Isoenzymes

• Definition

• They are isomers of the same enzyme,

have the same catalytic activity but differ
in physical, molecular weight, and
electrophoretic mobility
 Lactate dehydrogenase (LD)
• Tetramer
• Two different chains (H - heart, M - muscle)
• Five isoenzymes:
LD1 (H4), LD2 (H3M), LD3 (H2M2), LD4 (HM3), LD5 (M4)
• Widely distributed in the human body
• LD1 + LD2 ….. markers of myocardial infarction (MI)

2
 LDH
isoforms
 Clinical significance of LDH

 In normal serum, LDH2 (H3M) predominant

isoenzyme & LDH5 is rarely seen.
 In myocardial infarction, LDH1(H4) levels are
greater than LDH2.
 Megaloblastic anemia (50 times upper limit of
LDH 1 and LDH 2)
 Muscular dystrophy, LDH5 (M4) is increased.
 Toxic hepatitis with jaundice (10 times more
LDH5)
 Creatine kinase (CK)
• Dimer
• Two different chains (M – muscle, B – brain)
• Three isoenzymes: MM (muscle), MB (heart), BB (brain)
• Major isoenzyme in blood is MM (95 %)
• MB form in blood: 0 – 6 %
• BB in blood: traces (BB cannot pass across blood-brain
barrier) zero in blood

• MB isoenzyme …. excellent marker of myocardial

infarction
5
 Creatinine phosphokinase
(CPK)
 It catalyses creatine to creatine phosphate.
 Normal serum value:
 15-100 U/L for males & 10-80 U/L for females.
 CPK consists of 3 isoenzymes.
 Each isoenzyme of CK is a dimer;
 Molecular weight of 40 kD.
 The subunits are called B for brain (chromosome
-14) & M for muscle (chromosome -19)
 Three Iso-enzymes are separated by

electrophoresis.

CPK-1 (also called CPK-BB) is found mostly in

the brain & lungs.

CPK-2 (also called CPK-MB) is found mostly in

the heart.

CPK-3 (also called CPK-MM) is found mostly in

skeletal muscle.
 Clinical significance
of CK
 CPK & heart attack: very sensitive

 CPK2 isoenzymes is very small, (2% of total

CPK activity) & undetectable in plasma.

 In myocardial infarction (MI), CPK2 levels are

increased within 4 hrs, then falls rapidly.

 Total CPK level is elevated upto 20-folds in MI.

 Regulation of enzyme activity
Methods of regulation of enzyme activity

1. Modulation of enzyme activity

(a) Covalent modification
(b) Allosteric modulation
2. Feedback inhibition
3. Enzyme induction
4. Compartmentation
5. Proteolytic cleavage of proenzymes
(zymogens)
 1-a).Covalent modification

Phosphorylation /dephosphorylation Some

enzymes are active when phosphorylated,
while others are inactive when
phosphorylated.

Active Inactive
 1-b). Allosteric regulation
Allosteric enzymes have a second regulatory
site (allosteric site) distinct from the active
site

Allosteric enzymes contain more than one

polypeptide chain (have quaternary structure).

Allosteric modulators bind noncovalently to

allosteric site and regulate enzyme activity via
conformational changes

Some allosteric modulators alter the Km, the

Vmax remains constant.
Allosteric activator/inhibitor
 2.Feedback inhibition

Feedback inhibition
Regulation of a metabolic pathway by using end
product as an inhibitor of the first pathway.

Significance: keeps cells from synthesizing more product

than necessary
 3.Enzyme induction

Enzyme synthesis can be induced or

decreased by hormonal activity that controls
the genes.

The anabolic insulin hormone induces

increased synthesis of glucokinase, glycogen
synthase and phosphofructokinase
 4.Compartmentation

Fatty acid biosynthesis

takes place
in cytoplasm
while F.A. oxidation
in mitochondria.
 5-Activation by proteolytic cleavage
• Many enzymes are synthesized as inactive precursors
(zymogens) that are activated by proteolytic cleavage
• Proteolytic activation only occurs once in the life of an
enzyme molecule

Examples of specific proteolysis

•Digestive enzymes
–Synthesized as zymogens in stomach and pancreas
•Blood clotting enzymes
–Cascade of proteolytic activations
•Protein hormones
–Proinsulin to insulin by removal of a peptide
 Naming Enzymes
• The name of an enzyme in many cases end in –ase
• For example, sucrase catalyzes the hydrolysis of sucrose

• The name describes the function of the enzyme

For example, oxidases catalyze oxidation reactions

• Sometimes common names are used, particularly for the

digestion enzymes such as pepsin and trypsin

• Some names describe both the substrate and the function

• For example, alcohol dehydrogenase oxides ethanol
 Enzymes Are Classified into six functional
Classes (EC number Classification) by the
International Union of Biochemists (I.U.B.).
on the Basis of the Types of
Reactions That They Catalyze
• EC 1. Oxidoreductases
• EC 2. Transferases
• EC 3. Hydrolases
• EC 4. Lyases
• EC 5. Isomerases
• EC 6. Ligases
 Principle of the international
classification

Each enzyme has classification number

consisting of four digits:
Example, EC: (2.7.1.1) HEXOKINASE
• EC: (2.7.1.1) these components indicate the following
groups of enzymes:

• 2. IS CLASS (TRANSFERASE)

• 7. IS SUBCLASS (TRANSFER OF PHOSPHATE)

• 1. IS SUB-SUB CLASS (ALCOHOL IS PHOSPHATE

ACCEPTOR)
• 1. SPECIFIC NAME
ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)
 6 CH2OH 6 CH OPO 2−
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

1. Hexokinase catalyzes:
Glucose + ATP → glucose-6-P + ADP
 EC 1.Oxidoreductases
• Biochemical Activity:
– Catalyse Oxidation/Reduction Reactions Act
on many chemical groupings to add or
remove hydrogen atoms.
• Examples:
– Lactate dehydrogenase.
– Glucose Oxidase.
– Peroxidase.
– Catalase.
– Phenylalanine hydroxylase.
 1. Oxidoreductases
• Catalyze oxidation-reduction reactions

- oxidases
- peroxidases
- dehydrogenases
 EC 2.Transferases
• Biochemical Activity:
– Transfer a functional groups (e.g. methyl or
phosphate) between donor and acceptor
molecules.
• Examples:
– Transaminases (ALT & AST).
– Phosphotransferases (Kinases).
– Transmethylases.
– Transpeptidases.
– Transacylases.
 2. Transferases

• Catalyze group transfer reactions

 EC 3. Hydrolases
• Biochemical Activity:
– Catalyse the hydrolysis of various bonds. Adds
water across a bond.
• Examples:
– Protein hydrolyzing enzymes (Peptidases).
– Carbohydrases (Amylase, Maltase, Lactase).
– Lipid hydrolyzing enzymes (Lipase).
– Deaminases.
– Phosphatases.
 3. Hydrolases

• Catalyze hydrolysis reactions where water

is the acceptor of the transferred group

- esterases
- peptidases
- glycosidases
Oxidoreductases, Transferases and Hydrolases
 EC 4. Lyases
• Biochemical Activity:
– Cleave various bonds by means other than
hydrolysis and oxidation. no ATP

– Add Water, Ammonia or Carbon dioxide

across double bonds, or remove these
elements to produce double bonds.
• Examples:
– Fumarase.
– Carbonic anhydrase.
4. Lyases
 EC 5. Isomerases
• Biochemical Activity:
– Catalyse isomerization changes within a
single molecule.
– Carry out many kinds of isomerization:
• L to D isomerizations.
• Mutase reactions (Shifts of chemical
groups).
• Examples:
– Isomerase.
– Mutase.
 5. Isomerases

• Catalyze isomerization reactions

 EC 6. Ligases
• Biochemical Activity:
– Join two molecules with covalent bonds
Catalyse reactions in which two
chemical groups are joined (or ligated)
with the use of energy from ATP.
• Examples:
– Acetyl~CoA Carboxylase.
– Glutamine synthetase
 6. Ligases (synthetases)

• Catalyze ligation, or joining of two substrates

• Require chemical energy (e.g. ATP)
Lyases, Isomerases and Ligases