Enzymology

- It refers to the science of enzymes to the

"eg. pepsinogon + HCI = popsin
diagnosis and treatment of diseases
Changes in enzyme concentration in tissue
. Greek words en which means in and zyme
which means yeast. cells reflect changes in states of health and
. Substance that catalyzes a given chemical disease of the tissue
reaction, cellular catalyst

. They are biologic catalysts that causes

reactions in the body to take place DEFINITION OF TERMS

. There are over 1.500 enzymes and many of

which catalyzes the same reactions . COENZYME

. These are non-protein biochemicals or ions

CHARACTERISTICS OF AN

ENZYME . Part of the enzyme reaction

One of the most complicated type of . Essential to the catalytic activity as a co-
proteins in terms of both structure and substrate
functions Organic molecules, smaller than proteins

. Proteins in nature; easily denatured with Often the coenzyme is derived from a vitamin.
varying molecular weight and masses and one coenzyme may be associated with
Enzymes are amphoteric

. Enzymes operate in high rates different enzymes.

CHARACTERISTICS OF AN .e.g. NAD, pyridoxal phosphate

ENZYME

Reactions catalyzed are frequently reversible ACTIVATORS

. These are inorganic ionic cofactors

Usually, it operates with assistance of a non- . Usually metal ions
protein cofactor

Enzymes are synthesized in an inactive . Cations of 2* species

state/zymogen state . Obligate to the enzymatic reaction
. Metabolic regulators of a given reaction ENZYME STRUCTURE

series . It refers to the molecular/structural

"e.g. Mg*2, Na'l, K*. Zn*2 arrangement of enzyme molecule

4 Existing Structures

HOLOENZYME

- Refers to the combined enzyme and coenzyme . Primary structure - refers to the sequence

of amino acids joined by peptide bonds

APOENZYME . Secondary structure - conformation of the

. Enzymes without the presence of a cofactor steric arrangement or conformation of the

. Apoenzyme + Coenzyme = Holoenzyme segments of polypeptide chain

. PROSTHETIC GROUP ENZYME STRUCTURE

. A coenzyme that cannot be removed from its . Tertiary structure - arises from the interactions

attachment with an enzyme using dialysis among side chains/groups of the polymer chain:

.e.g. pyridoxal PO4 in transaminase reaction folded structure of an enzyme

. Quaternary Structure - refers to the

relationship
METALLOENZYME
between subunits
Inorganic activators existing as a part of the

enzyme molecule
Subunits perform specific functions
SUBSTRATE
. The secondary and tertiary structures are the

most important configurations of the enzyme

. Substances which are acted upon by an
enzyme because these structures are responsible for

and is converted into a new substance the characteristics such as coiling and folding

. PRODUCT resulting to conformational structure

. Substances derived from a transformed

substrate
Active Site

Refer to the area or portion of

on enzyme in which the

 Older names are dehydrogenases and oxidases

substrate is attached with the Assayed for investigation or gives information

regarding
enzyme molecule
heart attacks and liver problems
it is where the transformation of

the substrate takes place

Classification of Enzymes

. Examples of oxidoreductases
Classification of Enzymes
Oxidases
. Based on catalytic activity of an
. Glucose oxidase
enzyme
Cytochrome oxidase
.The International Union of
. Dehydrogenase

Biochemistry (IUB) enzyme

. LDH
commission categorized all enzymes
. Iditol DH
into 6 classes
. MDH
Oxidoreductases
. Isocitrate DH
Transferase's
Glucose-6-PO4 DH
Hydrolases
Hydroxybutyrate DH
Lyases

Isomerases
Oxidoreductases acts on
Ligases
Secondary alcohols

. Ketones
1. Oxidoreductases
. Alkenes
Enzymes catalyzing oxidation and reduction
reactions

Reduction - addition of hydrogen to a . Primary amines

double bond . Secondary amines

. NADH. NADPH

. Oxidation - removal of hydrogen to Example:

cleave a double bond * Lactate + NAD. LDH = pyruvate + NADH

Il. Transferases . Ornithine carbamyl transferase

. Enzymes that move and interact group of . Example:

atoms from one molecule to another . ATP + creatine + CK = ADP + creatine

phosphate
(Amine or PO, group)

Ill. Hydrolases
. Catalyzes the transfer of a functional
Enzymes involved in the splitting of
group from one molecule to another
molecules with water as a part of the
. Gives important information about liver
reaction process
Damage

Catalyze a hydrolysis reaction in which

. 2 major subtypes:
the addition of a water to a bond cause
. Transaminases which catalyze the
the bond to break.
transfer of an amino group from one

molecule to another.
3 groups of hydrolases

Esterases
. Kinases which catalyze the transfer of a
Peptidases
phosphate group from ATP to give ADP
Glycosidases
and a phosphorylated product.

ACP
. Examples of transferases
Leucine aminopeptidase
. Aspartic aminotransferase (AST)
Amylases
- Serum glutamate oxaloacetate transaminase
(SGOT)

. Alanine aminotransferase (ALT) ALP

. Serum glutamate pyruvate transaminase Trypsin

(SGPT]
Amylo-6-

glucosidase
. Creatine kinase

. Gamma glutamyl transferase

Cholinesterase . Glutamate decarboxylase

Pepsin . Tryptophan decarboxylase

Glucoside . Dehydratase

. Hydratase

Lipase

V. Isomerases

Galactosidase Enzymes responsible for the conversion of

one isomer to another

It involves the transformation from

IV. Lyases

Enzymes responsible for splitting molecules . Cis-trans

(lysis)
. L-D forms
. Catalyze the addition of a group to a double
. Aldehyde to ketone
bond or the removal of a group to form a
All reactions are reversible
double

bond in a manner that does not involve

No clinical importance
hydrolysis or oxidation.
Examples

. Glucose PO, isomerase

. Bonds broken are
. Ribose PO, isomerase
. C to C bond

. C to O bond

C to N bond

. Others

IV. Lyases

Assayed in disorders of skeletal muscles

Examples

. Aldolase
ENZYME NOMENCLATURE of 4 members separated by periods)

. Each enzyme was designated a name . Prefixed with letter E.C. (Enzyme Commission)

according to the reaction it catalyzes E.C. 3.1.3.2 - ACP

. Example: oxidation of glucose - glucose E.C. 3.1.3.1 - ALP

oxidase

. Ist digit - denotes the class of the enzyme

. It is costumary to identify enzymes by adding
. 2nd digit - denotes the sub-class of the
the suffix "ase" to the group upon which the
enzyme
enzyme acts
. 3rd digit - denotes the subsub-class
. Example: Urea - urease

. 4th digit - specific serial number given to each

Uric - uricase
enzyme within its subsub-class
Phosphate esters - phosphatase
. This approach removes all ambiguity about
Lipid - lipid esterase
the enzyme's identity
Lactose - lactase
. Trivial names
Starch - amylase
. A.K.A. non-specific, practical name, working

name
Naming of enzymes by the use of their
. Identical to systematic name and often a
empirical names

. Example: trypsin, pepsin

simplification of it

. Suitable for everyday use

Standard system of identifying enzymes
. Uses acronyms and abbreviations
. Formulated by IUB and IUPAC
. Orthophosphoric monoester phosphorylase

- ALP
Systematic name

Describe the nature of the reactions

Predict the function of the ff.
catalyzed
enzymes:

. Cellulase
Unique numerical code designation (consists
. L-amino acid oxidase the same specie

Sucrase Example: Lactate Dehydrogenase (LDH)

. Aspartate aminotransferase . Contains H & M sub-units (polypeptide

chains)

Maltase - catalyzes hydrolysis of maltose . The sub-units combine to form different

Lactate dehydrogenase - catalyzes the removal

of hydrogen from lactate ion
isoenzymes:
Fructose oxidase - catalyzes the oxidation of
H4 - LD1 - heart, RBC & renal tubules
fructose
H3M - LD2 - heart, RBC & renal tubules
Maleate isomerase - catalyzes the
isomerization/rearrangement of maleate ion H2M2 - LD3 - Lungs, Lymphocytes, spleen,
Lipase - catalyzes the hydrolysis of lipids pancreas
Malate dehydrogenase - catalyzes the removal HM3 - LD4 - Liver, skeletal muscles
of hydrogen from malate ion
M4 - LD5 - Liver, skeletal muscles
Tryptophan decarboxylase - catalyzes the
removal of carbon dioxide from tryptophan CHARACTERISTICS of

Glycogen synthetase - catalyzes the synthesis of ISOENZYMES

glycogen 1. Charged molecules - as shown in

Glucose oxidase - catalyzes the oxidation of electrophoresis

glucose 2. Mobility in ion Exchange Resin
Kinase - catalyzes the transfer of phosphate 3. Response to activation & inactivation process
group between substrates
E.g. ACP (RBC) - not inactivated by
ENZYME VARIANTS
2% Formalin
. Several distinct forms of enzymes
ACP (Prostate) - inactivated by
. Important in the diagnosis of specificity
2% Formalin
A. ISOENZYMES
4. Relative substrate specificities

E.g. ACP (RBC) is less sensitive to

Multichained enzymes
a-naphthyl PO
Enzymes of similar activity
5. Response to Inhibitors
Typically appearing in specific tissue, organ
E.g. ACP is inhibited by fluoride,
& Cell organelle of organisms belonging to
heparin, oxalates
 B.1 Enzymes of Secretion

B. HETEROENZYME - normally secreted in plasma at a high rate

-Enzymes of similar catalytic activity but but are rapidly disposed of to normal

are specie specific excretory channels

- concentration in plasma is maintained at

constantly low level
E.g. LDH of rabbits & humans

C. ALLOENZYME
Enzymes based on distribution
. Genetically-transmitted enzyme
Unilocular Enzymes
. Important in defining the biochemical
. These are enzymes found only in the cell
characteristics of an individual
sap
. Present only in selected individuals of
. Found only in one location
the same specie

Practical value in forensic medicine & genetics

Bilocular enzyme

. These are enzymes that are found in the

ORIGIN OF PLASMA ENZYMES:
mitochondria and cell sap
CLASSIFICATION OF ENZYMES IN BLOOD

A. PLASMA-SPECIFIC ENZYMES
ENZYME KINETICS
1. Generally secreted by the liver
E+S ES P+E
2. Enzymes that exert their function in
Where: E = enzyme that represents a true
plasma
catalyst
3. Examples: Coagulation factors
= not changed in the reaction
Fibrinolytic factors Complement system
S = substrate upon which the enzyme acts
B. NON-PLASMA SPECIFIC ENZYMES

1. Enzymes that do not have specific

P = the reacted substrate
functions in plasma
= represents a changed molecular
2. Plasma lacks activators or coenzymes w/C
species of
are necessary for enzyme activity
substrate

ES = postulated enzyme-substrate complex

3. Two Classes:
A. Factors that Affect Binding 1. TIME - refers to the condition when

of Enzyme to Substrate the enzymatic reaction is allowed to take place

Energy - refers to activation energy . If the catalytic activity of an enzyme on the

substrate is fast = shorter reaction time
. Molecular Compatibility -
The enzyme is freed & can act again on the
commonness/sameness between
remaining substrate
Enzyme & Substrate

. SUBSTRATE CONCENTRATION - direct

Space Availability - refers to the # of relationship

enzymes or substrates that can be . MICHAELIS-MENTEN CURVE - shows the

reacted relationship of the reaction velocity to the

. Specificity - refers to the particular substrate concentration

enzyme acting on a specific substrate . Two Phases:

a. First Order Kinetics

B. Factors Affecting E-S Combination -Enzyme conc. is fixed; Substrate conc. is varied

Rate of reaction is almost directly proportional

to
. Lock & Key - refers to the active site
substrate conc. at low values
being complementary in shape & size
Rate of reaction is dependent on substrate
to the substrate
conc.

. Induced Fit Model - the enzyme

Substrate concentration: Enzymatic reactions

Faster reaction but it reaches a saturation point

changes in shape during binding to be able to when all the enzyme
accommodate the substrate
molecules are occupied.

if you alter the concentration of the enzyme

C. Factors Influencing the then Vmax will change too

Enzymatic Reaction b. Zero Order Kinetics

-Reaction rate is unaffected by . Optimum pH - the point at w/c the

increased substrate concentration reaction rate is greatest

-Dependent on enzyme concentration . At pH 7.0-8.0, many enzymes show

-When maximum velocity is reached, maximum activity

the rate of increase in velocity is "O" . Pepsin is active at 1.5 and ALP at 10.5

(Zero order reaction)

6. Activators

3. Enzyme Concentration . Required for full enzyme activity

. Direct relationship . Help bind the substrate to the active site by

forming
. An increase in enzyme concentration results
ionic bridges
to an increase in the catalytic activity of the
. Help orient the substrate so it is attached to
enzyme
the enzyme in the correct configuration

. Examples
4. Temperature
Divalent Cations Mono Anions
. Optimum temperature - the temperature
- Fett
w/c is considered as favorable for the
- Cat
catalytic activity of the enzyme (30 - 37 C or
- Zn+ +

Mono Cations
37 - 40.C)
- K+
. Q10 value - refers to the increased reaction
-Nat
rate for every 10C increase (rate of reaction

is doubled)
CI
50 - 60 C: enzyme undergoes inactivation
- Br
and denaturation
- NO 3
Further increase in T leads to loss of

enzymatic activity
7. Inhibitors
. Some enzymes are stored at refrigerator. T
-Substances that decrease the rate of enzyme
(2-8.C)
reaction
5. pH - Hydrogen ion Concentration
 binding site

Functions: 2. Product of reaction - may itself be an

a. Binds to the active site, blocking access of the inhibitor of the forward reaction

substrate to the enzyme (Competitive 3. E-S complex does not break to yield
Inhibition)
products
b. Binds elsewhere on the enzyme causing
4. Chemical substances
change
E.g. Fluoride, Oxalate, Heparin, Cupric ion,

Tartrate
in shape that interferes w/ substrate binding
8. Coenzyme concentration
(Non-competitive Inhibition)
. Many enzymes require a coenzyme for the
c. Binds w/ the E-S complex; no product formed
reaction to proceed (Vitamins, Coenzyme A
(Uncompetitive Inhibition)
or B, NAD)

. These must be present in proper concentration

TYPES of INHIBITION
for many enzyme reactions to take place
a. Reversible Inhibition
9. Prosthetic Group
Inhibitors are possible removed from the
Coenzyme that firmly binds with the enzyme
system & enzyme is fully restored

ENZYME DENATURATION
Physical processes that remove inhibitors:
. Refers to the disruption of the 3-
dialysis, gel filtration
dimensional structure of the enzyme molecule
b. Irreversible Inhibition that leads to the loss of enzyme activity

Denaturation may be reversed if the reaction

process has not gone too far & if the denaturing
Inhibitors covalently combine w/ the enzyme
agent is removed
Physical methods are ineffective in

separating inhibitors from the enzymes

DENATURING CONDITIONS

. Elevated temperature - beyond 50 -

Examples of Inhibitors
60 C; weakens the stabilizing bonds
1. Excess substrate - causes competition
. Extremes in pH - causes unfolding of
between substrate molecules for a single
enzyme molecule
. Radiation

. Frothing . Thiamin - TPP (Thiamin pyrophosphate)

. Strong salt solution . Riboflavin - FMN/FAD

. Mechanical trauma . Niacin - NAD/NADH

. Time of exposure . Pyridoxine - PLP (pyridoxal phosphate)

. Chemicals . Folate - THE (tetrahydrofolate)

. B5 - COA

TYPES of ENZYME SPECIFICITY . Biotin - Bitotin

1. ABSOLUTE SPECIFICITY - when an enzyme can . B12 - B12

act and catalyze one unique reaction

2. GROUP SPECIFICITY - when some enzymes act

MOST COMMON DISTURBANCES
on different substrates belonging to the group
. 1. Kwashiorkor - disease resulting from a
3. STEREOISOMERIC - when an enzyme
deficiency of dietary protein relative to caloric
acts only on the specific isomer intake (protein-energy malnutrition).

. 2. Marasmus - results from slow starvation

caused by deficiency not only of protein but
UNITS of MEASUREMENT
also, of calories and other nutrients.
1. IU - International Unit Proposed by the
.3. Anemia - blood condition involving an
Commission on Enzymes (IUB)
abnormal reduction in the number of RBCs or in
-Defined as the quantity of enzyme that will their hemoglobin content.
catalyze the reaction of 1 umol of substrate per
. 4. Gout - caused by faulty metabolism of uric
minute
acid produced in the body by breakdown of
-Expressed in terms of U/L or mU/L proteins.

2. Katal .5. Phenylketonuria (PKU) - this is due to the

absence of the enzyme phenylalanine hydrolase
-a unit of enzyme activity w/c converts 1 mol of which converts phenylalanine to tyrosine.
substrate per second .6. Albinism - this is due to the absence of
-conforms w/ the Systeme International (SI) tyrosinase, which is necessary for the formation
scheme of units of melanin.

Other units: .7. Alkaptonuria - this is due to the absence of

the homogentisic acid oxygenase (homogentisic
3. Bodansky acid acetate and fumarate).
4. Gutman-Gutman 8. Tyrosinosis - this is due to the absence of
5. BLB (Bessiy Loury Brock) hydroxyphenylpyruvic acid oxygenase which
converts hydroxyphenylpyruvic acid to
homogentisic acid.

a. Infant’s exhibit diarrhea, vomiting, and a

"cabbage- like" odor and fail to thrive.

b. Without treatment, death from liver failure

ensues 6-8 months.

c. Treatment: diet low in tyrosine,

phenylalanine, and methionine.