ENZYMES

Introduction
 Enzymes are biological catalysts that
speed up the rate of the biochemical
reaction.

 Most enzymes are three dimensional

globular proteins (tertiary and quaternary
structure).

 Some special RNA species also act as

enzymes and are called Ribozymes e.g.
hammerhead ribozyme.

Hammerhead enzyme
STRUCTURE OF ENZYMES
 ACTIVE SITE

 The active site of an enzyme is the region that binds

substrates, co-factors and prosthetic groups and contains
residue that helps to hold the substrate.

 Active sites generally occupy less than 5% of the total

surface area of enzyme.

 Active site has a specific shape due to tertiary structure

of protein.

 A change in the shape of protein affects the shape of

active site and function of the enzyme.
 ACTIVE SITE
o Active site can be further divided into:

Active Site

Binding Site Catalytic Site

It chooses the substrate It performs the catalytic

and binds it to active site. action of enzyme.
COFACTORS (COENZYME AND ACTIVATOR)
Some enzymes require an additional nonprotein component for its optimum
activity.

This additional component is called cofactor which may be either loosely or

tightly bound to the protein portion of the enzyme.

• These cofactors may be:

– Organic compounds, called coenzymes
– Inorganic ions, called activators.

• Enzymes without its cofactor is referred to as an apoenzyme; the complete

catalytically active enzyme is called holoenzyme.

Apoenzyme(Protein part ) + cofactor (Non-protein part ) = holoenzyme.

 INORGANIC CO-FACTORS
o These are the inorganic molecules required for the proper
activity of enzymes.
Examples:
➢ Enzyme carbonic anhydrase requires Zn++ for it‟s activity.
++
➢ Hexokinase has co-factor Mg

ORGANIC CO-FACTORS
o These are the organic molecules required for the proper
activity of enzymes.
Example:
➢ Glycogen phosphorylase requires the small organic
molecule pyridoxal phosphate.
 SUBSTRATE
 The reactant in biochemical reaction is termed as substrate.

 When a substrate binds to an enzyme it forms an enzyme-

substrate complex.

Substrate Joins Enzyme

 SITES OF ENZYME SYNTHESIS
o Enzymes are synthesized by ribosomes which are attached to the
rough endoplasmic reticulum.

o Information for the synthesis of enzyme is carried by DNA.

o Amino acids are bonded together to form specific enzyme

according to the DNA‟s codes.
 INTRACELLULAR AND
EXTRACELLULAR ENZYMES
o Intracellular enzymes are synthesized and retained in the cell for
the use of cell itself.
o They are found in the cytoplasm, nucleus, mitochondria.
Example :
➢ Oxydoreductase catalyses biological oxidation.
➢ Enzymes involved in reduction in the mitochondria.

o Extracellular enzymes are synthesized in the cell but

secreted from the cell to work externally.
Example :
➢ Digestive enzyme produced by the pancreas, are not used by the
cells in the pancreas but are transported to the duodenum.
 NOMENCLATURE OF ENZYMES
o An enzyme is named according to the name of the substrate it catalyses.
o Some enzymes were named before a systematic way of
naming enzyme was formed.
Example: pepsin, trypsin and rennin
o By adding suffix -ase at the end of the name of the
substrate, enzymes are named.
o Enzyme for catalyzing the hydrolysis is termed as hydrolase.
Example :

maltose + water maltase glucose + glucose

 EXAMPLES
Substrate Enzymes Products
Lactose Lactase Glucose + Galactose
Maltose Maltase Glucose
Cellulose Cellulase Glucose
Lipid Lipase Glycerol + Fatty Acid
Starch Amylase Maltose
Protein Protease Peptides +
Polypeptide
CLASSIFICATION OF ENZYMES
 A systematic classification of enzymes has been
developed by
International Enzyme Commission i.e International Union
of Biochemistry (IUB).

 This classification is based on the type of reactions

catalyzed by enzymes.

 There are six major classes of Enzymes.

ENZYME CLASS REACTION TYPE EXAMPLES
Oxidoreductases Reduction-oxidation Lactate
(redox) dehydrogenase
Transferases Move chemical group Hexokinase
Hydrolases Hydrolysis; bond Lysozyme
cleavage with transfer of
functional group of
water
Lysases Non-hydrolytic bond Fumarase
cleavage
Isomerases Intramolecular group Triose phosphate
transfer (isomerization) isomerase

Ligases Synthesis of new RNA polymerase

covalent bond between
substrates, using ATP
hydrolysis
MECHANISM OF ENZYME ACTION
Formation of an enzyme-substrate
(ES) complex is the first step in
enzymatic catalysis

Two models for substrate binding to

the active site of the enzyme, have
been proposed to explain the
specificity that an enzyme has for its
substrate:

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
 Proposed by EMIL FISCHER in 1894.

 Lock and key hypothesis assumes the active site of an

enzymes are rigid in its shape.

 There is no change in the active site before and after a

chemical reaction.
 INDUCED FIT MODEL
 More recent studies have revealed that the process is much more likely to
involve an induced fit model(proposed by DANIAL KOSH LAND in 1958).

 According to this exposure of an enzyme to substrate cause a change in

enzyme, which causes the active site to change it‟s shape to allow enzyme and
substrate to bind.
FACTORS AFFECTING RATE OF
ENZYME ACTIVITY
1. Substrate concentration
2. Enzyme concentration
3. pH i.e. H+ ion concentration
4. Temperature
5. Time
EFFECT OF SUBSTRATE CONCENTRATION
• For a given quantity of
enzyme, the velocity of the
reaction increases as the
concentration of the
substrate is increased.

• At first, this relationship is

almost linear but later, the
reaction curve becomes
hyperbolic in shape.
 EFFECT OF ENZYME CONCENTRATION
• The velocity of a reaction is
directly proportional to the
amount of enzyme present as
long as the amount of substrate is
not limiting.
• The substrate must be present at a
concentration sufficient to ensure
that all of the enzyme molecules
have substrate bound to their
active site
 EFFECT OF 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, e.g. optimum pH for:
– Pepsin = 1.2
– Trypsin = 8.0

• A bell shaped curve is obtained when

enzyme velocity is plotted against pH
 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.
• A bell shaped curve is obtained when we
plot the enzyme velocity Vs. temperature
EFFECT OF TIME
• Under optimum conditions
of pH and temperature, 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.
 SPECIFICITY OF ENZYME ACTION
• Specificity is the ability of an enzyme to choose exact substrate
from a group of similar chemical molecules.

• The specificity is actually a molecular recognition mechanism.

• It operates through the structural and conformational

complementarity between enzyme and substrate.
Types of Specificity
1. Substrate specificity

2. Reaction specificity

3. Stereo specificity.
1. Substrate Specificity
• A feature of an enzyme activity with regard to the
kind of substrate reacting with an enzyme to yield a
product.
• There are following types of substrate specificity:
i. Absolute substrate specificity
ii. Relative substrate specificity
iii. Broad substrate specificity
i. Absolute substrate specificity
Certain enzymes will act on only one substrate and catalyze one
reaction, e.g. Glucokinase, lactase, urease, etc.
ii. Relative substrate specificity
In this case, enzyme acts on more than one substrate.
The relative substrate specificity is of two types:
a. Group specificity
b. Bond specificity.

a) In group specificity, an enzyme acts on more than one substrate containing

a particular group, e.g. chymotrypsin acts on several proteins by
hydrolyzing peptide bonds attached to aromatic amino acids.

b) In bond specificity, an enzyme acts on more than one substrate containing

a particular kind of bond, e.g. salivary α-amylase cleaves α-(1→4)
glycosidic bonds of carbohydrates, lipase hydrolyzes ester bonds of lipids
iii. Broad substrate specificity
• In this case, an enzyme acts on more than one
structurally related substrates.
• Examples:- hexokinase catalyzes the phosphorylation
of more than one kind of hexoses such as glucose,
fructose and mannose.
2. Reaction Specificity
• In this case, an enzyme is specific to a particular reaction but
not to substrate (s) and catalyzes only one type of reaction.
• For example, pyruvate can undergo several reactions.
• Each reaction is catalyzed by a separate enzyme, which
catalyzes only that reaction.
3. Stereo Specificity
Many enzymes show specificity towards stereoisomers, i.e.
they act on only one type of isomer.
For example,
• L-lactate dehydrogenase will act only on L-lactic acid and
not D-lactic acid.
• D-glucose oxidase can similarly act only on D-glucose and
not on L-glucose.
• Salivary α-amylase acts on the α-1,4 glycoside linkage and
is inactive on β-1,4 glycoside linkage.
 ENZYME INHIBITION
o The prevention of an enzyme process as a result of interaction of
inhibitors with the enzyme is known as enzyme inhibition.

➢ INHIBITORS:
Any substance that can diminish the velocity of an
enzyme catalyzed reaction is called an inhibitor.
TYPES OF ENZYME INHIBITION
 1.Reversible Inhibition
• Reversible inhibitors bind to enzymes through noncovalent

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 inhibition

ii. Noncompetitive inhibition

iii. Uncompetitive inhibition

 i. Competitive or substrate analogue inhibition
• A competitive inhibitor is
usually a structural analogue of
the substrate.

• The chemical structure of the

inhibitor (I) closely resembles
that of the substrate (S) and
binds to the enzyme at the active
site, forming an EI complex
rather than ES-complex
• Eg:-Succinate dehydrogenase is
inhibited by Malonate.
➢ Malonate is a competitive inhibitor
of => succinate dehydrogenase .
➢ The enzyme uses succinate as its
substrate but inhibited by malonate
=> which is structurally similar to
succinate and => differs in having
=> one rather than two methylene
groups.
 ii. Noncompetitive inhibition
• As the name implies, in this type of inhibition 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 and thus
there is no competition between inhibitor and substrate.
• Since inhibitor and substrate may bind at different sites; formation of both EI and
EIS-complexes is possible.
• Since EIS may breakdown to form product at a slower rate than does ES, the reaction
is slowed but not halted.
• Eg:-Alanine inhibits Pyruvate kinase.
iii. Uncompetitive inhibition

• Uncompetitive inhibitor can bind only to the enzyme-substrate

(ES) complex.

• It does not have affinity for free enzyme.

• This form of inhibitor is rare with single substrate but more

common in multiple substrate reaction.

• Eg:-Phenylalanine inhibits placental ALP.

 2.Irreversible Inhibition
• 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:
i. Group specific inhibition
ii. Substrate analogue inhibition or affinity labels
iii. Suicide inhibition or mechanism based inactivation.
i. Group specific inhibition
These inhibitors react with specific R-groups (side chain) of amino acid residues
in the active site of enzyme.
Examples -
Di-isopropylphosphofluoride (DIPF)
• 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
ii. Substrate analogue inhibition 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, e.g. 3-Bromoacetol phosphate (BAP).

• BAP is a substrate analogue of the normal substrate dihydroxyacetone

phosphate (DHAP) for the enzyme phosphotriose isomerase of glycolysis.
iii. Suicide inhibition 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.

• The enzyme literally commits suicide. These are also called mechanism
based inactivation because they utilize the normal enzyme reaction
mechanism to inactivate the enzyme.

• These inhibitors act as drugs for example- Penicillin, Aspirin,

 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.
• Like all enzymes, allosteric enzymes have active site for binding of the
substrate but they also have one or more regulatory (or allosteric) sites for
binding regulatory metabolites which is called modulator
• 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.
• Just as the active site of an enzyme is specific for its
substrate, the allosteric site is specific for its modulators.
• Allosteric enzymes are generally larger and more complex
than those of simple enzymes.
Feedback Allosteric Inhibition
• It is inhibited by the end product of the multienzyme
system whenever the end product of such metabolic
reaction produced in excess of the cells needs.
• The end product of the pathway acts as a specific
inhibitor of the first or regulatory enzyme in the
pathway.
• The whole enzyme system thus slows down to bring
the rate of production of its end product back into
balance with the cell’s needs.
• This type of regulation is called feedback inhibition.
• For example, first enzyme δ-aminolevulenic acid
synthase is an allosteric enzyme in heme synthesis is
inhibited by its end product heme.
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 but have same functions.

Not all enzymes have isoenzymes. In fact, it was found that only those enzymes,
which are active in polymeric form demonstrate isoenzyme.
For example:
1. Lactate dehydrogenase (LDH)
2. Creatine kinase (CK) (formerly called creatine phosphokinase (CPK).
Lactate Dehydrogenase (LDH)
Lactate dehydrogenase is a tetrameric enzyme that catalyzes the oxidation of
L-lactate to pyruvate.
• LDH has five isoenzymes:
– LDH1
– LDH2
– LDH3
– LDH4
– LDH5.
• Since LDH is a tetramer, made up of two types of polypeptide M (muscle)
type and H (heart) type, five combinations are possible with varying ratios of
two kinds of polypeptides
 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.
Some more examples of isoenzymes are-
CLINICAL SIGNIFICANCE OF ENZYMES

1. For the diagnosis of the disease

2. As therapeutic agents

3. As analytical reagents
1. Diagnostic Use of Enzymes
2. Therapeutic Use of Enzymes
• Some enzymes are used in the treatment of some diseases of human being.
For example:
1. Bacterial asparginase is used in the treatment of some types of
leukemia
2. Chymotrypsin: Used for dissolving ligaments of the lens during the
extraction of cataract.
3. Collagenase: Used for debridement (cleaning of wound by removing
dead tissue) of dermal ulcers and severe burns.
4. Fibrinolysin: It is used in the venous thrombosis, pulmonary and arterial
embolism (blood clot).
5. Hyaluronidase: It is used to promote the rapid absorption of drugs
injected subcutaneously. It acts by increasing tissue permeability. It is
used in the treatment of postoperative edema.
3.Analytical Use of Enzymes
• Enzymes can be used as reagents and labels.