ENZYMES

Introduction to enzyme
• Enzymes are biological catalysts that speed
up the rate of the biochemical reaction.

• All enzymes are proteins in nature with

large molecular weight

• Ribozymes however are an exception , being

RNA molecules with enzymatic activity

• Enzymes are specific due to tertiary

structure of protein.

2
Enzymes have three distinctive characteristics
1. High specificity
• The ability to promote a particular chemical reaction
2. High reaction rate
• The ability to enhance the reaction rate by factors as high
as 1012
• Facilitate the formation of the TS from the S to the P
 by lowering the AE
• Do not alter the position of equilibrium of reversible
reaction but accelerate the establishment of the
equilibrium position.
3. High capacity for regulation
• The ability to modify their catalytic activity
 In response to changing cellular and physiological
demands
Example: Feed back inhibition, Reversible phosphorylation
Definitions of Some Terms
• The active site of an enzyme is the special pockets or clefts
region that binds substrates.
• Apoenzyme is the protein part of an enzyme without any c
ofactors or prosthetic groups that may be required for the
enzyme to be functional.
• Cofactors are small organic or inorganic molecules that an
apoenzyme requires for its activity.
• A prosthetic group is similar to a cofactor but is tightly
bound to an apoenzyme. Examples: NAD+, Vitamins, FAD
• Addition of a cofactor or prosthetic group to the
apoenzyme yields the holoenzyme, i.e, the active enzyme.
4
Definitions of Some Terms
• The molecule acted upon by the enzyme to form product is t
he substrate.
❑ When d/t enzyme catalyzing sites are located in the
same macromolecule  multienzyme complex
• Fatty acid synthetase system,
• Pyruvate dehydrogenase System
 Co-factor is the non-protein molecule which carries
out chemical reactions.
 Co-factors are of two types:
1. Inorganic cofactors: Enzyme carbonic anhydrase
requires Zn++ for it’s activity
2. Organic co-factors:
• Coenzymes are small organic molecules, often
derivatives of vitamins, that function with the
enzyme in the catalytic process.
5
 Isoenzymes
• Enzymes which occur in a number of different molecular
forms but catalyze the same reaction.
• Made up of several subunits encoded by different genes
leading to d/t structures each of which can be tissue specific
• Examples:
 CK has 3 isoenzymes

 LD has 5 isoenzymes

6
Enzyme–Substrate complex
❑The reactant in biochemical reaction is termed as
substrate.

❑When a substrate binds to an enzyme it forms an

Enzyme-Substrate complex (ES).
❑ES is converted to Enzyme–Product (EP), which
subsequently dissociates to Enzyme & Product.

7
1 Enzyme available
with empty active
site
Active site

Enzyme
(sucrase)

8
Figure 5.14_s2
1 Enzyme available
with empty active
site
Active site Substrate
(sucrose)

2 Substrate binds
to enzyme with
induced fit
Enzyme
(sucrase)

9
Figure 5.14_s3
1 Enzyme available
with empty active
site
Active site Substrate
(sucrose)

2 Substrate binds
to enzyme with
induced fit
Enzyme
(sucrase)

H2O

3 Substrate is
converted to
products

10
Figure 5.14_s4
1 Enzyme available
with empty active
site
Active site Substrate
(sucrose)

2 Substrate binds
to enzyme with
induced fit
Enzyme
Glucose (sucrase)

Fructose
H2O

4 Products are
released
3 Substrate is
converted to
products

11
NOMENCLATURE OF ENZYMES
• Types of names: Trivial, Recommended & Systematic names
• An enzyme is named according to the name of the
substrate it catalyzes.
• Some enzymes were named before a systematic way of
naming enzyme was formed. E.g. pepsin, trypsin and
rennin
• BySubstrate
adding suffix Enzymes
-ase at the end of the name of the
Products
substrate, enzymes are named.
lactose lactase glucose + galactose
maltose maltase Glucose
cellulose cellulase Glucose
lipid lipase Glycerol + fatty acid
starch amylase Maltose
protein protease Peptide + polypeptide 12
CLASSIFICATION OF ENZYMES
 A systematic classification of enzymes has been developed
by International Enzyme Commission.
 This classification is based on the type of reactions
catalyzed by enzymes.
 There are six major classes.
 Each class is further divided into sub classes etc. to
describe the huge number of different enzyme- catalyzed
reactions.

13
CLASSIFICATION OF ENZYMES….cont’d
ENZYME CLASS REACTION TYPE EXAMPLES
Oxidoreductases Reduction-oxidation Lactate
(redox) dehydrogenase
Transferases Move chemical group Hexokinase
Hydrolases Hydrolysis; bond cleavage Lysozyme
with transfer of functional
group of water

Lysases Non-hydrolytic bond Fumarase

cleavage
Isomerases Intramolecular group Triose
transfer (isomerization) phosphate
isomerase
Ligases Synthesis of new covalent RNA polymerase
bond between substrates,
using ATP hydrolysis
14
MECHANISM OF ENZYME ACTION

15
 1. 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.

16
 2. 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.

17
Factors Affecting Rate of Enzyme Catalyzed
Reactions

1. Temperature

2. Hydrogen ion concentration(pH)

3. Substrate concentration

18
 1. EFFECT OF TEMPERATURE
 Raising the temperature increases the rate of enzyme
catalyzed reaction by increasing kinetic energy of reacting
molecules.
 Enzymes work maximum over a particular temperature
known as optimum temperature. Enzymes for humans
generally exhibit stability temperature up to 35-40 ᵒ C.

19
Enzymes and temperature: a tale of two effects

Collision rate of enzymes

Reaction rate / arbitrary units

and substrates

Number of enzymes
remaining Undenatured

Temperature / oC
Enzymes and temperature

Increasing kinetic
energy increases
successful collision rate
Reaction rate / arbitrary units

Temperature / oC
Enzymes and temperature

Permanent disruption
of tertiary structure
leads to loss of active
Reaction rate / arbitrary units

site shape, loss of

binding efficiency and
activity

Temperature / oC
 2. EFFECT OF PH
Rate of almost all enzymes catalyzed reactions depends
on pH
Most enzymes exhibit optimal activity at pH value
between 5 and 9.
High or low pH value than optimum value will cause
ionization of enzyme which result in their denaturation.

23
Enzymes and pH

Optimum pH Either side of the optimum pH,

the gradual ionising of the side-
chains (R-groups) results in loss
Reaction rate / arbitrary units

of H-bonding, 3o structure,
active site shape loss of binding
efficiency and eventually
enzyme activity

pH
Enzymes and pH

Optimum pH This loss of activity is only

Reaction rate / arbitrary units

truly denaturation at extreme

pH since between optimum
and these extremes, the loss of
activity is reversible

pH
Enzymes and pH
3. EFFECT OF SUBSTRATE CONC.

As soon as a reaction begins,

[S] begins to fall and so it is
important that initial
Initial reaction rate /

reaction rates are measured

arbitrary units

[S]
 Initial reaction rate /
arbitrary units
Enzymes and [S]

[S]
Enzymes and [S]

Increasing[S]
increases collision
rate and increases
Initial reaction rate /

reaction rate
arbitrary units

[S]
Enzymes and [S]

All active sites are

occupied. Enzymes
are working at
maximum rate.
Initial reaction rate /
arbitrary units

All active sites

are not occupied

[S]
Enzymes and [S]

Maximum
turnover number
or Vmax has been
reached
Initial reaction rate /
arbitrary units

[S]
Enzyme inhibitors or poisons
 INHIBITION
• The prevention of an enzyme process as a result of
interaction of inhibitors with the enzyme.

 INHIBITORS:
• Any substance that can diminish the velocity of an
enzyme catalyzed reaction is called an inhibitor.

32
 TYPES OF INHIBITION

Inhibition

Reversible Irreversible

Competitive Uncompetitive Non-

competitive

33
 REVERSIBLE INHIBITION
• It is an inhibition of enzyme activity in which the
inhibiting molecular entity can associate and
dissociate from the protein’s binding site.

34
COMPETITIVE INHIBITION
 In this type of inhibition, the inhibitors compete with the
substrate for the active site.
 Formation of E.S complex is reduced while a new E.I
complex is formed.
 These compete with the substrate molecules for the
active site
 Increasing substrate concentration will reduce effects of
inhibitor & reversible.
 Resembles the substrate’s structure closely.
 Statin Drug As Example Of Competitive Inhibition:
 Statin drugs compete with HMG-CoA(substrate) &
inhibit the active site of HMG CoA-REDUCTASE (that
bring about the catalysis of cholesterol synthesis).

35
 Non COMPETITIVE INHIBITION
 Inhibitor does not compete with the substrate for the active
site of enzyme instead it binds to another site known as
allosteric site.
 Another separate site where the modifier binds (allo =
other)
 These are not influenced by the concentration of the
substrate & irreversible.
 They alter the overall shape of the enzyme.
 Examples A variety of poisons, such as
 Cyanide inhibits cytochrome oxidase (Fe).
 Fluoride inhibits the enzyme, enolase (Mg).
 Iodoacetate inhibits enzymes having –SH group in their
active site.

36
 Uncompetitive INHIBITION
 Here inhibitor does not have any affinity for free
enzyme.
 Inhibitor binds to Enzyme–Substrate Complex; but
not to the free enzyme.
 placental alkaline phosphatase by phenylalanine is an
example of uncompetitive inhibition.

37
Figure 5.15A

Substrate
Active site

Enzyme

Allosteric site

Normal binding of substrate

Competitive Noncompetitive
inhibitor inhibitor

Enzyme inhibition

38
IRREVERSIBLE INHIBITION
 This type of inhibition involves the covalent attachment of
the inhibitor to the enzyme.
 Combine with the functional groups of the amino acids in
the active site, irreversibly
 The catalytic activity of enzyme is completely lost.

 It can only be restored only by synthesizing molecules.

 Example

 Aspirin which targets and covalently modifies a key

enzyme involved in inflammation is an irreversible
inhibitor.

39
 Clinical Use of Enzyme

Plasma functional enzymes Vs Non-plasma functional enzymes

 present in plasma and have  Present in plasma in very low conc..
specific function  Their substrate is absent in plasma.
 Activities are higher in  Increased levels in plasma indicate
plasma than tissues. tissue damage.
 These enzymes can be used for
diagnosis. 40
Clinical Use of Enzyme
• In disease of tissues and organs, the non plasma specific
enzymes are most important.
• Normally, the plasma levels of these enzymes are low to
absent.
• A disease process may cause changes in cell membrane
permeability or increased cell death, resulting in release of
intracellular enzymes into the plasma.
• The measurement of serum levels of certain enzymes in
plasma is used as indicator of disease of particular organ.
• An ideal biomarker:
1. will not be present in the blood of normal, healthy
individuals.
2. will be specific to a particular type of tissue.
3. Any changes in the concentration in clinical samples
must be directly related to disease.
41
42
43