Ashok Vardhan N

 Enzymes are biological catalyst produced by living

tissues.

 They are proteins with the exception of few classes of

RNA molecules called ribozyme.

 They accelerate specific chemical reactions without

being consumed in the process.

 They function in aqueous solutions under very mild

conditions of temperature and pH.
 The catalytic activity of enzyme depends on their native
protein conformation. If an enzyme is denatured or
dissociated into its subunits, catalytic activity is usually
lost.

 Thus, the primary, secondary, tertiary, and quaternary

structures of protein enzymes are essential to their
catalytic activity
 Enzyme Classification
 Enzymes are classified according to the type
of reaction they catalyse.

 All enzymes have formal ‘EC’ (Enzyme

Commission) number and names, and most
have trivial names.
 According to the International Union of Biochemistry
and Molecular Biology (IUBMB) system, enzymes are
classified into seven major classes

 Enzymes are classified according to the type of reaction

they catalyse.

 Each enzyme is assigned a four-digit ‘EC’ (Enzyme

Commission) number, the first three digits of which
define the reaction catalyzed and the fourth of which is a
unique identifier (serial number).
The seven classes as per IUBMB are as follows:

1. EC-1: Oxidoreductase

2. EC-2: Transferase

3. EC-3: Hydrolase

4. EC-4: Lyase

5. EC-5: Isomerase

6. EC-6: Ligase

7. EC-7: Translocases
 EC-1 Oxidoreductases

Catalyzes oxidation-reduction reactions.

Enzymes in this category include :

 Dehydrogenases

 Reductases

 Oxidases

 Peroxidases.
 EC-2 Transferases

Catalyses the transfer of a group such as, amino,

carboxyl, methyl or phosphate, etc. from one
molecule to another
 Enzymes in this category include :

 Amino transferase or transaminase

 Kinase: catalyzes the transfer of phosphate groups

 Transcarboxylase.
 EC-3 Hydrolases

Catalyze the cleavage of C-O, C-N, C-C and some

other bonds with the addition of water.
 Enzymes in this category are:

 All digestive enzymes like:

- α-amylase,
- pepsin,
- trypsin,
- chymotrypsin, etc.

 Acid phosphatase
EC-4 Lyases

Catalyze the cleavage of C-O, C-C and C-N bonds by means

other than hydrolysis or oxidation, giving rise to compound
with double bonds or catalyze the reverse reaction, by the
addition of group to a double bond.
In cases where reverse reaction is important, then
synthase, (not synthetase of group EC-6) is used
in the name.
EC-5 Isomerases

Catalyze intramolecular struc-tural rearrangement in a

molecule. They are called epimerases, isomerases or

mutases, depending on the type of isomerism involved.

EC-6 Ligases (Synthetases)

Catalyze the joining of two molecules coupled with the

hydrolysis of ATP.
EC-7: Translocases (A new EC Class)

Translocases catalyze the movement of ions or

molecules across membranes or their separation

within membranes.
.
Examples are:
 Enzymes catalyzing the translocation of:
Hydrons (H+), inorganic cations, inorganic anions, amino
acids and peptides, and carbohydrates and their derivatives.

 Enzymes of the reaction that provided the driving

force for the translocation linked to:
Oxidoreductase reactions, hydrolysis of a nucleoside
triphosphate, hydrolysis of a diphosphate, and
decarboxylation reaction.
 Zymogen OR Proenzyme

 Enzymes found in in an inactive (precursor)

form, called zymogen or Proenzyme.

 Zymogen have the prefix “pro” or suffix “ogen”.

 For example,

- prothrombin,

- proelastase

- chymotrypsinogen,

- trypsinogen,

- pepsino-gen
 Cofactors (Coenzyme And Activator)

 Some enzymes require an additional non-protein

component for its activity. This additional

compo-nent is called cofactor

– Inorganic ions, called activators.

– Organic compounds, called coenzymes

 Enzymes without its cofactor is referred to as

an apoenzyme

 The complete catalytically active enzyme is

called holoenzyme.

 Apoenzyme + Cofactor = Holoenzyme.

 MECHANISM OF ENZYME ACTION

Formation of an enzyme-substrate (ES) complex is

the first step in enzymatic catalysis which is

subsequently converted to product and free enzyme.

Substrate is bound through non-covalent

interactions at the active site of the enzyme.

The active site of an enzyme is the region that

binds the substrate and which contains the

specific amino acid residues.

 Models for binding of substrate to enzyme

1. Lock and key model or rigid template model of

Emil Fisher.

2. Induced fit model or hand-in-glove model of

Daniel E Koshland
 Lock and Key Model or Rigid Template Model
of Emil Fisher

 Enzyme is pre-shaped and the active site has a

rigid structure, complementary to that of the

substrate.

 Substrate fits into the active site in much the

same way that a key fits into a lock.

Representation of Fisher’s lock and key model.
This model explains all mechanisms but do not
explain the changes in the enzyme activity in the

presence of modulator.
 Induced Fit Model or Hand-in-glove Model of

Daniel Koshland

Enzymes are flexible

Shapes of active site can be modified by the

binding of the substrate.

Substrate induces a conformational change in

enzyme.
Conformational change in enzyme induces
reciprocal changes in its bound substrate that
alters their orientation and configuration and
strains the structure of the bound substrate.

intrinsic binding energy is liberated.

Intrinsic binding energy converts substrate into

product.
Schematic representation of induced fit model of Koshland
 SPECIFICITY OF ENZYME ACTION

 Ability of enzyme to discriminate between two substrates.

 Enzymes are highly specific both in the reaction catalyzed

and in their choice of substrates.

 Specificity makes it possible for number of enzymes to

co-exist in cell without interfering in each other’s actions.

 Types of Specificity

1. Substrate specificity

2. Reaction specificity

3. Stereo specificity
 Substrate Specificity

i. Absolute substrate specificity

ii. Relative substrate specificity

iii. Broad substrate specificity.

 Absolute substrate specificity

Certain enzymes will act on only one substrate

and catalyze one reaction, e.g. Glucokinase,

lactase, urease, etc.

 Relative substrate specificity

Enzyme acts on more than one substrate.

 Group specificity

 Bond specificity.
Chymotrypsin acts on several proteins by
hydrolyzing peptide bonds attached to aromatic
amino acids.

Trypsin hydrolyzes peptide linkages involving

arginine or lysine.
α-amylase, cleaves α-(1→4) glycosidic bonds
of carbohydrates.

Lipase hydrolyzes ester bonds of lipids.

 Broad substrate specificity
 Enzyme acts on more than one structurally
related substrates.

 hexokinase catalyzes the phosphorylation of

more than one kind of hexoses such as glucose,

fructose and mannose.

 Reaction Specificity

Enzyme is specific to a particular reaction but

not to substrate (s) and catalyzes only one type

of reaction.
Figure 6.5: Example of reaction specificity.
 Stereo Specificity
 L-lactate dehydrogenase will act only on

L-lactic acid and not D-lactic acid.

 L-amino acid oxidase and D-amino acid

oxidase act only on L and D-amino acids.

 Salivary α-amylase acts on the α-1,4

glycoside linkage and is inactive on β-1,4
glycoside bond
Factors Affecting The Velocity Of

Enzyme Reaction
 Substrate concentration

 Enzyme concentration

 pH i.e. H+ ion concentration

 Temperature

 Product concentration

 Activators and coenzymes

 Time

 Physical agents
Effect of Substrate Concentration
V0 : initial velocity

Vmax : maximum velocity

Km : 1/2 Vmax = Michaelis Menten constant

[S] : substrate concentration

Effect of Enzyme Concentration
 Effect of Hydrogen Ion Concentration pH
 Each enzyme has an optimum pH, i.e. a pH at which
the enzyme activity is maximum.
 Below or above this pH, enzyme activity is
decreased.
 The optimum pH differs from enzyme to enzyme.
– Pepsin = 1.2
– Trypsin = 8.0
Effect of pH on enzyme activity
Changes in pH can alter the following:

 Ionization state of the amino acids

present in the active site of the enzyme.

 The ionization state of the substrate.

 Drastic change in pH denatures enzyme

 Effect of Temperature
 Enzyme catalyzed reactions show an increase in rate with

increasing temperature only within a relatively small and

low temperature range.

 Each enzyme shows the highest activity at a particular

temperature called optimum temperature.

 The activity progressively declines both above and below

this temperature.
Effect of temperature on enzyme activity
Increase in velocity is due to the increase in the

kinetic energy.

Further elevation of the temperature results in a

decrease in reaction velocity due to denaturation

of the enzyme protein.

Low temperature also decreases enzyme activity

and enzymes may be completely inactive at

temperature of 0°C and below.

The inactivity at low temperature is reversible.

Most of the body enzymes have the optimum

tempe-rature close to 37°C to 38°C and have

progressively less activity as the temperature

rises.
Effect of Product

Accumulation of products of the reaction causes the

inhibition of enzyme activity for some enzymatic

reactions.
Effect of Activators and Co-enzymes

In absence of activators and coenzymes, enzymes

become functionally inactive.

Effect of Time

Under optimum conditions of pH and temp, time

required for an enzyme reaction is less.

The time required for the completion of an

enzyme reaction increases with changes in
temperature and pH from its optimum.
 ENZYME KINETICS

 The study of enzyme reaction rates and how they change

in response to changes in experimental parameters is
known as kinetics.

 One of the key factors affecting the enzyme reaction rates

is the concentration of substrate [S].
 Effect of Substrate Concentration
V0 : initial velocity
Vmax : maximum velocity
Km : 1/2 Vmax = Michaelis Menten constant
[S] : substrate concentration
 Significance of Km (Michaelis Constant)

1. Km provides a amount of the substrate required for

significant catalysis to occur.

2. It is a measure of the affinity of the enzyme for its

substrate, a high Km indicates weak binding and a

low Km indicates strong binding with its substrate.
 Significance of Vmax

 The Vmax of a reaction is an index of the catalytic

efficiency of an enzyme.

 The Vmax is useful in comparing the activity of one

enzyme with that of another.

 Lineweaver-Burk plot
(Double reciprocal plot)
 ENZYME INHIBITION

Any substance that can diminish the velocity of

an enzyme reaction is called inhibitor.

Two general classes of inhibitors are:

1. Reversible inhibitor

2. Irreversible inhibitor.
 REVERSIBLE INHIBITOR

Reversible inhibitors bind to enzymes through

non-covalent bonds and the activity of the enzyme

is restored fully when the inhibitor is removed

from the system.

Different types of reversible inhibitors are:

i. Competitive or substrate analogue inhibitor

ii. Non-competitive inhibitor

iii. Uncompetitive inhibitor.

 Competitive or Substrate Analogue Inhibitor

A competitive inhibitor is a structural analogue of

the substrate.

Chemical structure of inhibitor (I) resembles that

of substrate (S) and binds to enzyme at active

site, forming EI complex rather than ES-complex.

Diagrammatic representation of competitive inhibition

E: Enzyme; S: Substrate; I: Competitive inhibitor; P: Product

The inhibition could be overcome by increasing

substrate concentration
Enzyme kinetics of competitive inhibitor
Enzyme kinetics of competitive inhibitor
Vmax is unaltered
Km is increased
Drugs act as competitive inhibitors
 Sulphonamide

Analogue of P- aminobenzoic acid (PABA) and inhibits

the synthesis of folic acid in microorganisms.

 Isoniazide [Isonicotinic acid hydrazine (INH)]

It is an anti-tuberculosis drug, inhibits the biosynthesis of

NAD and restrict the growth of the organisms that cause

tuberculosis.
 Dicumarol

It is an anticoagulant drug structurally similar to vitamin

K. It inhibits the vitamin K activity and inhibits the

formation of prothrombin.

 Physostigmine

It inhibits acetylcholinesterase and use to treat glaucoma

and myasthenia gravis.

Drugs such an ibuprofen (anti-inflammatory drug),

statin (cholesterol lowing drug) are competitive

inhibitors of enzymes, that involved in the

prostaglandins and cholesterol synthesis

respectively.
 Non-competitive Inhibitors

 No competition occurs between substrate and

inhibitor.

 Inhibitor is usually structurally different from the

substrate.

 It binds at a site on the enzyme molecule other

than the substrate-binding site.
 Noncompetitive inhibitor can bind free enzyme
(EI) or the enzyme substrate complex (EIS)

 However, EIS complex does not continue to form

product.

 Noncompetitive inhibitor lowers the concentration

of functional enzymes.

 Noncompetitive inhibition cannot be overcome by

increasing the substrate concentration
 Diagrammatic representation of noncompetitive inhibition.

(E = Enzyme; S = Substrate; I = Non-competitive inhibitor; P = Product)

 Examples of non-competitive inhibitors are:

– Ethanol or certain narcotic drugs are non-com-petitive

inhibitor of acid phosphatase.

– Trypsin inhibitors occur in soybean and raw egg

white, inhibit activity of trypsin.

– Ascaris parasites (worm) contain pepsin and trypsin

inhibitors, inhibit action of pepsin and trypsin.
For non-competitive inhibition, the Km value is unchanged while
Vmax is lowered
 Uncompetitive Inhibitor

 Uncompetitive inhibitor can bind only to the

enzyme-substrate (ES) complex.

 It does not have affinity for free enzyme.

 Enzyme-substrate-inhibitor complex, ESI does

not continue to form any product.

 Uncompetitive inhibition cannot be overcome by

the addition of more substrate.

 Consequently Vmax cannot be attained, even at

high substrate concentration.

Uncompetitive inhibitor binds only to enzyme-substrate
complex.
Uncompetitive inhibitor decreases both Vmax and Km.
 The herbicide glyphosate, also known as

Roundup, is an uncompetitive inhibitor of an

enzyme in the biosynthetic pathway for aromatic

amino acids in bacteria

 Nontoxic in animals because they lack the

enzyme.
IRREVERSIBLE INHIBITOR
An irreversible inhibitor binds with an enzyme
tightly covalently and forms a stable complex.

An irreversible inhibitor cannot be released by

dilution or dialysis or simply by increasing the
concentration of substrate.
Irreversible inhibitors can be divided into three

categories:

 Substrate analogue inhibitor or affinity labels

 Group specific inhibitors

 Suicide inhibitor or mechanism based

inactivation.
In terms of enzyme kinetics, the effect of an

irreversible inhibitor is like that of the reversible

non-competitive inhibitors resulting in a decreased

in Vmax but having no effect on the Km

 Substrate Analogue Irreversible
Inhibitor or Affinity Labels

 Substrate analogues or affinity labels are molecules

that are structurally similar to the substrate.

 These substrate analogues possess a highly reactive

group which is not present in the natural substrate.

The reactive group of substrate analogues
covalently reacts with amino acid residues of the
active site of the enzyme and permanently block
the active site of the enzyme

3-Bromoacetal phosphate (BAP) inhibits enzyme

phosphotriose isomerase of glycolysis.

 Group Specific Irreversible Inhibitor

 These inhibitors react with specific R-groups

(side chain) of amino acid residues in the

active site of enzyme.

 Examples of group specific irreversible inhibitors:

– Di-isopropylphosphofluoride (DIPF)

– Iodoacetamide

– Heavy metals
 DIPF can inhibit an enzyme acetylcholine

esterase by covalently reacting with hydroxyl

group of a serine residue present at the active site

of the enzyme

 DIPF has also been found to inhibit trypsin,

chymotrypsin, elastase and phosphoglucomutase

Figure 6.20: Irreversible inhibition of acetylcholinesterase by a
group-specific inhibitor, diisopropylphosphofluoride (DIPF).
 Iodoacetamide and heavy metals like, Pb2+, Ag+,

Hg2+, etc. which react with sulfhydryl (-SH) group

of cysteine residues present at the active site of the

enzyme and makes them inactive.

 Suicide Inhibitor or Mechanism Based inactivation

 These compounds are relatively unreactive until they bind

to the active site of a specific enzyme.

 On binding to the active site of the enzyme they carry out

the first few catalytic activities of the normal enzyme

reaction.
 Instead of being transformed into a normal product,

however, the inhibitor is converted to a very reactive

compound that combines irreversibly with the enzyme

leading to its irreversible inhibition

 These are also called mechanism based inactivation

because they utilize the normal enzyme reaction

mechanism to inactivate the enzyme

Example Suicide Inhibitor
Penicillin

Inactivates bacterial enzyme glycopeptidyl

transpeptidase involved in the formation of bacterial
cell wall.

Aspirin

Inactivates an enzyme cyclo-oxygenase required for

the synthesis of prostaglandins .
 Disulfiram (antabuse)

 Inhibits aldehyde dehydro-genase enzyme resulting in

accumulation of acetaldehyde.

 Antabuse, or disulfiram is, a medicine for treatment of

alcohol abuse and alcohol dependence

 Antabuse is prescribed to people who want to quit

drinking.
Clinical Application of Enzyme Inhibitor

 Enzyme inhibitors have therapeutic applications.

 Most antibiotics and anticancer drugs that are used

therapeutically are either competitive inhibitor or

mechanism based suicide inhibitor.

 ALLOSTERIC ENZYME

Allosteric enzyme is a regulatory enzyme.

The term allosteric derives from Greek word,

allo means other and steros means space or site.
Allosteric enzymes are those having other site in
addition to active site for binding of modulator
(regulatory metabolites).

Allosteric enzymes may be inhibited or

stimulated by their modulators
Modulators that inhibit enzyme activity are termed

negative modulators. Whereas those that increase

enzyme activity are called positive modulators.

 ISOENZYME

 Isoenzymes or isozymes are multiple forms (isomers)

of the same enzyme that catalyze the same biochemical
reaction.

 Isoenzymes show different chemical and physical

properties like electrophoretic mobility and kinetic
properties.

 Only those enzymes, which are in polymeric form

demonstrate isoenzyme.
 For example:

1. Lactate dehydrogenase (LDH)

2. Creatine kinase (CK)

 Lactate Dehydrogenase (LDH)

 Lactate dehydrogenase is a tetrameric enzyme that

catalyzes the oxidation of L-lactate to pyruvate.

 LDH is made up of two types of polypeptide M (muscle)

type and H (heart) type.

 LDH has five isoenzymes:
– LDH1
– LDH2
– LDH3
– LDH4
– LDH5.
 Five isoenzymes of LDH can be detected by
electrophoresis as they have different electrophoretic
mobilities.
 LDH1 is the fastest moving fraction towards the anode
and LDH5 is the slowest moving isoenzyme of LDH.

 LDH1 predominates in cells of cardiac muscle, and

erythrocytes and LDH5 is the most abundant form in
the liver and in skeletal muscle
 Clinical Applications of LDH
1. Significant elevation of LDH1 and LDH2 occurs within
24 to 48 hours after myocardial infarction.

2. Predominant elevation of LDH2 and LDH3 occur in

leukaemia.

3. LDH3 elevated in malignancy of many tissues.

4. Elevation of LDH5 occurs after damage to the liver or

skeletal muscle.
 Creatine Kinase (CK)
Creatine kinase isoenzymes are dimer that are made up of
two types of polypeptide chains, which may be either M
(muscle) type or B (brain) type, generating three
isoenzymes.

1 CK1 (BB) : present in the brain

2 CK2 (MB) : present only in Cardiac tissue

3 CK3 (MM) : present in skeletal muscle

 Clinical Application

1. CK1 may be elevated in neonates particularly in damaged brain

or very low birth weight new-born

2. Increased level of CK2 occurs in myocardial infarction Cardiac

tissue is the only tissue which has mixed MB (CK2) isoenzyme.

3. CK-MB isoenzyme starts to increase within 4 hours after an acute

myocardial infarction (AMI) and reaches a maximum within 24 hrs.

4. Elevated levels of CK3 in serum occur in dystrophies and

myopathies.
 CLINICAL SIGNIFICANCE OF ENZYMES

Certain enzymes are used:

 For the diagnosis of the disease

As therapeutic agents

As analytical reagents.

 Diagnostic Use of Enzymes

The enzymes that are found in plasma can be

categorized into two major groups:

 Plasma specific enzyme

 Plasma nonspecific enzyme.

The plasma specific enzymes are:

– The enzymes involved in blood coagulation

– Ferroxidase

– Pseudocholinesterase

– Lipoprotein lipase.

These enzymes are clinically of interest when their

concentration decreases in plasma.

 The plasma nonspecific enzymes are present in very high
concentration in tissues than in the plasma.

 Estimation of plasma nonspecific enzymes is very

important for the diagnosis of several disease.
 Enzymes useful for the diagnosis of diseases

Alanine transaminase (ALT)

 Alanine transaminase was known formerly as glutamate

pyruvate transaminase (GPT).

 The plasma ALT normal value for adult is 10 to 40 U/L.

 ALT level is elevated in liver diseases (viral or toxic

hepatitis), jaundice and cirrhosis of liver.
 Aspartate transaminase (AST)

 It was known formerly as glutamate oxaloacetate

transminase (GOT).

 The plasma AST normal value for adults is 10 to 30 U/L.

 Increased AST level occurs after myocardial infarction.

 It is moderately elevated in liver disease.

The plasma AST level starts increasing after 6 to 8 hours

after the onset of chest pain with peak values 18 to 24 hours

and the values fall to normal level by the fourth or fifth day.
Alkaline phosphatase (ALP)

 ALP hydrolyzes organic phosphate at alkaline pH.

 Normal serum level for adults is 3-13 KA units/dl.

 It is elevated in certain bone and liver disease.

 Very high levels may be noticed in obstructive

jaundice, bone diseases such as Paget’s disease, rickets,
osteomalacia, carcinoma of bone and
hyperparathyroidism
Acid phosphatase (ACP)
 It hydrolyzes phosphoric acid ester at pH 5 to 6.

 Normal serum value for ACP is 0.5 to 4 KA units/dL.

 Acid phosphatase enzyme is useful for the diagnosis

and prognosis of prostate cancer. ACP is therefore an
important tumor marker.
Amylase

 It catalyzes hydrolysis of starch and glycogen.

 Normal serum value is 50-120 U/L.

 The activity of serum amylase is increased in acute

pancreatitis, chronic pancreatitis, mumps and obstruction
of pancreatic duct.
Creatine kinase (CK) : Refer isoenzyme.

Lactate dehydrogenase (LDH) : Refer isoenzyme.

 Enzymes as Tumor Marker

Elevated enzyme levels may signal the presence of

malignancy.
Enzyme Assays in Myocardial Infarction/Cardiac Markers

Diagnostic enzymes include:

 Creatine kinase

 Lactate dehydrogenase

 Serum aspartate aminotransferase, also called

 serum glutamate oxaloacetate transaminase.

Nonenzyme proteins includes :

 Myoglobin (Mb)

 Cardiac troponin T and I (cTnT and cTnI).

Figure 6.28: Various enzyme assays and their time
course after onset of acute myocardial infarction.
 Enzyme Assays in Liver Diseases
1. Enzymes in hepatocyte damage:
 Aspartate aminotransferase
 Alanine aminotransferase.
ALT is the more liver-specific enzyme.
2. Enzymes in cholestasis:
 Alkaline phosphatase
 5’-nucleotidase
 γ-glutamyl transferase.
 Enzyme Assays in Pancreatitis

 Serum Amylase

 Urine amylase

 Lipase
 Use Of Enzymes In Laboratory Investigations
(Enzyme-based Assays)

Enzymes can be used as analytical laboratory reagents

 Therapeutic Use of Enzymes

Some enzymes are used in the treatment of some diseases

of human being