., switching hemoglobin types).
* **X-Chromosome Inactivation:** Silencing of one X chromosome in females.

* **Pathological Consequences (Aberrant Epigenetics):**

* **Fragile X Syndrome:** Hypermethylation and silencing of the *FMR1* gene.
* **Cancer:**
* Hypomethylation (less methylation) of oncogenes -> activation -> cancer promotion.
* Hypermethylation (more methylation) of tumor suppressor genes -> silencing -> cancer promot
 * HDACs are often overexpressed in cancers (drugs like Vorinostat target this).
* **Prader-Willi & Angelman Syndromes:** Result from deletion or defect in an imprinted region on

* **Detection Methods for Epigenetic Modifications:**

* **Methylation-Specific PCR (MSP):** Detects methylation status of specific CpG sites.
* **Chromatin Immunoprecipitation (ChIP):** Uses antibodies to isolate DNA regions bound to speci
* **ChIP-seq:** Sequencing the isolated DNA.
* **ChIP-on-chip:** Hybridizing the isolated DNA to a microarray.
* **Bisulfite Sequencing:** Treats DNA with sodium bisulfite, which converts **unmethylated** cyt

## VI. Important Points to Remember

* Gene expression is tightly regulated at multiple levels (DNA, transcription, post-transcription).

* The **Lac Operon** is a classic example of transcriptional regulation (induction/repression) in pro
* **Catabolite repression** (via CAP/cAMP) ensures glucose is prioritized over lactose in E. coli.
* **Gene amplification** (e.g., DHFR/Methotrexate) and **gene rearrangement** (e.g., VDJ/Immunoglobul
* **Transposons** are mobile DNA elements that can alter gene expression and cause mutations.
* **Epigenetics** refers to heritable changes in gene function **without** changing the DNA sequence,
* **DNA methylation** (at CpG islands, by DNMTs) typically **silences** genes.
* **Histone acetylation** (by HATs) typically **activates** genes (euchromatin).
* **Histone deacetylation** (by HDACs) typically **silences** genes (heterochromatin).
* Epigenetic misregulation is implicated in diseases like **cancer**, **Fragile X**, **Prader-Willi/A
* Epigenetic marks can be detected by **MSP**, **ChIP-seq/ChIP-on-chip**, and **Bisulfite Sequencing*
* Drugs targeting epigenetic enzymes (DNMT inhibitors like **Azacitidine**, HDAC inhibitors like **Vo

Okay students, here is a detailed summary of the lecture on Hybridization Techniques for your revision.

Hybridization Techniques Summary

This lecture covered several key molecular biology techniques based on the principle of hybridization
(binding of complementary nucleic acid strands) or specific molecular interactions (antigen-antibody).

1. Blotting Techniques
Blotting involves separating molecules (DNA, RNA, or Protein) by electrophoresis, transferring (blotting)
them onto a solid membrane, and then detecting a specific molecule using a labeled probe.

a) Southern Blot

• Named After: E.M. Southern (1975).

• Target Molecule: DNA.
• Principle: DNA-DNA Hybridization.
• Probe: Labeled (radioactive or fluorescent) single-stranded DNA probe, complementary to the target
DNA sequence.
• Key Steps:
1. Isolate DNA from the sample (e.g., patient blood containing human and potentially bacterial DNA).
2. Fragment DNA using Restriction Endonucleases.
3. Separate DNA fragments by size using Agarose Gel Electrophoresis.
4. Denature DNA (make single-stranded) using NaOH.
5. Blot (Transfer) the single-stranded DNA fragments from the gel to a Nitrocellulose or Nylon
membrane.
6. Hybridize the membrane with the labeled DNA probe. The probe binds only if the complementary
target DNA sequence is present.
7. Wash away unbound probe.
 8. Detect the bound probe (e.g., using Autoradiography for radioactive labels or fluorescence detection).
• Example Application: Detecting Mycobacterium tuberculosis DNA in a suspected TB patient's blood
sample.
• Other Applications:
◦ Detecting viral or bacterial DNA pathogens.
◦ Screening for inborn errors of metabolism (detecting gene mutations, large deletions, single gene
deletions, point mutations).
◦ Forensic medicine (analyzing DNA samples from crime scenes).
• Mnemonic: Southern = DNA (Sun Dial)

b) Northern Blot

• Target Molecule: RNA (specifically mRNA to study gene expression).

• Principle: RNA-DNA Hybridization.
• Probe: Labeled (radioactive or fluorescent) single-stranded cDNA probe (complementary DNA made
from an RNA template using reverse transcriptase), complementary to the target RNA sequence.
• Key Steps:
1. Isolate total RNA from the sample.
2. Separate RNA fragments by size using Agarose Gel Electrophoresis (under denaturing conditions
to prevent secondary structure formation - added for clarity).
3. Blot (Transfer) RNA to a Nitrocellulose or Nylon membrane.
4. Hybridize the membrane with the labeled cDNA probe.
5. Wash away unbound probe.
6. Detect the bound probe.
• Example Application: Detecting HIV viral RNA in a patient sample.
• Applications:
◦ Detecting specific RNA molecules (viral, bacterial, or cellular).
◦ Studying Gene Expression (quantifying the amount of specific mRNA, indicating how active a gene
is).
• Mnemonic: Northern = RNA (No Rain)

c) Western Blot (Immunoblot)

• Target Molecule: Protein (Antigen).

• Principle: Antigen-Antibody Interaction (Immune Reaction).
• Probe: Labeled (e.g., fluorescent or enzyme-linked - added for clarity) Antibody specific to the target
protein. Often uses a primary antibody that binds the target protein, followed by a labeled secondary
antibody that binds the primary antibody for signal amplification - added for clarity.
• Key Steps:
1. Isolate Proteins from the sample.
2. Separate Proteins by size using Protein Electrophoresis (commonly SDS-PAGE - added for
clarity).
3. Blot (Transfer) proteins to a Nitrocellulose or Nylon membrane.
4. Block the membrane to prevent non-specific antibody binding (common step, added for clarity).
 5. Incubate (Hybridize) the membrane with the labeled primary Antibody (or primary then labeled
secondary antibody).
6. Wash away unbound antibody.
7. Detect the bound antibody (fluorescence, chemiluminescence from enzyme reaction, etc.).
• Example Application: Detecting HBsAg (Hepatitis B surface antigen, a protein) in a patient post-
transfusion.
• Applications: Detecting specific proteins in a sample.
• Mnemonic: Western = Protein (Wet Paint)
Combined Mnemonic (Commonly Used): SNOW DROP Southern = DNA Northern = RNA O = O
(Nothing specific / Often skipped) Western = Protein

d) Other Blot Techniques (Briefly Mentioned)

• Southwestern Blot: Detects DNA-Protein interactions. (Overlay Blot)

• Slot Blot / Dot Blot: Samples are applied directly to the membrane in slots/dots without prior
electrophoretic separation. Useful for screening many samples quickly for the presence/absence of a
target.
• Zoo Blot: A type of Southern blot used to compare DNA sequences between different species to study
evolutionary relationships.

2. Microarray Techniques
Microarrays allow for the simultaneous analysis of thousands of targets. Known molecules (probes) are
immobilized in an ordered grid (array) on a small surface (chip/slide), and a labeled sample containing
unknown molecules is hybridized to it.

• Key Concept: Massively parallel analysis. Thousands of known oligonucleotides/cDNAs/proteins are

fixed onto a slide. An unknown, labeled sample is added. Hybridization occurs at spots corresponding to
complementary sequences/interactions. Computer analysis identifies the components of the unknown
sample.
• Advantage over Blots: Can detect thousands of targets simultaneously, whereas blots typically detect
one or a few targets per experiment.

a) DNA Microarray

• Array Contains: Thousands of known single-stranded DNA oligonucleotides.

• Sample Added: Unknown DNA (e.g., genomic DNA fragments), labeled with a fluorescent tag.
• Principle: DNA-DNA Hybridization.
• Applications:
◦ Genotyping: Identifying variations in DNA sequence.
◦ Gene Sequencing: Determining the sequence of the unknown DNA by identifying which known
probes it binds to.

b) cDNA Microarray

• Array Contains: Thousands of known cDNAs (representing specific genes).

• Sample Added: Unknown RNA (typically mRNA converted to labeled cDNA, or directly labeled
RNA), usually labeled with a fluorescent tag.
• Principle: RNA-cDNA Hybridization.
 • Applications:
◦ Gene Expression Profiling: Determining which genes are active (transcribed into mRNA) in a
particular cell type or condition (e.g., comparing cancer tissue vs. normal tissue).
◦ Detecting specific RNAs (e.g., viral RNAs).

c) Protein Microarray

• Array Contains: Thousands of known Proteins (Antigens) OR known Antibodies.

• Sample Added: Unknown Antibodies (if array has antigens) OR Unknown Antigens/Proteins (if array
has antibodies), labeled fluorescently.
• Principle: Antigen-Antibody Interaction.
• Applications:
◦ Proteomics: Studying the entire protein complement of a cell or organism.
◦ Detecting multiple specific proteins or antibodies simultaneously.

d) Array Comparative Genomic Hybridization (aCGH)

• Concept: Compares the entire genome of a test sample against a reference (normal) genome to find
differences in DNA copy number (deletions or duplications/amplifications).
• Array Contains: Genome Chip - Probes representing sequences spanning the entire reference genome.
• Samples Added:
◦ Test Genomic DNA, labeled with one fluorescent color (e.g., Red).
◦ Normal/Reference Genomic DNA, labeled with a different fluorescent color (e.g., Green).
◦ Both samples are added simultaneously to the genome chip.
• Principle: Competitive DNA-DNA Hybridization. The relative amount of red vs. green fluorescence
binding to each spot on the chip indicates the copy number of that specific genomic region in the test
sample compared to the normal sample.
• Interpretation of Spot Color:
◦ Yellow: Equal binding of Red and Green = Normal copy number in the test sample.
◦ Green: Predominant binding of Green = Deletion in the test sample (less Red DNA available to
bind).
◦ Red: Predominant binding of Red = Amplification/Duplication in the test sample (more Red DNA
available to bind).
• Applications:
◦ Detecting gene deletions and gene amplifications (Copy Number Variations - CNVs) across the
genome.
◦ Analyzing genomic abnormalities in complex diseases like cancer, autism, syndromes with
dysmorphic features, diseases with unknown etiology.
• Limitation: Cannot detect balanced structural rearrangements (e.g., balanced translocations where
no net gain or loss of genetic material occurs). Cannot detect point mutations.

3. Fluorescent In Situ Hybridization (FISH)

FISH detects specific DNA sequences within intact cells or chromosomes ("in situ") using fluorescently
labeled probes.

• Concept: Visualize the location of specific genes or DNA sequences directly on chromosomes or within
cells.
 • Principle: DNA-DNA Hybridization using fluorescently labeled DNA probes.
• Key Feature: Maintains morphological context (cell/tissue structure).
• General Steps: Sample preparation -> Denaturation (of DNA within the cell/chromosome) -> Probe
hybridization -> Washing -> Fluorescence microscopy detection.

a) Multicolor FISH (mFISH) / Spectral Karyotyping (SKY)

• Performed On: Metaphase chromosomes (condensed chromosomes from dividing cells).

• Method: Uses combinations of 5 basic fluorescent dyes to create unique "color signatures" for each of
the 23 pairs of human chromosomes.
• Applications:
◦ Detailed karyotyping with color identification for each chromosome.
◦ Detecting numerical abnormalities (e.g., trisomies like Trisomy 21 - three chromosomes of the
same color; monosomies - only one chromosome of a specific color).
◦ Detecting structural abnormalities (e.g., deletions - missing color segment; amplifications -
brighter/larger color segment; translocations - a color from one chromosome appearing on a different
chromosome).
◦ Locating newly identified genes on specific chromosomes.

b) Interphase FISH (Nuclear FISH)

• Performed On: Interphase nuclei (non-dividing cells or cells not in metaphase). Chromosomes are not
condensed and visible individually; target sequences appear as fluorescent dots/signals within the
nucleus.
• Advantages:
◦ Rapid results (no need for cell culture to get metaphase spreads).
◦ Can be more sensitive for certain applications.
◦ Can be performed on non-growing cells (e.g., archival tissue).
• Applications: Rapid prenatal diagnosis, analysis of tumor cells (e.g., detecting gene amplification like
HER2 in breast cancer), situations requiring quick analysis.

c) Chromosome Painting

• Method: Uses probes that cover (paint) an entire chromosome with a single fluorescent color.
• Limitation: An older technique; fewer dyes are used, so different chromosome pairs might share the
same color (unlike the unique colors in mFISH/SKY).

Key Points to Remember

• Southern Blot: Detects DNA using a DNA probe.

• Northern Blot: Detects RNA using a cDNA probe. Studies gene expression.
• Western Blot: Detects Protein using an Antibody. Principle: Antigen-Antibody interaction.
• Microarrays: Allow high-throughput analysis of thousands of targets (DNA, RNA, Protein)
simultaneously. Known probes are on the array; unknown sample is added.
• aCGH: Compares two whole genomes (test vs. reference) using competitive hybridization on a genome
chip to detect copy number variations (deletions/amplifications). Cannot detect balanced
rearrangements.
 • FISH: Detects specific DNA sequences within intact cells/chromosomes ("in situ") using fluorescent
probes. Can detect numerical and structural chromosomal abnormalities and locate genes.
◦ mFISH/SKY: Uses unique colors for each chromosome pair on metaphase spreads.
◦ Interphase FISH: Faster analysis on non-dividing cells (signals appear as dots).
Remember the specific target molecule and probe type for each blotting technique. Understand the
difference between blotting (separate first, then probe) and microarrays (fixed probes, add sample).
Recognize that aCGH and FISH are powerful cytogenetic tools for detecting genomic changes.
Okay students, here is a summary of the lecture on Recombinant DNA Technology:
Recombinant DNA Technology (RDT)

• Definition: An in vivo (inside the cell) amplification technique used to create clones (multiple identical
copies) of a desired DNA fragment.
• Classification: Falls under Amplification Techniques.
• Prerequisites: Understanding Restriction Endonucleases and Vectors is essential.
1. Restriction Endonucleases (REs)

• What they are: Enzymes found inside bacteria.

• Discovery: Werner Arber.
• Function:
◦ Cut double-stranded DNA (dsDNA).
◦ Specifically cleave the 3' to 5' phosphodiester bond (a covalent bond).
◦ Belong to the Hydrolase enzyme class (Class III).
• Naming ("Restriction"): They restrict the entry and replication of foreign DNA, like bacteriophages
(viruses infecting bacteria), within the bacterial cell.
• Types:
◦ Type 1: Cut dsDNA at random sites.
◦ Type 2: Cut dsDNA at specific palindromic sites.
▪ Mnemonic: Type 2 cuts at 2 identical sequences (palindromes, read the same forwards/backwards
on opposite strands).
▪ Molecular Biology Focus: When "Restriction Endonuclease" is mentioned in molecular biology
techniques, it typically refers to Type 2.
▪ Key Scientists (Type 2 Use): Hamilton Smith and Daniel Nathans.
▪ Nickname: "Molecular Scissors".
• Action & Palindromic Sites:
◦ A palindromic site reads the same forwards on one strand as it does backwards on the complementary
strand (e.g., 5'-GAATTC-3' / 3'-CTTAAG-5').
• Types of Cuts/Ends Produced:
◦ Sticky Ends (Cohesive/Staggered Ends): Result from an offset cut, leaving single-stranded
overhangs.
▪ Example: EcoR1 (from E. coli) recognizes GAATTC and cuts between G and A on both strands,
leaving AATT overhangs. (Sequence: G | AATTC and CTTAA | G).
◦ Blunt Ends: Result from a direct cut across both strands at the same position, leaving no overhangs.
▪ Example: HpaI recognizes GTTAAC and cuts directly between T and A (GTT | AAC and CAA |
TTG).
 • Bacterial Self-Protection:
◦ Bacteria prevent their own REs from cutting their own DNA.
◦ Mechanism: An enzyme called Site-Specific Methylase adds methyl groups (-CH3) to bases within
the RE recognition sites on the bacterial DNA.
◦ This methylation blocks the RE from binding and cutting its own genome.
2. Vectors

• Definition: A DNA molecule used as a vehicle to carry a foreign DNA fragment (the fragment to be
cloned) into a host cell.
• Essential Properties: (Mnemonic: ARA - Autonomous Replication, Restriction site(s), Antibiotic
resistance gene)
◦ Autonomous Replication: Can replicate independently within the host cell (often has an Origin of
Replication - Ori).
◦ At least one Restriction Site: A specific sequence recognized and cut by an RE, allowing insertion
of the foreign DNA.
◦ At least one Selectable Marker: Typically an antibiotic resistance gene, allowing identification and
selection of cells that have successfully taken up the vector.
• Types of Vectors & DNA Insert Sizes:
◦ Plasmids:
▪ Naturally occurring, circular, double-stranded, extra-chromosomal DNA in bacteria (8-10 copies/
cell).
▪ Function in bacteria: Often confer antibiotic resistance.
▪ Used as vectors. Have an Ori.
▪ Insert Size: 0.01 - 10 kbp (kilobase pairs).
◦ Phages (Bacteriophages):
▪ Viruses that infect bacteria.
▪ DNA is typically linear.
▪ Used as vectors.
▪ Insert Size: 10 - 20 kbp.
◦ Cosmids:
▪ Hybrid vectors: Plasmids containing a phage cos site (gene/sequence required for packaging DNA
into phage particles).
▪ Combine properties of plasmids (e.g., replication) and phages (e.g., packaging efficiency).
Circular dsDNA.
▪ Insert Size: 30 - 50 kbp.
◦ Artificial Chromosomes:
▪ Artificially constructed vectors with high capacity, mimicking chromosome properties.
▪ BAC (Bacterial Artificial Chromosome): Based on bacterial components. Insert Size: 50 - 250
kbp. (Note: Text mentioned 52-250 kbp).
▪ PAC (Phage Artificial Chromosome): Based on phage components. Insert Size: 50 - 250 kbp.
(Note: Text mentioned 52-250 kbp).
▪ YAC (Yeast Artificial Chromosome): Based on yeast components. Insert Size: 250 - 3000 kbp
(Highest capacity).
▪ HAC (Human Artificial Chromosome): Mentioned as based on human chromosomes.
3. Steps of Recombinant DNA Formation (Synthesis of Chimeric DNA)

1. Select: Choose a plasmid (vector) and a source DNA containing the desired gene/fragment. Both must
have compatible restriction sites.
2. Cut: Treat both the plasmid and the source DNA with the same Type 2 Restriction Endonuclease. This
creates complementary sticky ends (or blunt ends, though sticky ends are often preferred for directional
cloning).
3. Anneal (Join): Mix the cut plasmid and the cut DNA fragment. The complementary sticky ends base-
pair with each other.
4. (Ligation - Implied Step): An enzyme called DNA Ligase is typically used to form phosphodiester
bonds, permanently sealing the gaps in the DNA backbone. (Note: Ligase wasn't explicitly mentioned in
this section of the provided text but is essential).
5. Result: A Recombinant Plasmid (also called Chimeric DNA) is formed, containing the desired DNA
fragment inserted into the vector.
6. Transformation & Amplification: This recombinant plasmid is introduced into a host cell (e.g.,
bacteria). When the host cell divides, the plasmid (with the inserted DNA) replicates along with it,
amplifying the desired fragment.
4. Recombinases

• Role: Enzymes that facilitate site-specific insertion of DNA fragments without necessarily requiring
restriction enzymes, acting as an adjunct or alternative.
• Mechanism: Mediate Homologous Recombination between specific recognition sites on the incoming
DNA and the target DNA.
• Examples (Enzyme -> Target Site):
◦ Cre Recombinase -> LoxP site (bacteria)
◦ Lambda Phage INT protein -> Lambda ATT sites
◦ Yeast FLP -> FRT sites
• Process: A recombinase attached to the desired DNA fragment recognizes its specific target site (e.g.,
LoxP) on the host genome and integrates the fragment there. Amplification occurs as the host cell
divides.
5. Gene Libraries

• Definition: A collection of cloned DNA fragments (recombinant clones) derived from a single specific
source organism or tissue. Analogous to a book library holding different books (clones).
• Types (based on source DNA):
◦ Genomic Library:
▪ Source: Total genomic DNA of an organism.
▪ Content: Represents the entire genome, including coding regions (exons), non-coding regions
(introns, regulatory sequences).
▪ Characteristics: Contains very large fragments, includes introns, may be difficult to replicate large
clones efficiently, less directly informative about gene expression patterns. (Like taking a scoop
from the ocean).
◦ cDNA Library:
▪ Source: Messenger RNA (mRNA) isolated from a specific cell type or tissue.
▪ Process: mRNA is converted into complementary DNA (cDNA) using the enzyme Reverse
Transcriptase. This cDNA is then cloned into vectors.
 ▪ Content: Represents only the genes that were being expressed (transcribed into mRNA) in the
source cells at the time of isolation. Contains only exons (no introns).
▪ Characteristics: Clones are smaller, lack introns, easier to replicate, more informative about gene
structure (coding sequence) and gene expression levels. Useful for studying expressed genes.
Key Points to Remember:

• RDT is an in vivo amplification method using vectors and host cells.

• Type 2 Restriction Endonucleases ("Molecular Scissors") are crucial for cutting DNA at specific
palindromic sites, creating sticky or blunt ends.
• Bacteria protect their own DNA from their REs using Site-Specific Methylases.
• Vectors (like plasmids, phages, cosmids, YACs) are essential vehicles for carrying DNA into host cells
and must have Autonomous replication, Restriction sites, and Antibiotic resistance genes.
• The same RE must be used on both the vector and the DNA insert to ensure compatible ends for joining.
• Recombinases (Cre-LoxP, FLP-FRT) allow site-specific integration via homologous recombination.
• cDNA libraries (from mRNA) lack introns and reflect expressed genes, making them smaller and more
informative for studying gene function/expression than genomic libraries (from total DNA).
• YACs have the largest insert capacity among the listed vectors.

PCR Lecture Summary

Introduction & Relevance

• PCR (Polymerase Chain Reaction): A fundamental molecular biology technique, widely known and
utilized.
• Invented: By Dr. Kary P. Mullis in 1989 (as stated in the lecture). Described as an "ingenious" technique
due to its long-standing relevance.
• Applications Demonstrated:
◦ Forensic Science: Identifying individuals from small biological samples (e.g., blood DNA) found at
crime scenes.
◦ Clinical Diagnostics: Detecting infectious agents, famously used for COVID-19 testing.

Classification of Amplification Techniques

1. Based on Location:
◦ In Vivo: Amplification occurs inside a living cell (Example: Recombinant DNA technology).
◦ In Vitro: Amplification occurs outside a cell, in a test tube (Example: PCR).
2. Based on Temperature Profile (for In Vitro):
◦ Thermal Cycling: Reaction temperature varies cyclically.
▪ Examples: PCR, LCR (Ligase Chain Reaction).
◦ Isothermal Cycling: Reaction temperature remains constant.
▪ Examples: NASBA (Nucleic Acid Sequence Based Amplification), bDNA (Branched DNA)
technology.
 3. Based on Component Amplified (for In Vitro):
◦ Target Amplification: The original nucleic acid sequence of interest (the target) is copied many
times.
▪ Examples: PCR, NASBA.
◦ Probe/Primer Amplification: A probe or primer that binds to the target is amplified.
▪ Examples: LCR, Q beta replicase.
◦ Signal Amplification: The signal generated from the probe-target interaction is amplified, not the
target or probe itself.
▪ Example: bDNA (Branched DNA) technology.

PCR: Definition & Principles

• Definition: An in vitro target amplification technique that uses a thermocycler (machine to change
temperatures).
• Etymology:
◦ "Polymerase": Requires a DNA polymerase enzyme.
◦ "Chain Reaction": Involves repetitive cycles of steps, leading to exponential amplification.

Prerequisites for PCR

• Pure DNA/RNA Sample: Quality assessed by A260/A280 ratio. (DNA absorbs light maximally at
260nm, proteins at 280nm; a high ratio indicates DNA purity).
• Primers: Short, single-stranded DNA sequences complementary to the regions flanking the target DNA.
Both forward and reverse primers are needed. DNA polymerase cannot start synthesis without a primer.
• Deoxyribonucleotides (dNTPs): Building blocks (dATP, dGTP, dCTP, dTTP) for the new DNA strands.
• Cations: Divalent cations like Magnesium (Mg2+) are essential cofactors for the polymerase enzyme.
Potassium (K+) is also mentioned.
• Thermostable DNA Polymerase: Typically Taq polymerase, isolated from the thermophilic bacterium
Thermus aquaticus (lives in hot springs). It can withstand the high temperatures used in PCR without
denaturing.

Steps of PCR (One Cycle)

Mnemonic: DAE - Denaturation, Annealing, Extension

1. Denaturation:
◦ Purpose: Separate the two strands of the double-stranded DNA template.
◦ Temperature: High, 90-96°C.
◦ Initial Denaturation: The very first denaturation step is often longer (~3 minutes) to ensure
complete separation of the initial template DNA. Subsequent denaturation steps within the cycles are
shorter (~1 minute or less).
2. Annealing:
◦ Purpose: Allow the forward and reverse primers to bind (anneal) to their complementary sequences
on the separated single strands. Primers bind to the 3' end of the flanking sequence adjacent to the
target region.
◦ Temperature: Lowered, 54-60°C. Temperature is crucial for specificity.
3. Extension/Elongation:
◦ Purpose: The DNA polymerase (Taq) synthesizes new DNA strands by adding dNTPs, starting from
the 3' end of the primers.
◦ Temperature: Raised to the optimal temperature for the polymerase, typically ~72°C.
4. Cycling: These three steps (Denaturation, Annealing, Extension) constitute one cycle. The cycle is
repeated many times (usually 20-40 cycles).
5. Amplification: Each cycle theoretically doubles the amount of the target DNA sequence. This leads to
exponential amplification.
6. Formula: Number of target copies ≈ 2^n, where 'n' is the number of cycles.
7. Duration: One cycle takes approximately 5 minutes on average.

Variants of PCR

1. Reverse Transcriptase PCR (RT-PCR):

◦ Purpose: To amplify RNA targets (e.g., mRNA, viral RNA).
◦ Key Enzyme: Reverse Transcriptase first converts RNA into complementary DNA (cDNA).
◦ Process: RNA → cDNA → standard PCR amplification of cDNA.
◦ RNase: An enzyme used to degrade the original RNA template after cDNA synthesis.
◦ Tth Polymerase: An enzyme from Thermus thermophilus possessing both reverse transcriptase and
DNA polymerase activities, simplifying the process. Notation: RT-PCR.
2. Simplex vs. Multiplex PCR:
◦ Simplex PCR: Amplifies a single target sequence using one pair of primers per reaction. (Higher
specificity, more time-consuming if multiple targets need analysis).
◦ Multiplex PCR: Amplifies multiple target sequences simultaneously in the same reaction tube using
multiple primer pairs. (Faster, cost-effective for multiple targets, but requires careful optimization to
avoid primer interference and ensure comparable efficiency; potentially lower specificity).
3. Real-Time PCR (qPCR - Quantitative PCR):
◦ Purpose: Monitors the amplification of DNA in real-time during the PCR process, allowing for
quantification.
◦ Mechanism: Uses fluorescent reporters:
▪ Dyes: Like SYBR Green, which binds to double-stranded DNA and fluoresces.
▪ Probes: Sequence-specific probes like TaqMan probes, Molecular Beacons, or using FRET
(Fluorescence Resonance Energy Transfer), which release fluorescence upon hybridization or
cleavage during amplification.
◦ Advantage: Amplification and detection occur simultaneously. Allows quantification of the initial
amount of target nucleic acid.
4. Real-Time Reverse Transcriptase PCR (qRT-PCR or RRT-PCR):
◦ Combines: RT-PCR with Real-Time PCR.
◦ Purpose: To detect and quantify RNA targets in real-time.
◦ Notation: Often written as qRT-PCR or rRT-PCR (small 'r' indicates real-time). Note: Ensure
distinction from standard RT-PCR.
◦ Application: Widely used for COVID-19 diagnosis and viral load quantification.
Applications of PCR

• Forensic Medicine: DNA fingerprinting from minimal samples.

• Microbiology: Detection and identification of pathogens (viruses like NIPAH, COVID-19; bacteria,
fungi).
• Mutation Studies: Detecting genetic mutations (e.g., if a mutation prevents primer binding, no product is
formed).
• Repeat Length Polymorphism (RLP) Analysis: Detecting variations in the length of DNA segments
(amplicons), which can indicate certain genetic variations or diseases. Amplicon length typically ~350
nucleotides (lecture example); variations alter this length.
• Preliminary Technique: PCR is often a crucial first step to generate sufficient DNA material for other
molecular techniques like DNA sequencing, cloning, etc.

Key Takeaways / Points to Remember

• PCR amplifies a specific target DNA sequence exponentially in vitro.

• It relies on thermal cycling (DAE: Denaturation, Annealing, Extension) and a thermostable DNA
polymerase (Taq).
• Requires primers, dNTPs, and Mg2+.
• RT-PCR is used for RNA targets, requiring an initial reverse transcription step.
• qPCR (Real-Time PCR) allows for simultaneous amplification and quantification using fluorescence.
• qRT-PCR quantifies RNA in real-time.
• PCR has wide applications in diagnostics, forensics, research, and mutation detection.
• Sample purity (A260/A280 ratio) and proper primer design are critical.
• Distinguish between RT-PCR (amplifies RNA) and qRT-PCR (quantifies RNA in real-time).

DNA Sequencing Summary

Introduction & Exam Relevance

• Exam questions on DNA sequencing are moving towards longer, vignette-type, and applied questions,
rather than simple one-liners.
• Understanding the basic principles underlying different techniques is crucial.
• This lecture focuses primarily on Sanger Sequencing, Pyrosequencing, and Next-Generation
Sequencing (NGS).
Overview of DNA Sequencing Methods

1. Maxam-Gilbert Method:
◦ Oldest method.
◦ Also known as the Chemical Cleavage Method.
◦ Suitable only for sequencing small fragments of DNA.
2. Sanger Sequencing:
◦ Invented by Frederick Sanger (who also developed amino acid sequencing).
◦ Also known as the Controlled Chain Termination Method.
◦ Still the most popular technique and considered the Gold Standard for mutation detection.
 ◦ Major Drawback: High Cost.
3. Pyrosequencing: A sequencing-by-synthesis approach.
4. Next-Generation Sequencing (NGS): High-throughput sequencing methodologies.

Sanger Sequencing (Controlled Chain Termination Method)

• Principle: Based on the incorporation of dideoxynucleotides (ddNTPs) during in vitro DNA synthesis.
◦ Normal DNA synthesis involves joining nucleotides via a 3'-5' phosphodiester bond. This requires a
free 3'-hydroxyl (-OH) group on the preceding nucleotide.
◦ Deoxynucleotides (dNTPs) have a -H at the 2' position but an -OH at the 3' position.
◦ Dideoxynucleotides (ddNTPs) lack the hydroxyl group at both the 2' and 3' positions (have -H at 3').
◦ Mnemonic: dd = double deoxy = dead dend (no 3'-OH for extension).
◦ When a ddNTP is incorporated into a growing DNA strand, synthesis terminates because there is no
3'-OH group to form the next phosphodiester bond.
• Requirements:
1. Sample DNA Template: Sufficient quantity needed for multiple reactions.
2. Primer: (Implicit, required to start synthesis - Added for clarity).
3. Normal Deoxynucleotides (dNTPs): dATP, dGTP, dCTP, dTTP.
4. Dideoxynucleotides (ddNTPs): ddATP, ddGTP, ddCTP, ddTTP (used in limited concentration).
5. DNA Polymerase: Klenow fragment (DNA Polymerase I lacking its 5'→3' exonuclease activity,
preventing degradation of the newly synthesized strand).
• Procedure:
1. Four separate reaction tubes are set up.
2. Each tube contains: template DNA, primer, Klenow polymerase, all four dNTPs.
3. Crucially, each tube also contains a small amount of one specific type of ddNTP (Tube 1: ddATP,
Tube 2: ddGTP, Tube 3: ddCTP, Tube 4: ddTTP).
4. DNA synthesis occurs. Randomly, instead of a normal dNTP, the corresponding ddNTP is
incorporated, causing chain termination.
5. Each tube produces a set of DNA fragments of different lengths, all ending with the specific ddNTP
added to that tube (e.g., Tube 1 fragments all end in 'A', Tube 2 fragments all end in 'G', etc.).
6. The fragments from all four tubes are separated by size using electrophoresis (traditionally gel, now
often capillary).
7. By reading the fragments from shortest to longest across the four "lanes" (or equivalent data output),
the sequence of the newly synthesized strand (complementary to the template) can be determined. For
example, if the shortest fragment is in the 'ddGTP' lane, the first base added after the primer is G. If
the next shortest is in the 'ddATP' lane, the next base is A, and so on.
• Automation: The process, especially using capillary electrophoresis, can be highly automated for faster
results.

Pyrosequencing

• Principle: Sequencing by Synthesis, based on detecting pyrophosphate (PPi) release during nucleotide
incorporation.
◦ Mnemonic: Pyro = Fire = Light (luminescence signals PPi release).
 ◦ When DNA polymerase incorporates a complementary dNTP into the growing strand, it releases PPi.
◦ dNTP + DNA(n) → DNA(n+1) + PPi
• Detection:
1. The released PPi is enzymatically converted (via ATP sulfurylase and luciferase in secondary
reactions) into a detectable light signal (bioluminescence or chemiluminescence).
2. The intensity of light is proportional to the amount of PPi released (can indicate multiple
incorporations of the same base).
• Procedure:
1. dNTPs (dATP, dGTP, dCTP, dTTP) are added sequentially, one at a time, to the reaction containing
the template, primer, and necessary enzymes.
2. If the added dNTP is complementary to the next base in the template, it's incorporated, PPi is
released, and light is detected.
3. If the added dNTP is not complementary, no incorporation occurs, no PPi is released, and no light is
detected. The unincorporated dNTP is degraded before the next dNTP is added.
4. The sequence is determined by recording which dNTP generates a light signal at each step.
• Advantage: More sensitive than Sanger sequencing.

Next-Generation Sequencing (NGS)

• Nature: A High-Throughput Sequencing (HTS) methodology, not a fundamentally new type of

sequencing chemistry (often uses principles similar to Sanger or Pyro). It's a new approach or platform.
• Key Feature: Massively Parallel Sequencing. Allows sequencing of millions or billions of DNA strands
simultaneously.
◦ Mnemonic: NGS = Numerous Genomes Simultaneously (or Now Generating Sequences...
massively!)
• Basic Steps:
1. Library Preparation: Sample DNA is fragmented. (Adapters are usually added - Added for clarity).
2. Spatial Separation: Fragments are spatially isolated (e.g., attached to a solid surface like a slide or
flow cell, or in nanowells).
3. Clonal Amplification: Each individual fragment is amplified in its location to create clusters of
identical fragments (clonal amplification), done simultaneously for all fragments.
4. Parallel Sequencing: All amplified clusters are sequenced at the same time using various specific
chemistries (e.g., sequencing by synthesis).
• Application: Ideal for large-scale projects like whole-genome sequencing, RNA-seq, population studies
(e.g., studying COVID-19 across large populations).

Comparison: Sanger vs. NGS

Feature Sanger Sequencing Next-Generation Sequencing (NGS)

Parallelism One sequence read per sample/reaction Massively Parallel (Millions simultaneous)

Throughput Lower (~1 million bases/day approx.) Very High (~2 billion bases/day approx.)

Cost Higher cost per base Lower cost per base

 Feature Sanger Sequencing Next-Generation Sequencing (NGS)

Primary Use Mutation detection (Gold Std), smaller projects Large-scale genomics, population studies

Key Points to Remember:

• Sanger Sequencing: Gold standard for mutation detection, uses ddNTPs for controlled chain
termination. Requires Klenow fragment. Drawback is cost.
• Pyrosequencing: Sequencing by synthesis, detects PPi release via luminescence. More sensitive than
Sanger.
• NGS: A high-throughput methodology characterized by massively parallel sequencing. Enables large-
scale projects at a lower cost per base. Key steps: Fragmentation, Spatial Separation, Clonal
Amplification, Parallel Sequencing.
• Understand the role of the 3'-OH group in DNA elongation and why its absence in ddNTPs is key to
Sanger sequencing.
• Know the different names: Maxam-Gilbert (Chemical Cleavage), Sanger (Controlled Chain Termination).

Summary: Mutations
1. Definition & Core Concepts

• Mutation: A permanent change in the nucleotide sequence of DNA, regardless of its functional effect.
◦ Key characteristic: Permanence.
• Frequency: Occurs in less than 1% of the population. Variations occurring in >1% are generally
considered polymorphisms.
• Mutation vs. Polymorphism vs. Epigenetics:
◦ Mutation: Abnormal, permanent DNA sequence change (<1% frequency).
◦ Polymorphism: Normal variation in DNA sequence (>1% frequency).
◦ Epigenetics: Reversible chemical modification (e.g., methylation) of DNA or chromatin proteins,
without altering the nucleotide sequence itself.
• Heritability:
◦ Germline Mutations: Occur in germ cells (sperm/egg) and can be transmitted to the next
generation.
◦ Somatic Mutations: Occur in non-germline body cells and are not transmitted to the next
generation.

2. Classification of Mutations
A. Point Mutations (Most Common Type)

• Involve a change in a single nucleotide base.

• Base Substitution: One base is replaced by another.
◦ Groups:
▪ Synonymous Mutation (Silent Mutation):
▪ The substituted base results in a codon that codes for the same amino acid.
▪ No change in the resulting polypeptide sequence.
 ▪ Reason: Degeneracy of the genetic code (multiple codons can specify the same amino acid,
often differing at the 3rd position).
▪ Example: UUU (Phe) -> UUC (Phe).
▪ Non-synonymous Mutation: The substitution results in a change in the amino acid sequence.
▪ Missense Mutation: The new codon codes for a different amino acid.
▪ Subtype 1 (Based on Property Change):
▪ Conservative: New amino acid has similar chemical properties (e.g., charge, polarity)
to the original. (e.g., GAA [Glu, acidic] -> GAC [Asp, acidic]).
▪ Non-conservative: New amino acid has different chemical properties. (e.g., GAG
[Glu, polar acidic] -> GUG [Val, non-polar] in Sickle Cell Anemia/HbS). Mnemonic:
CONserve = CONtinue similar properties.
▪ Subtype 2 (Based on Clinical Impact):
▪ Acceptable: No significant clinical symptoms or functional defect. (e.g., Hb Hikari -
may only alter electrophoretic mobility).
▪ Partially Acceptable: Clinical symptoms present, but protein function is largely
maintained. (e.g., HbS - oxygen transport okay, but sickling occurs).
▪ Unacceptable: Clinical symptoms present AND protein function is significantly
impaired. (e.g., HbM [Methemoglobin] - iron is Fe3+, cannot bind O2 effectively).
Mnemonic: Acceptable = All okay; Partially = Problems, function okay;
Unacceptable = Utterly impaired function.
▪ Nonsense Mutation: The new codon is a stop codon (UAA, UAG, UGA).
▪ Leads to premature termination of translation.
▪ Results in a truncated, usually non-functional protein.
▪ Example: UGU (Cys) -> UGA (Stop); CAG -> UAG causing Beta-0 Thalassemia.
Mnemonic: NONsense = NO more protein synthesis.
◦ Types (Based on Chemical Nature):
▪ Transition: Substitution of a purine for a purine (A ↔ G) or a pyrimidine for a pyrimidine (C ↔
T/U). Mnemonic: TransiTion = Two rings to Two rings, or Thin ring to Thin ring (Same type).
▪ Transversion: Substitution of a purine for a pyrimidine, or vice versa (A/G ↔ C/T/U).
Mnemonic: TransVersion = ConVersion between types.

B. Insertions and Deletions (Indels)

• Addition (insertion) or removal (deletion) of one or more nucleotides.

• Can range from a single base to large gene segments.
• Often highly deleterious.
• Frameshift Mutation:
◦ Caused by indels where the number of inserted/deleted bases is not a multiple of three.
◦ Alters the triplet reading frame from the point of mutation onwards.
◦ Leads to a completely different downstream amino acid sequence and often an early stop codon.
◦ Exception: If an indel involves multiples of three bases, the reading frame is maintained, but amino
acids are added or removed. (e.g., Deletion of the Phe codon [3 bases] at position 508 in the CFTR
gene causes Cystic Fibrosis without a frameshift).

C. Other Mutation Types

• Truncated Polypeptide: A shorter-than-normal protein, typically resulting from a nonsense mutation.

 • Run-on Polypeptide: A longer-than-normal protein, resulting from a mutation that changes a stop codon
into a coding codon, causing translation to continue past the normal termination point.
◦ Example: Hemoglobin Constant Spring.
• Trinucleotide Repeat Expansion: Amplification of a 3-base repeat sequence within a gene.
◦ Example: CGG repeats in the FMR1 gene causing Fragile X Syndrome.
◦ Characteristic: Dynamic mutation - the number of repeats can change (usually increase) between
generations, especially during gametogenesis.

3. Mutation Detection Techniques

• Detecting Chromosomal Abnormalities (Numerical/Structural):

◦ Cytogenetic Analysis (Karyotyping)
◦ Fluorescence In Situ Hybridization (FISH)
• Detecting Point Mutations, Small Deletions/Insertions:
◦ DNA Sequencing (Sanger Sequencing): Gold Standard method; determines the exact nucleotide
sequence. Can be expensive.
◦ Restriction Fragment Length Polymorphism (RFLP): Detects mutations that create or abolish
restriction enzyme recognition sites. Requires PCR amplification first.
◦ Single-Strand Conformation Polymorphism (SSCP): Detects differences in electrophoretic
mobility of single-stranded DNA caused by conformational changes due to mutation.
◦ Denaturing Gradient Gel Electrophoresis (DGGE): Separates DNA fragments based on melting
properties in a denaturing gradient gel; mutations alter melting behavior and mobility.
◦ Oligonucleotide Specific Hybridization (OSH) / Allele-Specific Oligonucleotide (ASO): Uses
labeled probes specific for either the normal or mutant sequence to detect presence/absence of
mutation via hybridization.
◦ RNA Cleavage Assays: Methods that detect mismatches in RNA:DNA or RNA:RNA hybrids.
◦ Microarray / DNA Chip: High-throughput method for detecting known mutations and genotyping
Single Nucleotide Polymorphisms (SNPs).
• Detecting Sequence Alterations (Robins/Emery Perspective):
◦ PCR analysis followed by:
▪ Sanger Sequencing (Gold Standard)
▪ Restriction Digest (RFLP)
▪ Single Base Primer Extension
• Detecting Length Alterations (Trinucleotide repeats, large indels, Copy Number Variations -
CNVs):
◦ Amplicon Length Analysis: Comparing the size of PCR products to expected size.
◦ Real-Time PCR: Quantifying PCR product in real-time; deviations from expected amplification
curves/quantities can indicate deletions (less template) or duplications (more template).
◦ Multiplex Ligation-dependent Probe Amplification (MLPA):
▪ Purpose: Detects copy number variations (large deletions/duplications of exons or entire genes).
▪ Mechanism: Involves simultaneous Hybridization of multiple (~40) probe pairs to adjacent
sequences on the target DNA, Ligation of adjacent probes, followed by PCR Amplification using
universal primers, and Detection/quantification of amplicons. Mnemonic: MLPA uses Hybridize,
Ligate, Amplify, Detect.
 ▪ Interpretation: Reduced/absent amplicon signal indicates deletion; increased signal indicates
duplication.

4. Example Revisited (AUG UUU UGG GAG -> AUG UUC UGG GAG)

• Amino Acid Sequence: Met-Phe-Trp-Glu remains Met-Phe-Trp-Glu.

• Classification:
◦ Silent Mutation: No change in amino acid sequence.
◦ Point Mutation: Single base change.
◦ Base Substitution: U replaced by C.
◦ Transition: Pyrimidine (U) replaced by another pyrimidine (C).

5. Important Points to Remember

• Mutation = Permanent DNA sequence change (<1% freq).

• Distinguish Mutation, Polymorphism (>1% freq, normal), and Epigenetics (reversible, no sequence
change).
• Germline = Heritable; Somatic = Not heritable.
• Point mutations (substitutions, indels) are common.
◦ Substitutions: Silent (same AA), Missense (different AA), Nonsense (STOP).
◦ Indels: Cause Frameshift IF NOT multiple of 3.
• Functional consequences of Missense: Conservative (similar AA) vs Non-conservative (different AA);
Acceptable/Partially/Unacceptable impact.
• Nonsense -> Truncated protein; Stop codon mutation -> Run-on protein.
• Trinucleotide Repeats are dynamic (e.g., Fragile X).
• Detection:
◦ Sanger Sequencing = Gold Standard for exact sequence.
◦ MLPA = Key for Copy Number Variations (large deletions/duplications).
◦ Know other methods like RFLP, SSCP, Microarray.

Okay students, here is a summary of our Krebs Cycle session:

Krebs Cycle (TCA Cycle / Citric Acid Cycle)
I. Introduction & Importance

• Central Role: The final common oxidative pathway for carbohydrates, lipids, and proteins.
• Convergence Point: All macronutrients break down to Acetyl-CoA (a 2-carbon compound), which
enters the cycle.
• Goal: Complete oxidation of Acetyl-CoA. The two carbons from Acetyl-CoA are released as two
molecules of CO2.
• Energy Production: Generates reducing equivalents (NADH, FADH2) and direct ATP (or GTP).
◦ NADH and FADH2 subsequently enter the Electron Transport Chain (ETC) for substantial ATP
synthesis via oxidative phosphorylation.
• Overall Function: Converts chemical energy from food into ATP.
II. Basic Characteristics

• Other Names:
◦ TCA Cycle: Tricarboxylic Acid cycle (Citrate is a tricarboxylic acid).
◦ Citric Acid Cycle: Citrate is the first product formed.
◦ Krebs Cycle: Named after discoverer Hans Krebs.
• Location: Exclusively in the mitochondrial matrix of cells containing mitochondria.
• Enzyme Location Exception: Succinate Dehydrogenase (SDH) is located in the inner mitochondrial
membrane (as it's part of ETC Complex II); all other enzymes are in the matrix.
III. Cycle Overview

• Starting Point: Acetyl-CoA (2C) combines with Oxaloacetate (OAA) (4C) to form Citrate (6C).
• First Half (Oxidative Decarboxylation):
◦ Citrate (6C) is progressively oxidized and decarboxylated (loses 2 CO2 molecules).
◦ Key intermediate formed: Succinyl-CoA (4C).
◦ Generates NADH.
• Second Half (Regeneration of OAA):
◦ Succinyl-CoA (4C) is converted back to Oxaloacetate (OAA) (4C).
◦ Generates FADH2, NADH, and ATP/GTP.
IV. Detailed Reactions & Enzymes
(Note: Steps 1, 3, 4 are generally considered the main irreversible/regulatory points)

1. OAA (4C) + Acetyl-CoA (2C) → Citrate (6C)

◦ Enzyme: Citrate Synthase
◦ Irreversible, Regulatory step.
◦ OAA binds first to the enzyme.
2. Citrate ↔ Isocitrate
◦ Enzyme: Aconitase
◦ Reversible.
◦ Requires Fe2+.
◦ Two-step: Dehydration → Rehydration.
◦ Class: Lyase.
◦ Moonlighting enzyme: Also involved in iron homeostasis.
3. Isocitrate (6C) → α-Ketoglutarate (α-KG) (5C)
◦ Enzyme: Isocitrate Dehydrogenase (IDH/ICDH)
◦ Oxidative Decarboxylation (1st one).
◦ Releases 1 CO2.
◦ Produces 1 NADH (→ 2.5 ATP via ETC).
◦ Regulatory step.
◦ Mitochondrial IDH (involved here) uses NAD+ → NADH.
◦ Cytoplasmic IDH (different function) uses NADP+ → NADPH.
4. α-KG (5C) → Succinyl-CoA (4C)
◦ Enzyme: α-Ketoglutarate Dehydrogenase (α-KGDH)
 ◦ Irreversible, Regulatory step.
◦ Oxidative Decarboxylation (2nd one).
◦ Releases 1 CO2.
◦ Produces 1 NADH (→ 2.5 ATP via ETC).
◦ Requires 5 Coenzymes (like Pyruvate Dehydrogenase): TPP (B1), CoA (B5), Lipoamide, FAD (B2),
NAD+ (B3).
▪ Mnemonic: Tender Loving Care For Nancy
◦ Inhibited in Thiamine (B1) deficiency.
5. Succinyl-CoA ↔ Succinate
◦ Enzyme: Succinate Thiokinase (or Succinyl-CoA Synthetase)
◦ Reversible.
◦ Substrate-Level Phosphorylation: Produces 1 ATP (or GTP).
◦ GTP produced in tissues active in gluconeogenesis (e.g., liver), as GTP is used by PEPCK.
6. Succinate ↔ Fumarate
◦ Enzyme: Succinate Dehydrogenase (SDH)
◦ Reversible.
◦ Produces 1 FADH2 (→ 1.5 ATP via ETC).
◦ Location: Inner mitochondrial membrane (Complex II of ETC).
7. Fumarate ↔ Malate
◦ Enzyme: Fumarase
◦ Reversible.
◦ Class: Lyase.
8. Malate ↔ Oxaloacetate (OAA)
◦ Enzyme: Malate Dehydrogenase (MDH)
◦ Reversible.
◦ Produces 1 NADH (→ 2.5 ATP via ETC).
V. Energetics (per 1 molecule of Acetyl-CoA)

• From NADH (via ETC): 3 NADH (1 from IDH, 1 from α-KGDH, 1 from MDH) → 3 x 2.5 = 7.5 ATP
• From FADH2 (via ETC): 1 FADH2 (from SDH) → 1.5 ATP
• From Substrate-Level Phosphorylation: 1 ATP/GTP (from Succinate Thiokinase) → 1 ATP
• Total: 7.5 + 1.5 + 1 = 10 ATP per Acetyl-CoA molecule.
VI. Inhibitors

• Fluoroacetate: Metabolized to Fluorocitrate, which inhibits Aconitase (Non-competitive).

• Arsenite: Inhibits α-KGDH (Non-competitive, targets -SH groups like lipoamide).
• Malonate: Competitive inhibitor of SDH (structurally similar to Succinate).
VII. Significances

• Complete Oxidation: Final pathway for oxidizing Acetyl-CoA derived from all macronutrients.
• Amphibolic Nature: Acts as both a catabolic and anabolic pathway.
◦ Catabolic: Breaks down Acetyl-CoA for energy.
 ◦ Anabolic (Provides Precursors):
▪ Citrate: Moves to cytosol → Acetyl-CoA for fatty acid synthesis.
▪ α-Ketoglutarate: Precursor for glutamate (→ GABA neurotransmitter, other amino acids).
▪ Succinyl-CoA: Used for heme synthesis (requires glycine too).
▪ Oxaloacetate (OAA): Precursor for glucose (gluconeogenesis) and aspartate (other amino acids,
purine/pyrimidine synthesis).
• Metabolic Traffic Circle: Intermediates can be drawn off for biosynthesis or replenished.
VIII. Anaplerotic Reactions ("Filling Up")

• Replenish TCA cycle intermediates that are removed for biosynthesis.

• Most Important: Pyruvate → Oxaloacetate (OAA)
◦ Enzyme: Pyruvate Carboxylase
◦ Requires: Biotin, ATP, CO2.
• Amino Acid Catabolism: Various amino acids can feed into the cycle at different points:
◦ → α-KG (e.g., Glutamate, Glutamine, Proline, Arginine, Histidine)
◦ → Succinyl-CoA (e.g., Methionine, Threonine, Valine, Isoleucine - Mnemonic: MTV enters at
Succinyl-CoA)
◦ → Fumarate (e.g., Phenylalanine, Tyrosine)
◦ → OAA (e.g., Aspartate, Asparagine)
IX. Recent Updates & Clinical Correlations (Onco-Metabolism)

• Mutant Isocitrate Dehydrogenase (IDH):

◦ Converts α-KG → 2-Hydroxyglutarate (2-HG) (instead of Succinyl-CoA).
◦ 2-HG is an Onco-metabolite.
◦ Mechanism: Inhibits TET enzymes → Alters epigenetics (DNA hypermethylation, histone
modification) → Promotes cancer.
◦ Associated Cancers: Gliomas, Acute Myeloid Leukemia (AML), Cholangiocarcinoma, Sarcomas.
◦ Therapeutic Target: Inhibitors of mutant IDH (e.g., Ivosidenib, Enasidenib - text mentioned
"evocydenibs").
• Succinate Dehydrogenase (SDH) Mutations:
◦ Mutations in SDH subunits (e.g., SDHB, SDHD).
◦ Associated Cancers (as per text): Familial Glioblastoma, Familial Pheochromocytoma (also
strongly linked to Paraganglioma, GIST).
X. Vitamins Involved

• B1 (Thiamine): As TPP (for α-KGDH).

• B2 (Riboflavin): As FAD (for SDH, α-KGDH).
• B3 (Niacin): As NAD+ (for IDH, α-KGDH, MDH).
• B5 (Pantothenic Acid): As part of Coenzyme A.
• Deficiency affects energy production.
XI. Regulation

• Key Regulatory Enzymes: Citrate Synthase, IDH, α-KGDH. (PDH complex regulating Acetyl-CoA
entry is also critical).
 • Primary Control: Cellular energy status.
◦ Inhibition: High ATP/ADP ratio, High NADH/NAD+ ratio (signal energy abundance).
◦ Activation: Low ATP/ADP ratio, Low NADH/NAD+ ratio (signal energy need).
• No Direct Hormonal Control: Unlike glycolysis/gluconeogenesis, TCA cycle activity is dictated by
substrate availability and energy demand, not directly by insulin/glucagon.
--- Important Points to Remember ---

• Central Hub: TCA cycle is the nexus of metabolism.

• Location: Mitochondrial matrix (except SDH).
• Key Output: 10 ATP equivalents (directly + via ETC), 2 CO2 per Acetyl-CoA.
• Amphibolic: Both breaks down fuel and provides building blocks. Remember Citrate→Fatty acids, α-
KG→Glutamate, Succinyl-CoA→Heme, OAA→Glucose/Aspartate.
• Anaplerosis: Pyruvate Carboxylase is the major replenishing reaction.
• Regulation: Primarily by energy charge (ATP/ADP, NADH/NAD+ ratios) at key irreversible steps
(Citrate Synthase, IDH, α-KGDH).
• Clinical Links: Understand the roles of mutant IDH (→ 2-HG, cancer) and SDH defects (→ cancer).
• Vitamins: B1, B2, B3, B5 are essential cofactors.

Electron Transport Chain (ETC) Summary

I. Fundamental Concepts

1. Oxidation: Loss of electrons.

2. Reduction: Gain of electrons.
3. Redox Couple: Compounds existing in both oxidized and reduced states.
◦ Examples: NAD+/NADH, FAD/FADH2, FMN/FMNH2.
4. Redox Potential: The ability of a redox couple to transfer electrons.
◦ Rule: Electrons flow from a redox couple with lower redox potential to one with higher redox
potential.
◦ Energy Release: This electron flow is an exothermic reaction, releasing energy. This explains the
unidirectional flow of electrons in the ETC.

II. ETC Overview

1. Definition: A series of redox couples arranged in ascending order of redox potential.

2. Location: Inner mitochondrial membrane.
3. Function: To harness the energy released from electron transfer to synthesize ATP (Oxidative
Phosphorylation).

III. Components of the ETC

(Remember: Complexes are arranged by increasing redox potential)

1. Complex I: NADH-Q Oxidoreductase

◦ Function: Accepts electrons from NADH (oxidizing it to NAD+) and transfers them to Coenzyme Q
(CoQ).
 ◦ Components: FMN (Flavin mononucleotide), Fe-S (Iron-Sulfur) centers.
◦ Proton Pumping: Pumps 4 H+ from the mitochondrial matrix to the intermembrane space.
◦ Mnemonic: Complex I takes electrons from NADH (the 1st major electron carrier).
2. Coenzyme Q (Ubiquinone / Q10)
◦ Nature: Small, lipophilic, mobile electron carrier within the inner mitochondrial membrane.
◦ Function: Accepts electrons from Complex I and Complex II, transfers them to Complex III.
3. Complex II: Succinate-Q Oxidoreductase
◦ Function: Accepts electrons from Succinate (converting it to Fumarate; this complex is Succinate
Dehydrogenase from the TCA cycle) using FAD (reducing it to FADH2) and transfers them to
Coenzyme Q (CoQ).
◦ Components: FAD, Fe-S centers.
◦ Proton Pumping: Pumps 0 H+. (Crucial point: FADH2 yields less ATP because it bypasses Complex
I's proton pumping).
◦ Mnemonic: Complex II links TCA Cycle (Succinate -> Fumarate) to ETC.
4. Complex III: Q-Cytochrome c Oxidoreductase
◦ Function: Accepts electrons from reduced CoQ (QH2) and transfers them to Cytochrome c.
◦ Components: Cytochrome b, Cytochrome c1, Rieske Fe-S center.
◦ Proton Pumping: Pumps 4 H+.
5. Cytochrome c
◦ Nature: Small, mobile electron carrier (protein) in the intermembrane space, transfers electrons
between Complex III and Complex IV.
6. Complex IV: Cytochrome c Oxidase
◦ Function: Accepts electrons from Cytochrome c and transfers them to the final electron acceptor,
Oxygen (O2), reducing it to Water (H2O).
◦ Components: Cytochrome a, Cytochrome a3 (Heme a, Heme a3), Copper centers (CuA, CuB).
◦ Proton Pumping: Pumps 2 H+.
◦ Key Feature: Considered the irreversible step in the ETC.
◦ Mnemonic: Complex IV involves 4 components needing electrons for O2 reduction (2 Cyt, 2 Cu) and
uses O2.
7. Complex V: ATP Synthase ("Smallest Motor")
◦ Function: Uses the energy stored in the proton gradient to synthesize ATP from ADP and Pi.
◦ Structure:
▪ F0 subunit: Embedded in the inner membrane, forms a proton channel (hydrophobic, contains
c-subunits). Allows H+ flow back into the matrix. (Mnemonic: O in F0 for the O-pening/hole for
protons).
▪ F1 subunit: Protrudes into the mitochondrial matrix (hydrophilic). Contains the catalytic sites for
ATP synthesis.
▪ Subunits: 3α, 3β, γ, δ, ε.
▪ β subunits: Catalytic sites where ATP is synthesized.
▪ γ subunit: Acts as a "bent axle" or stalk, connects F0 to F1, rotates as protons flow through
F0.
IV. Mechanism of ATP Synthesis: Oxidative Phosphorylation

1. Proton Pumping: Electron flow through Complexes I, III, and IV drives pumping of H+ from the matrix
to the intermembrane space.
2. Electrochemical Gradient (Proton Gradient): Creates a higher concentration of H+ (lower pH) and
positive charge in the intermembrane space compared to the matrix.
3. Proton Motive Force (PMF): The potential energy stored in the proton gradient.
4. ATP Synthesis: H+ flows back down its gradient into the matrix through the F0 channel of ATP
synthase. This flow drives the rotation of the γ subunit.
5. Conformational Changes: Rotation of the γ subunit causes conformational changes in the β subunits of
F1, cycling them through states that bind ADP + Pi, synthesize ATP, and release ATP.
6. Theories:
◦ Chemiosmotic Theory (Peter Mitchell): Explains how electron transport and ATP synthesis are
coupled by the proton gradient across the inner mitochondrial membrane.
◦ Binding Change Mechanism (Paul Boyer): Describes how the rotational energy causes
conformational changes in the F1 β subunits to drive ATP synthesis.

V. Energy Yield (P/O Ratio)

• P/O Ratio: Moles of ATP synthesized per mole of Oxygen atoms reduced.
• NADH: Enters at Complex I -> Pumps 10 H+ (4+0+4+2) -> Yields ~2.5 ATP.
• FADH2: Enters at Complex II (bypassing Complex I) -> Pumps 6 H+ (0+0+4+2) -> Yields ~1.5 ATP.
◦ This difference arises because FADH2 bypasses the proton pumping of Complex I.

VI. Inhibitors of Respiratory Chain

1. Inhibitors of Electron Flow (Complex Specific):

◦ Complex I: Rotenone (insecticide, fish poison), Amobarbital (barbiturate), Piericidin A.
(Mnemonic: R.A.P. stops Complex I)
◦ Complex II: Malonate (competes with succinate), TTFA (Thenoyltrifluoroacetone - iron chelator),
Carboxin.
◦ Complex III: Antimycin A, BAL (British Anti-Lewisite / Dimercaprol). (Mnemonic: Anti-BAL
blocks 3)
◦ Complex IV: Cyanide (CN-), Carbon Monoxide (CO), Hydrogen Sulfide (H2S), Sodium Azide
(NaN3). (Mnemonic: Gases + Azide block the final O2 step at IV)
2. Inhibitors of Oxidative Phosphorylation (ATP Synthase):
◦ ATP/ADP Translocase: Atractyloside (blocks ADP entry into matrix / ATP exit). (Mnemonic: A-
tract-ylo-side stops transport)
◦ F0 Subunit: Oligomycin, Venturicidin (block proton flow through F0). (Mnemonic: O-ligomycin
blocks F-O)
◦ F1 Subunit: Aurovertin (inhibits the catalytic part).
3. Uncouplers:
◦ Mechanism: Disrupt the coupling between electron transport and ATP synthesis by dissipating the
proton gradient. Electron transport continues (O2 consumption increases), but energy is released as
heat instead of being used for ATP synthesis.
 ◦ Chemical Uncouplers: DNP (2,4-Dinitrophenol), Dinitrocresol, FCCP (Carbonyl cyanide-p-
trifluoromethoxyphenylhydrazone), Aspirin (high doses).
◦ Physiological Uncouplers:
▪ Thermogenin (UCP1 - Uncoupling Protein 1): Found in Brown Adipose Tissue (BAT).
Responsible for non-shivering thermogenesis in newborns (explains chubby cheeks for
insulation/heat) and hibernating animals. UCP1 acts as a regulated proton channel.
▪ Others mentioned: Thyroxine, Long-Chain Fatty Acids (LCFA), Unconjugated Bilirubin.

VII. High Energy Phosphates

• Compounds releasing > 7 kcal/mol upon hydrolysis of their phosphate bond(s).

• Examples: ATP (terminal two ~P bonds), ADP, Phosphoenolpyruvate (PEP), 1,3-
Bisphosphoglycerate (1,3-BPG), Creatine Phosphate, Carbamoyl Phosphate.
◦ Note: PEP and 1,3-BPG are used in Substrate-Level Phosphorylation.

VIII. Clinical Correlations Recap

• Newborn Chubby Cheeks: Due to Brown Adipose Tissue containing UCP1 (Thermogenin) for non-
shivering thermogenesis.
• Carbon Monoxide Poisoning: CO inhibits Complex IV, blocking electron transfer to O2, halting ATP
synthesis and causing death (as seen in the defective heater scenario).

Key Takeaways to Remember:

• Electrons flow from low to high redox potential, releasing energy.

• ETC is located in the inner mitochondrial membrane.
• Complexes I, III, IV pump protons; Complex II does not.
• Oxygen is the final electron acceptor (reduced to water at Complex IV).
• Proton Motive Force (PMF) couples electron transport (oxidation) to ATP synthesis (phosphorylation)
via ATP Synthase (Chemiosmotic Theory).
• NADH yields ~2.5 ATP, FADH2 yields ~1.5 ATP.
• Know the specific inhibitors for each complex and ATP synthase components.
• Uncouplers (like DNP, UCP1) dissipate the proton gradient, generating heat instead of ATP.
• UCP1/Thermogenin in BAT is crucial for non-shivering thermogenesis.
• CO and Cyanide are deadly inhibitors of Complex IV.