Enzymes are protein catalysts that accelerate biochemical reactions without being consumed. They achieve this by lowering the activation energy of reactions and stabilizing transition states. Enzymes require cofactors like metal ions or organic coenzymes to function. The active site of an enzyme binds specifically to substrates to catalyze reactions. Many factors influence enzyme activity, such as temperature, pH, substrate and product concentration.

1. ENZYMES
MAN BAHADUR RANA
BPH
ACAS,NEPAL

2. Enzymes: Introduction
Enzymes may be defined as biocatalysts
synthesized by living cells. They are protein in
nature (exception - RNA acting as ribozyme),
colloidal and thermolabile in character, and
specific in their action.
Enzyme or catalysts accelerates reaction by
lowering energy of activation of the reacting
molecules but do not influence their
thermodynamic characteristics. Similarly catalyst
or enzymes do not change the equilibrium
constants of reactions.

3. Catalysts or enzymes increase product formation by
(1) lowering the energy barrier (activation energy) for the
product to form
(2) increases the favorable orientation of colliding
reactant molecules for product formation to be
successful (stabilize transition state intermediate)

4. Structural Organization of Enzyme
• Some enzymes require an additional chemical
component called a cofactor for their activity
• holoenzyme= Apoenzyme or Apoprotein+ Co-
factors
(Active) (Inactive)
• Haloenzyme: A complete catalytically active
enzyme with its cofactor is called Holoenzyme.
• Apoenzyme: The protein part of the holoenzyme is
called apoenzyme.

5. Structural Organization of Enzyme
• Cofactors are non-protein molecules that help enzymes
function and associated with the active site of enzyme.
• Organic co-factors – thiamin, riboflavin, niacin, biotin
are called Co-enzymes. Coenzymes are often regarded
as the second substrates or co-substrates.
• Inorganic co-factors – Mg ++, Fe++, Zn++, Mn++ are
called activators.
• In some enzymes, Co-enzyme or metal ion is tightly
and permanently bound to the enzyme called a
prosthetic group.

6. Structural Organization of Enzyme
Active Centre/Active Site
• As the substrate molecules are comparatively much smaller than
the enzyme molecules, there should be some specific regions or
sites on the enzyme for binding with the substrate. Such sites of
attachment are variously called as ‘active sites’ or ‘catalytic sites’
or ‘substrate sites’.

7. Structural Organization of Enzyme
Isozyme:
• Many enzymes occur in more than one molecular
form in the same species, in the same tissue or
even in the same cell and catalyze the same
reaction at different rates.
• Their activities towards different substrates will
also vary.
• Lactate dehydrogenase-have 4 subunits hence 5
isozymes
LDH1 and LDH2 have greater activity towards
Beta-Hydroxy butyrate than others.

8. Classification of Enzymes
Since 1964, the IUB system of enzyme
classification has been in force. Enzymes are
divided into six major classes (in that order).
Each class on its own represens the general
Type of reaction brought about by the
enzymes of that class.

9. Classes of enzymes
1. Oxidoreductases = catalyze oxidation-reduction
reactions e.g. Succinate dehydrogenase, Alcohol
dehydrogenase, dehydrogenase of NADH and NADPH
2. Transferases = catalyze transfer of functional groups
from one molecule to another.
eg. Phosphotransferase, aminotransferase, Acyl
transferase
3. Hydrolases = catalyze hydrolytic cleavage.
eg. Peptidase, Glycosidase, Esterase.

10. Classes of enzymes
4. Lyases = catalyze removal of a group from or
addition of a group to a double bond, or other
cleavages involving electron rearrangement.
eg. Decarboxylases, Aldolase, Dehydratase,
Deaminase.
5. Isomerases = catalyze intramolecular
rearrangement.
Eg. Glucose-6- phosphoisomerase,
Phosphoglycerate phosphomutase.
6. Ligases = catalyze reactions in which two
molecules are joined.
Eg. Amino acetyl tRNA synthetase, Glutamine
synthetase.

11. Mechanism of Enzymes Action
• For any chemical reaction to occur, the reactants have
to be in an activated state or transition state which
depends upon mainly two factors.
• 1. Lowering of Activation energy: The energy
required by the reactants to undergo the reaction is
known as activation energy.
• The reactants when heated attain the activation
energy. The catalyst (or the enzyme in the biological
system) reduces the activation energy and this causes
the reaction to proceed at a lower temperature.

12. Mechanism of Enzymes Action
• Enzymes do not alter the equilibrium constants, they
only enhance the velocity of the reaction. The role of
catalyst or enzyme is comparable with a tunnel made
in a mountain to reduce the .barrier

13. Mechanism of Enzymes Action
2. Enzyme Substrate Complex Formation
Michaelis Menten have proposed a hypothesis for enzyme action,
which is most acceptable. According to their hypothesis, the
enzyme molecule (E) first combines with a substrate molecule
(S) to form an enzyme-substrate ES complex which further
dissociates to form product (P) and enzyme (E) back. Enzyme
once dissociated from the complex is free to combine with
another molecule of substrate and form product in a similar
way.
The ES complex is an intermediate or transient complex and the
bonds involved are weak non-covalent bonds such as H-bond,
Van der waals forces, hydrophobic interations. Sometimes two
substrates can bind to an enzyme molecule and such reactions
are called as bisubstrate reactions.

14. Mechanism of Enzymes Action
A few theories have been put forth to explain mechanism
of enzyme-substrate complex formation.
1. Template or Lock and key model or Fischer's
template theory:
It states that the active site already exists in proper
conformation even in absence of substrate. Thus the
active site by itself provides a rigid, pre-shaped template
fitting with the size and shape of the substrate molecule.
Substrate fits into active site of an enzyme as the key fits
into the lock and hence it is called lock-and-key model.
But this can not explain change in enzymatic activity in
presence of allosteric modulators.

15. Mechanism of Enzymes Action

16. Mechanism of Enzymes Action
2. Induced fit theory or Koshland's model
• Koshland, in 1958, proposed a more acceptable and
realistic model for enzyme substrate complex
formation.
• The important feature of this model is the flexibility of
the region of active site. According to this active site
does not possess a rigid preformed structure on
enzyme to fit the substrate.
• On the contrary, the substrate during its binding
induces conformational changes in the active site to
attain the final catalytic shape and form.
• This explains several matters related to enzyme actions
such as enzymes become inactive on denaturation,,
allosteric modulation and competitive inhibition.

17. Mechanism of Enzymes Action
3. Substrate strain theory
• In this model, the substrate is strained due to the
induced conformation change in the enzyme.
• It is also possible that when a substrate binds to the
preformed active site, the enzyme induces a strain to
the substrate. The strained substrate leads to the
formation of product.
• In fact, a combination of the induced fit model with the
substrates train is considered to be operative in the
enzymatic action.

18. Factors Affecting Enzyme Action
a.Temperature :A bell-shaped curve is
usually observed. Temperature
coefficient or Q10 is defined as
increase in enzyme velocity when
the temperature is increased by
10oC.
1.Increase of velocity with
temperature: The reaction velocity
increases with temperature until a
peak velocity is reached. This
increase is the result of the
increased number of molecules
having sufficient energy to pass
over the energy barrier and form
the products of the reaction.

19. Factors Affecting Enzyme Action
2. Decrease of velocity with higher temperature:
Further elevation of the temperature results in a
decrease in reaction velocity as a result of
temperature-induced denaturation of the enzyme

20. Factors Affecting Enzyme Action
b.pH
Increase in the hydrogen ionconcentration (pH) considerably
influences the enzyme activity and a bell-shaped curve is
normally obtained

21. Factors Affecting Enzyme Action
1. Effect of pH on the ionization of the active site:
The concentration of H+affects reaction velocity in several
ways. First, the catalytic process usually requires that the
enzyme and substrate have specific chemical groups in either
an ionized or unionized state in order to interact. For example,
catalytic activity may require that an amino group of the
enzyme be in the protonated form –NH3
+
At alkaline pH this group is deprotonated, and the rate of the
reaction, therefore, declines.
2. Effect of pH on enzyme denaturation: Extremes of
can also lead to denaturation of the enzyme, because the
structure of the catalytically active protein molecule depends
on
the ionic character of the amino acid side chains

22. Factors Affecting Enzyme Action
3.The pH optimum varies for different enzymes: The pH at
which maximal enzyme activity is achieved is different for
different enzymes, and often reflects the [H+] at which the
enzyme functions in the body. For example, pepsin, a
digestive enzyme in the stomach, is maximally active at pH
2, whereas other enzymes, designed to work at neutral pH,
are denatured by such an acidic environment .

23. Factors Affecting Enzyme Action
c. Effect of Enzyme Concentration
As the concentration of the enzyme
is increased, the velocity of the
reaction proportionately increase as
enzyme is the limiting factor in the
enzyme substrate Reaction. This
property of enzyme is made use in
determining the serum enzymes for
the diagnosis of diseases. By using
a known volume of serum, and
keeping all the other factors
(substrate, pH, temperature etc.) at
the optimum level, the enzyme
could be assayed in the laboratory.

24. Factors Affecting Enzyme Action
d. Effect of Substrate Concentration:
Increase in the substrate concentration gradually increases the
velocity of enzyme reaction within the limited range of
Substrate levels. A rectangular hyperbola is obtained when
velocity is plotted against the substrate concentration, Three
distinct phases of the reaction are observed in the graph (A-
linear; B-curve; C-almost unchanged).
Order of reaction : When the velocity of the reaction is almost
proportional to the substrate concentration( i.e. [S] is less than
Km),the rate of the reaction is said to be first order with
respect
to substrate. When the [S] is much greater than Km, the rate of
reaction is independent of substrate concentration, and the
reaction is said to be zero order.

25. Factors Affecting Enzyme Action

26. Factors Affecting Enzyme Action
e. Effect of Product Concentration:
• The accumulation of reaction products generally decreases
the enzyme velocity. For certain enzymes, the products
combine with the active site of enzyme and form a loose
complex and, thus, inhibit the enzyme activity.
• It is also possible that under certain conditions of high
concentration of products a reverse reaction may be favored
forming back the substrate.
• In the living system, this type of inhibition is generally
prevented by a quick removal of products formed.

27. Factors Affecting Enzyme Action
f. Effect of time:
• Under ideal and optimal conditions (like pH,
temperature etc.), the time required for an
enzyme reaction is less.
• Variations in the time of the reaction are generally
related to the alterations in pH and temperature.
g. Other factors: Effect of Activators and Coenzymes,
Effect of Modulators and Inhibitors, Effect of light
and radiation.