FACTORS AFFECTING ENZYME

ACTIVITY
Part Two
 Vmax
• The active site of all enzyme molecule is occupied
by substrate molecule.
• The entire enzyme is in the form of enzyme
substrate complex.
• All the substrate are about to be converted to
product.
 ½ Vmax
• At this point half of the enzyme molecules are
bound with substrate.
• Which means 50% of substrate are in the form of
enzyme substrate complex
 Km
• The substrate concentration at half maximal
velocity.
 Significance of Km value

• Km value is characteristic feature of a particular

enzyme for a specific substrate.
• It is the signature of the enzyme.
• It is constant
• Km is independent of enzyme concentration.
• If enzyme concentration is doubled Vmax is
doubled.
• But ½ Vmax remains same.
• Irrespective of enzyme concentration 50% of
enzyme molecules are bound to substrate at that
particular substrate concentration.
• Km is independent of enzyme concentration.
• If enzyme concentration is doubled Vmax is
doubled.
• But ½ Vmax remains same.
• Irrespective of enzyme concentration 50% of
enzyme molecules are bound to substrate at that
particular substrate concentration.
• Km denotes affinity of the enzyme for the
substrate
• Low Km value indicates a high affinity of the
enzyme for the substrate and high Km value
indicates low affinity
• For example, hexokinase has more affinity for
glucose than glucokinase.
 Affinity

• Affinity is the tendency of the enzyme to bind to

the substrate.
• A high affinity means it binds readily even at low
concentration.
• A low affinity means it binds reluctantly even at
higher concentration.
 Double reciprocal plot

• At higher substrate concentrations determining Km

and Vmax was difficult.
• Hence experimental data at lower concentration is
plotted as reciprocals. The straight line thus
obtained is extrapolated to get the reciprocal of Km
• This is called Lineweaver – Burk plot or double
reciprocal plot where intercept in X axis is taken as
minus 1/Km
 1 Km
V Vmax

1
Vmax

--1
1
Km
[S]
 Dixon plot

• The velocity is measured at several concentrations

of inhibitor when the substrate concentration is
kept constant.
• It is used for determining inhibition constant.
• If 1/V is plotted against I a straight line is obtained.
1/V

I-Inhibitor
 Cooperative binding

• When the enzyme has many subunits the binding

of substrate to one unit enhances the affinity for
binding to other subunits. This is called cooperative
binding.
• Here a sigmoid shaped saturation curve is obtained.
• The determination of Km value is invalid and
instead Hill equation is employed.
FACTORS AFFECTING ENZYME
ACTIVITY
Part Three
 3. Concentration of products
• When equilibrium is attained in a reversible
reaction the rate of the reaction slows down.
• As the product is further accumulated the reaction
either stops or is reversed.
• So when the final product of a pathway is
accumulated it inhibits the whole pathway. This is
called feedback inhibition
 E1 E2 E 3
A B C D
 4. Effect of temperature

• When the temperature of the medium is increased,

the velocity of the reaction also increases and then
reaches a maximum after which it falls.

• A bell shaped curve is obtained if a graph is plotted

with velocity against temperature
 Optimum temperature

• The temperature at which maximum amount of the

substrate is converted to product per unit time is
called optimum temperature

• Human enzymes have optimum temperature around

37 degree centigrade
 Temperature coefficient

• It is the factor by which the rate of catalysis is

increased by a rise of 10 degree celsius.
• It is denoted by Q10
 Increased temperature

Impart kinetic energy to molecules

Increased number of molecules enter in to

transition state by overcoming energy barrier

Reaction rate is increased

 Higher temperature

Denaturation of enzyme proteins

Consequent loss of tertiary structure of protein

Activity of the enzyme is decreased

 5. EFFECT OF pH

• Each enzyme has optimum pH

• pH decides the charge on amino acid residues at the
active site.
• The net charge of enzyme protein influences
substrate binding and catalytic activity.
 5. EFFECT OF pH

• The graph plotted gives a bell shaped curve.

• The enzyme activity is reduced if the pH is lowered
or raised than optimum pH.
• Most human enzymes have optimum pH between 6
to 8.
• Exceptions are
➢ Pepsin with optimum pH between 1 and 2
➢ Alkaline phosphatase between 9 and 10.
 6. Enzyme activation

A) Presence of inorganic ions that can activate

enzymes
• Examples:
➢ Chloride ions activating salivary amylase
➢ Calcium ions activating lipase
 6. Enzyme activation
B) Conversion of inactive proenzyme or zymogen to
active enzyme
• This is done by splitting of peptide bond or removal
of small polypeptide which will either unmask active
site or bring conformational orientation.
• Examples:
➢ Trypsinogen to trypsin
➢ Chymotyrpsinogen to chymotrypsin.
➢ Coagulation factors
➢ Complement cascade pathway.
• All gastrointestinal enzymes are synthesized in the
form of proenzymes and they are activated only
after they are secreted in to alimentary canal.
• This prevents autolysis of cellular structural proteins.
FACTORS AFFECTING ENZYME
ACTIVITY
Part Four
 Enzyme inhibition
1) Competitive inhibition
2) Noncompetitive inhibition
3) Uncompetitive inhibition
4) Suicidal inhibition
5) Allosteric inhibition
6) Feedback inhibition
 a) COMPETITIVE INHIBITION

• The inhibitor is structurally similar to the substrate

• Hence it competes with the substrate for binding to the
active site on enzyme.
• Since the formation of enzyme–substrate complex is
decreased and so the product obtained is also
decreased.
 CHANGES IN ENZYME KINETICS
• The binding of substrate and inhibitor to the active
site depends upon their relative concentration.
• So when the substrate concentration is gradually
increased, keeping the inhibitor concentration as
constant, the enzyme activity is gradually increased.
• Hence, at higher substrate concentration [S], the
effect of inhibitor is overcome.
 CHANGES IN ENZYME KINETICS
• Vmax remains the same
• Km of the enzyme is increased.
• The affinity of enzyme towards substrate is
apparently decreased in the presence of inhibitor.

• So the velocity of the reaction is also decreased.

 Examples

1. MALONIC ACID is the competitive inhibitor of

succinate dehydrogenase in TCA cycle, because
malonic acid resembles succinate
 Examples

2. SULPHUR DRUGS AND PABA

Sulphur drugs contain sulphanilamide which is similar
to Para-amino benzoic acid
 Examples

2. METHOTREXATE
➢ Methotrexate is a structural analog of folic acid and
act as competitive inhibitor for dihydrofolate
reductase enzyme.
➢ Hence THFA formation and DNA synthesis are
affected.
➢ So cancer cells fail to thrive and die.
 Examples

➢ The enzyme Dihydrofolate reductase (DHFR) reduce

Folic acid to dihydrofolic acid (DHF) and further
reduce it to Tetrahydro Foilc acid (THF)
➢ This THFA helps in the formation of DNA
➢ In cancer cells this activity is very fast as they are
rapidly dividing.
 Examples

3. DICOUMAROL
➢ Vitamin K is required for gamma carboxylation of
glutamic acid residues of clotting factors like
prothrombin which is necessary for blood
coagulation.
➢ Vitamin K, in this process is changed to epoxide
form and is reconverted back quinone form by
reductase enzyme.
 Examples

3. DICOUMARAL
➢ Dicumeral and its synthetic analog called warfarin
are structural analog of vitamin K. Hence they act as
competitive inhibitor for reductase enzyme which
cause decreased regeneration of vitamin K.
➢ This results in decreased blood clotting.
➢ Dicumeral is used in the treatment of
thromboembolic diseases like cerebral stroke.
FACTORS AFFECTING ENZYME
ACTIVITY
Part Five
 Enzyme inhibition

• Competitive inhibition
• Noncompetitive inhibition
• Uncompetitive inhibition
• Allosteric inhibition
• Suicidal inhibition
 b) Non-competitive inhibition

• The inhibitor is not structurally similar to the

substrate
• There is NO competition with the substrate for
binding to the active site on enzyme.
• This is also called as mixed inhibition.
 b) Non-competitive inhibition

• The inhibitor binds to different domain on the

enzyme and not on active site.
• The inhibitor combine with the enzyme by
forming a covalent bond and then the reaction
becomes irreversible.
 b) Non-competitive inhibition

• It induces a conformational change in the enzyme

causing a decrease in catalytic activity.
• Substrate can still bind to the enzyme but the
reaction cannot be catalyzed at optimal rate. This
causes decreased product formation.
 CHANGES IN ENZYME KINETICS
• An increase in substrate concentration will not
relieve the inhibition.
• V max is reduced.
• Km value is not changed.
• (because the remaining enzyme molecules have
same affinity towards the substrate)
 EXAMPLES
1. Fluoride inhibit the enzyme enolase by removal of
magnesium and manganese ions.
2. EDTA(Ethylene diamine tetra-acetic acid) remove
metal ions required for the action of enzymes like
calcium for pancreatic lipase and blood coagulation
3. BAL (British Anti Lewisite or dimercaprol) is used as
an antidote for heavy metal poisoning.
4. Heavy metal ions like mercury, lead and silver react
with SH group of enzyme and inactivate them
5. Cyanide inhibiting cytochrome oxidase
 c) Uncompetitive reversible inhibition

• The inhibitor binds to enzyme-substrate complex

and not to the enzyme.
• Both Vmax and Km are reduced
 EXAMPLES

1. Inhibition of placental alkaline phosphatase by

phenyl alanine
2. Sodium valproate inhibit gamma-aminobutyric
acid (GABA) transaminase.
3. Campothecin inhibiting topoisomerase1 and
used in cancer therapy
REGULATION OF ENZYME ACTIVITY
 Regulation of enzyme activity

1. Coarse regulation
2. Fine regulation
 1. Coarse regulation

• Coarse regulation of enzyme activity is a process in

which the activity of an enzyme is controlled by
regulating the amount of enzyme present.
• This is done by altering the
➢ transcription
➢ translation of the enzyme
➢ altering the stability of its protein structure.
 2. Fine regulation

• Fine regulation of enzyme activity is the ability to

precisely control the activity of an enzyme in
response to a variety of internal and external
factors.
 1. Coarse regulation

a) Induction
b) Repression
b) Repression
 b) Repression

• Repressor acts at the level of genes.

• When heme is not available the operator site is free
and the enzyme is synthesized.
• When heme is surplus, it acts as corepressor and in
combination with aporepressor it represses the
operator site. The enzyme could not be synthesized.
 a) Induction

• Induction is effected through the process of de-

repression.
• The inducer will relieve the repression on the
operator site and will remove the block on the
biosynthesis of the enzyme molecules.
 a) Induction

• In bacteria when the media contains only lactose

and absence of glucose, there will be induction of
lactose utilizing enzymes.
 2. Fine regulation

• It is executed by
a) Allosteric regulation
b) Covalent modification
c) Feedback regulation
d) Compartmentalization
e) Stabilization
 a) Allosteric regulation

• Allosteric enzymes have one active site for

substrate binding and another separate allosteric
site for modifier binding.
 a) Allosteric regulation

• The binding of regulatory molecule can increase the

enzyme activity called as allosteric activation. The
regulatory molecule is called positive modifier.
• If the regulatory molecule inhibit the enzyme
activity it is called allosteric inhibition and the
molecule is called negative modifier.
 b) Covalent modification

• Change in activity of enzyme due to chemical

changes involving covalent bonds is called covalent
modification.
• This is achieved by adding new groups by forming
covalent bond or removing groups by cleaving
covalent bond
 b) Covalent modification

• There are three methods mainly for covalent

modification.

i. Protein phosphorylation
ii. ADP ribosylation
iii. Zymogen activation
 i) Protein phosphorylation
• Hormone binds to the membrane receptor to form
hormone receptor complex.
• This activates adenyl cyclase which is mediated
through G protein
• The active adenyl cyclase converts ATP to cyclic AMP
which acts as second messenger.
 i. Protein phosphorylation
• cAMP activate protein kinase.
• The protein kinases in turn phosphorylate the
enzyme.
• Enzymes can become active or inactive on
phosphorylation or dephosphorylation.
Enzyme Phosphorylated enzyme
Examples of Covalent Modification
Acetyl-CoA carboxylase Inactive
Glycogen synthase Inactive
Pyruvate dehydrogenase Inactive
HMG-CoA reductase Inactive
Pyruvate kinase Inactive
PFK2 Inactive
Glycogen phosphorylase Active
Citrate lyase Active
Phosphorylase b kinase Active
HMG-CoA reductase kinase Active
Fructose-2,6-bisphosphatase Active
 ii. ADP ribosylation
• When ADP ribose is added to alpha subunit of G
protein it cause inhibition of GTPase activity.
• So G protein remains active.
• ADP ribosylation of glyceraldehyde -3-phospahte
dehydrogenase results in inhibition of glycolysis
 iii. Zymogen activation
• Conversion of inactive proenzyme or zymogen to
active enzyme
• This is done by splitting of peptide bond or removal
of small polypeptide which will either unmask
active site or bring conformational orientation.
 Examples
➢ Trypsinogen to trypsin
➢ Chymotyrpsinogen to chymotrypsin.
➢ Coagulation factors
➢ Complement cascade pathway.
c) Feed back regulation
 E1 E2 E 3
A B C D
• Feedback regulation is a process by which the
output of a system is used to maintain a desired
output level in response to changes in its
environment.
• For example, dietary cholesterol decrease the
hepatic synthesis of cholesterol by enzyme
repression at genetic level by cholesterol.
 d) Compartmentalization
• Some reactions in a given metabolic pathway occurs
in different organelles.
• The metabolic intermediates which have to be
shuttled across membranes provide a control point
for regulation.
• For example, in heme synthesis takes place partly in
mitochondria and partly in cytoplasm
 e) Stabilization
• Enzyme molecules worn out gradually and finally
become degraded.
• The overall enzyme activity can be enhanced if such
degradation is prevented. This is called stabilization
of enzyme
• Examples are:
➢ Phosphofructokinase stabilized by growth
hormone.
➢ Tryptophan pyrrolase stabilized by tryptophan.
FIRST SLIDE
FACTORS AFFECTING ENZYME
ACTIVITY
Part Six
 Suicidal inhibition

• It is also called as mechanism based inactivation

• The inhibitor is structural analog of the substrate
• It undergoes the reactions that the substrate
would normally undergo by attaching to the active
site
• In that process it is converted in to a intermediate.
 Suicidal inhibition

• The intermediate forms irreversible covalent

complex with the amino acid intermediates at
active site which will affect the configuration of
the enzyme.
• This will affect substrate binding and inhibit
enzyme activity.
 Examples

❖ Inhibition of Xanthine oxidase by Allopurinol

• Allopurinol is acted upon by Xanthine oxidase
and gets converted to Alloxanthine
• Alloxanthine binds to the xanthine oxidase and
cause effective and irreversible inhibition
 Examples

❖ Inhibition of prostaglandin synthesis by Aspirin

• Arachidonic acid is acted upon by Cyclooxygenase
(COX) to form prostaglandins
• Aspirin cause acetylation of serine residues in the
active centre of enzyme Cyclooxygenase and
prostaglandin synthesis is inhibited.
 Allosteric inhibition

• Allosteric enzymes are those where catalysis at

their active site is modified by the presence of
effectors at the allosteric site.
• Allosteric enzymes have one active site for
substrate binding and another separate allosteric
site for modifier binding.
• These modulator binding changes the conformation
of the enzyme leading to change in its activity.
• If the regulatory molecule inhibit the enzyme
activity it is called allosteric inhibition and the
molecule is called negative modifier
• The binding of regulatory molecule can increase the
enzyme activity called as allosteric activation. The
regulatory molecule is called positive modifier.
• When the enzyme has many subunits the binding of
substrate to one unit enhances the affinity for
binding to other subunits. This is called positive
cooperativity.

• If the binding of substrate to one unit decreases the

affinity for binding to other subunits. This is called
negative cooperativity.
 TYPES

➢ Homotropic allosteric enzymes are those in which

the substrate itself acts as allosteric modulator. So
the catalytic site functions as the allosteric site.
• Example: Aspartate binding to aspartate
transcarbomyolase

➢ Heterotropic allosteric enzymes are those in which

the substrate and allosteric modulator are different.
So the catalytic site and allosteric site lie separate
on enzyme.
 Enzyme kinetics

• Here a sigmoid shaped saturation curve is obtained.

• Vmax is reduced because inhibitor causes
modification in the configuration of catalytic site
• Km is usually increased.
 Examples

➢ Phosphofructokinase in glycolysis

✓ Allosteric inhibitor is CTP and citrate

✓ Allosteric activator is AMP and Fructose 2,6
bisphosphate
 FEEDBACK INHIBITION

• The activity of the enzyme is inhibited by the

product of the enzyme
• Example is inhibition of hexokinase by Glucose-6-
phosphate
 End product inhibition

E1 E2 E 3
A B C D
• D inhibiting E1 is called end product inhibition.
• Because of this unnecessary execution of a pathway is
prevented thus saving energy
This is the first step in heme synthesis.
The end product heme will inhibit ALA synthase
LDH & CPK
 LDH

• Lactate dehydrogenase
 i) Function

• LDH catalyze reversible inter-conversion reaction

of pyruvate and lactate.
• The conversion of pyruvate to lactate takes place
in anaerobic condition.
• NADH/NAD are the coenzymes.
 ii) Isoenzyme forms

• LDH enzyme is a tetramer with 4 subunits.

• The subunits are made up of either one or both of
polypeptide chains – H(Heart) and M(Muscle)
• H and M subunits are encoded by different genes
in heart and muscle respectively.
• So there are five isoenzyme forms of LDH
• They are H4,H3M1, H2M2, H1M3 and M4.
iii) Distribution
Subunit Electrophore Activity Tissue of Percentage in
Characteristic Featuresatof60°C
make up of tic
LDH Isoenzymes.
for origin human
serum (Mean)
isoenzyme mobility at 30 min
pH 8.6

H4 Fastest Not Heart 30%

destroyed muscle

H3M1 Faster Not RBC, Brain 35%

destroyed

H2M2 Fast Partially Brain, 20%

destroyed Leukocytes

H1M3 Slow Destroyed Liver, 10%

Leukocytes

M4 Slowest Destroyed Skeletal 5%

muscle
 iv) Separation

• The isoenzymes are separated by cellulose acetate

electrophoresis.
• The bands are identified by staining with nitroblue
tetrazolinum
 v) Normal value

• 100 – 200 U/L

 vi) Measurement

• RBC has high LDH activity. So sample should not

be hemolysed. Otherwise false high value will be
obtained.
 vii) Biochemical importance

1. Myocardial infarction
2. Other diseases.
Subunit Electrophore Activity
No. of Tissue of Percentage in
Characteristic Featuresatisoenzyme
make up of tic
of60°C
LDH Isoenzymes.
for origin human
serum (Mean)
isoenzyme mobility at 30 min
pH 8.6

H4 Fastest Not Heart 30%

LDH-1
destroyed muscle

H3M1 Faster Not RBC, Brain 35%

LDH-2
destroyed

H2M2 Fast Partially Brain, 20%

LDH-3
destroyed Leukocytes

H1M3 Slow Destroyed Liver, 10%

LDH-4
Leukocytes

M4 Slowest Destroyed
LDH-3 Skeletal 5%
muscle
 Myocardial infarction

• The damage of myocardium release large quantities

of LDH1 nearly up to 10 times in serum. So the total
LDH concentration becomes very high.
 Time course in MI

• Blood levels of LDH starts increasing by 10-12

hours after the onset of MI.
• The peak values are attained after 40 to 48 hours
and then returns to normal by 5 to 6 days.
 a) Myocardial infarction

• Normally LDH2 is maximal in activity. So the ratio

of LDH1:LDH2 is less than 1.
 Flipped ratio
• So the normal LDH1: LDH2 ratio which is less than
one in healthy individuals gets reversed in MI due
to increase in LDH1.
• This reversal of ratio in MI is called flipped ratio.
 2. Other diseases

• LDH2 is increased in hemolytic anemias

• High levels of LDH4 is seen in hepatocellular
damage.
• LDH5 is increased in muscular dystrophies.
Subunit Electrophore Activity
No. of Tissue of Percentage in
Characteristic Featuresatisoenzyme
make up of tic
of60°C
LDH Isoenzymes.
for origin human
serum (Mean)
isoenzyme mobility at 30 min
pH 8.6

H4 Fastest Not Heart 30%

LDH-1
destroyed muscle

H3M1 Faster Not RBC, Brain 35%

LDH-2
destroyed

H2M2 Fast Partially Brain, 20%

LDH-3
destroyed Leukocytes

H1M3 Slow Destroyed Liver, 10%

LDH-4
Leukocytes

M4 Slowest Destroyed
LDH-3 Skeletal 5%
muscle
• Since total LDH is increased in other conditions
also, it is not used as cardiac marker now and is
replaced by other specific markers.
 CPK

• Creatine kinase or Creatine Phosphokinase

 a) Function

• It catalyse the reaction where creatine is

phosphorylated to creatine phosphate .
• Creatine phosphate is a very high energy
compound which help in maintaining
concentration of ATP.
 b) Isoenzyme forms

• CPK has three isoenzymes (CPK 1,2,3) due to

differential composition of two subunits- B and M.
c) Distribution

% in serum

1%

5%

80%
 d) Normal concentration

• Males- 20 to 80 IU/L
• Females- 20 to 50 IU/L
f) Biochemical significance
 i) Myocardial infarction

• In myocardial infarction CPK-MB starts raising by 3

hours, peaks by 30 hours and returns to normal by
3 days.
• CPK-MB is most useful when ECG changes are not
clearly indicative of MI.
• The magnitude of the rise is proportional to the
cardiac muscle infarction indicating severity of MI
 ii) Other diseases

• CPK can raise in crush injuries, fractures and

muscle dystrophies.
• It can rise in cerebral stroke.
ISOENZYMES
Part two
 HEPATIC MARKERS

1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Nucleotide phosphatase
5. Gamma glutamyl transferase
 HEPATIC MARKERS

1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Gamma glutamyl transferase
 1.ALT

• Alanine Transaminase
• SGPT
• Serum Glutamate Pyruvate Transaminase
 Transamination

COOH COOH COOH COOH

CH-NH2 C=O C=O CH-NH2
ALT
CH2 + CH3 CH2 + CH3
CH2 CH2
COOH COOH

Glutamate + Pyruvate ➔ α keto glutarate + Alanine

Alanine Amino Transferse

Serum Glutamate Pyruvate Transaminase
 NORMAL SERUM LEVEL

• Males - 13 to 35 U/L
• Females - 10 to 30 U/L
 RAISED LEVELS
• More specific for liver disease as marked rise in
hepato cellular damage.
• Moderate increase is seen in cirrhosis, Hepatitis C
and NASH (Non alcoholic steatohepatitis)
• Very high levels (300 to 1000 U/L) are seen in acute
hepatitis caused by virus or toxins
 2. AST

• Aspartate Transaminase
• SGOT
• Serum Glutamate Oxaloacetate Transaminase
 Transamination

COOH COOH COOH COOH

CH-NH2 C=O C=O CH-NH2
AST
CH2 + CH2 CH2 + CH2
CH2 COOH CH2 COOH
COOH COOH

Glutamate + Oxaloacetate ➔ α keto glutarate + Aspartate

Aspartae Amino Transferse

Serum Glutamate Oxaloacetate Transaminase
 NORMAL SERUM LEVEL
• 8 to 20 IU/L
 RAISED LEVELS
• Moderate to severe increase in parenchymal liver
disease like hepatitis, liver malignancy and in
hemolytic anaemia
• Previously it was also used as a marker for cardiac
injury.
• In necrosis or hepatic malignancy AST is greater than
ALT due to release of mitochondrial AST. It is also
higher than ALT in alcoholic hepatitis
 DE RITIS RATIO
• AST/ALT ratio is known as ‘De Ritis ratio’ and ratio
greater than 2:1 is suggestive of alcoholic liver
disease.
• This ratio is greatly elevated (3:1) in hepatitis C virus
infection with cirrhosis.
 3. ALP

• Alkaline phosphatase
• It is produced in bones, liver, placenta and
intestine.
• It is localized in cell membranes as ectoenzyme
and is associated with transport mechanisms.
 REFERENCE RANGE

• 40 to 125 u/l or 3 to 13 KA u/dl

 ISOENZYME FORMS
• Alpha 1
• Alpha 2 heat labile
• Alpha 2 heat stable
• Pre beta
• Gamma
 ALPHA 1 ALP
• It is also called biliary ALP as it is synthesized by
epithelial cells of biliary canaliculi.
 ALPHA 2 ALP

• There are two types

1. Alpha 2 heat labile ALP
2. Alpha 2 heat stable ALP
 ALPHA 2 HEAT LABILE ALP
• It is also called hepatic ALP since it is produced by
hepatic cells.
• It loses its activity by heating at 65 degree celsius
for 30 minutes.
• This forms 25% of total ALP
 REGAN ISOENZYME

• It is also called Carcino placental isoenzyme.

• It resembles placental/ heat stable alpha 2 ALP
• It is found in carcinoma of lungs, liver and
intestine.
• It constitute 1% of total ALP.
 BIOCHEMICAL SIGNIFICANCE
1. Moderate increase about 2 to 3 times is seen in
hepatic diseases like infective hepatitis, alcoholic
hepatitis and hepatocellular carcinoma.
2. Very high levels about 10 to 12 times is seen in
extrahepatic obstruction or intrahepatic cholestasis.
• Extrahepatic obstruction is caused by
cholelithiasis or carcinoma of pancreatic head
causing obstructive jaundice.
• Intrahepatic cholestasis is caused by viral
hepatitis or by drugs like chlorpromazine
 4. GGT

• Gamma Glutamyl Transferase

• Also called as Gamma glutamyl transpeptidase
• It can tansfer gamma glutamyl residues to substrate
• It is used in the synthesis of glutathione
• It has 11 isoenzymes
 REFERENCE RANGE
• 10 to 30 U/L
 DIAGNOSTIC USES
• It is routinely measured in patients with metabolic
syndrome, nonalcoholic fatty liver disease
(NAFLD) and non alcoholic steatohepatitis (NASH)
to detect deficiency in liver function.
• It is moderately increased in infective hepatitis,
prostate cancers.
• It is sensitive marker for alcohol abuse and the
elevation is proportional to amount of alcohol
consumption.
• Very high levels indicate alcoholic liver cirrhosis
 PROSTATE MARKER

• Prostate specific antigen

 PROSTATE SPECIFIC ANTIGEN
• It is produced from secretory epithelium of
prostate gland and secreted in to seminal fluid for
its liquefaction.
• It is a serine protease which is bound to alpha-2
macroglobulin and alpha-1 antitrypsin.
• The reference serum value is 1-5 units/L
• It is mildly increased in benign prostate
enlargement. The values above 10 units/L indicate
prostate cancer.
 G6PD
• Glucose 6 phosphate dehydrogenase
 G6PD
• It is an important enzyme in the Hexose
monophosphate shunt pathway of glucose.
• It is mainly used for production of NADPH.
 G6PD
• In GPD deficient cells the level of NADPH is low,
leading to increased accumulation of peroxides
resulting in premature cell lysis.
• This drug-induced hemolytic anemia is
characteristic of GPD deficiency.
 G6PD
• When certain drugs like aspirin, mepacrine,
primaquine and sulpha are taken by G6PD
deficient individuals, there will be sudden damage
to RBCs as these drugs stimulates peroxide
formation.

• Fava beans may also induce hemolytic anemia

which is called favism.
 G6PD
• GPD deficiency seems to protect the person from
falciparum malaria. The malarial parasites require
NADPH for optimal growth. Thus, GPD deficiency
has a selective protection in malarial endemic
regions
 G6PD
• NADPH is also necessary for reduction of met-
hemoglobin (oxidized form) to hemoglobin. Hence
in GPD deficient individuals, met-hemoglobinemia
may also be manifested.