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ENZYME INHIBITION &

REGULATION

Dr. Atif Amin Baig


Department of biochemistry
UNISZA.
RECALL
 The relationship between reaction velocity and substrate concentration of an enzyme catalyzed reaction.

 What is enzyme kinetics

 Substrate specificity and enzyme kinetics

 Michaelis-Menten equation

 Ping Pong Mechanism

 Factors effecting Enzyme Activity


LEARNING OUTCOMES
• What are the enzyme inhibitors?

• Need to study the enzyme inhibitors.

• Types of enzyme inhibitors?

• Classification of enzyme inhibitors

• Reversible enzyme inhibitors and its types

• Irreversible enzyme inhibitors

• Mechanism of enzyme inhibition

• Types and mechanisms of reversible enzyme inhibitors

• Le Chatelier's principle

• Mechanisms of irreversible enzyme inhibitors

• Inhibition and types of enzymatic inhibitions

• Substrate analogs
WHAT ARE ENZYME
INHIBITORS?
Substances that can affect metabolic processes by
influencing the activity of enzymes

A large proportion of medicines act as enzyme inhibitors

Enzyme- kinetic experiments are therefore an important aspect of drug development


and testing procedures

Natural metabolites are also involved in regulatory processes as inhibitors


WHY TO STUDY ENZYME
INHIBITORS
• Most drugs are enzyme inhibitors

• Almost all therapeutic drugs are enzyme inhibitors

• Old medicine box standards such as aspirin and penicillin to the newest
compounds used to treat HIV infection

• Enzyme kinetics plays an outstanding role in this effort to produce effective


therapeutics

• For kinetic studies can quantify the degree that inhibitors inactivate or slow
down the targeted enzyme’s catalytic rate and describe its potential efficacy
as a drug
WHY TO STUDY ENZYME
INHIBITORS

Many naturally produced toxic substances and chemical warfare


agents are also enzyme inhibitors

For example,

1. A peptide found in snake venom became a lead compound in the development of a class of
hypertension drugs that inhibit angiotensin-converting enzyme (ACE inhibitors).
2. The nerve gas sarin is a potent inhibitor of an enzyme important for synaptic transmission in
nerve tissue.

CAN WE CONTROL METABOLISM THROUGH ENZYME INHIBITORS?


WHAT INHIBITORS DOES?

1. Decrease enzyme ability to bind substrate

2. Lower the enzyme’s catalytic activity

Do both
HOW TO PICK AN ENZYME
INHIBITOR?

1. Biochemical environment of the target enzyme

2. Specificity of action

3. The time period for which an enzyme is blocked .


INHIBITION
ENZYME INHIBITION

• The process of deactivation, deformation or


decrease in enzyme activity is called enzyme
inhibition
TYPES OF ENZYME
INHIBITIONS

• Type of enzyme inhibition is based on type of


inhibitors for enzymes
TYPES OF ENZYME
INHIBITORS
• There are two main types:

1. Reversible Enzyme Inhibitors

2. Irreversible Enzyme Inhibitors

• Differentiate based on the magnitude of the affinity for


enzyme
REVERSIBLE ENZYME
INHIBITORS

Reversible enzyme inhibitors bind and dissociate with their


enzyme in a equilibrium process

What it means?
IRREVERSIBLE ENZYME
INHIBITORS

Irreversible inhibitors bind tightly to an enzyme to


form an essentially permanent complex

What it means?
REVERSIBLE ENZYME
INHIBITORS
They do not cause any permanent changes in the enzyme

Types of Reversible Enzyme Inhibitors

1. Competitive Inhibitors
2. Uncompetitive inhibitors
3. Non-competitive inhibitors
4. Mixed inhibitors
TYPES OF REVERSIBLE
ENZYME INHIBITORS

1. COMPETITIVE INHIBITORS

The inhibitor reacts reversibly with an enzyme to form an


enzyme-inhibitor complex

The inhibitor must possess structural similarity with the natural


substrate to act as competitive enzyme inhibitor

substrate and inhibitor cannot bind to the enzyme at the same


time

This usually results from the inhibitor having an affinity for the
active site

substrate and inhibitor compete for access to the enzyme's


active site

If substrate will be added

Vmax = Constant
Km = High
TYPES OF REVERSIBLE
ENZYME INHIBITORS

2. UNCOMPETITIVE INHIBITORS
Inhibitor binds only to the substrate-enzyme complex, it should
not be confused with non-competitive inhibitors

The inhibitor combines with enzyme-substrate complex rather


than with the free enzyme to give inactive enzyme inhibitor
complex.

Vmax will decrease as a result of removing activated complex


Km will decrease due to better binding efficiency as a result of
Le Chatelier's principle and the effective elimination of the ES
complex thus decreasing the Km which indicates a higher
binding affinity

Le Châtelier's principle, also called Chatelier's principle or


"The Equilibrium Law”,

The principle is named after Henry Louis Le Châtelier and


sometimes Karl Ferdinand Braun who discovered it
independently

“When any system at equilibrium is subjected to change in


concentration, temperature, volume, or pressure, then the
system readjusts itself to (partially) counteract the effect of the
applied change and a new equilibrium is established”.
TYPES OF REVERSIBLE ENZYME
INHIBITORS

3. NON- COMPETITIVE INHIBITORS


A non-competitive inhibitor can combine with either the free
enzyme or the enzyme-substrate complex , interfering with the
action of both

Binding of the inhibitor to the enzyme reduces its activity but


does not affect the binding of substrate

The extent of inhibition depends only on the concentration of


the inhibitor.

Vmax will decrease due to the inability for the reaction to proceed
as efficiently

Km will remain the same as the actual binding of the substrate,


by definition, will still function properly.

Deforms the shape of enzyme

The altered shape and conformation of the enzyme slow down


both , the rates of formation and dissociation of enzyme-
substrate complexes
TYPES OF REVERSIBLE ENZYME
INHIBITORS

4. MIXED COMPETITIVE INHIBITORS

Inhibitor can bind to the enzyme at the same time as the


enzyme's substrate

Binding of the inhibitor affects the binding of the substrate, and


vice versa.

This type of inhibition can be reduced, but not overcome by


increasing concentrations of substrate

Although it is possible for mixed-type inhibitors to bind in the


active site, this type of inhibition generally results from an
allosteric effect where the inhibitor binds to a different site on an
enzyme.

Inhibitor binding to this allosteric site changes the conformation

Deforms the shape of enzyme


affinity of the substrate for the active site is reduced.
IRREVERSIBLE ENZYME
INHIBITION

They form tight complex with the enzyme

They permanently remove its catalytic activity


MECHANISM OF ENZYME INHIBITION
No. Mechanism
The blockade of an enzyme , catalysing a particular biochemical reaction leads to accumulation of the substrate. This results into a desired
1 biological response

2 The inhibition of an enzyme that catalyses the rate- limiting step decreases the production of a metabolite . (Multistep biochemical pathway)

3 Most of the enzymes need cofactors to catalyse the biochemical pathway . Inhibitors can be developed selectively for the cofactor involved

Two inhibitors may be employed simultaneously to achieve a great therapeutic effects . e.g. co-trimoxazole ( trimethoprim and
4 sulphamethoxazole ).

5 If the metabolism of the end-product is minimised , the accumulated end-product decreases the activity of an enzyme on its substrate

Inhibition of metabolizing target enzyme permits higher plasma levels as well as an increase in the plasma half-life of the drug .e.g. clavulanic
6 acid inhibits beta-lactamase enzymes

Multisubstrate analogs : A multisubstrate compound consists the features of binding sites of two or more different substrates in the same
7 molecule . e.g. pyridoxyl alanine has more binding ability towards pyruvate transaminase than pyridoxal and alanine .

Transition state analogs : An enzyme interacts with a substrate to form enzyme substrate complex through a tetrahedral transition state . A
8 transition state analog binds more tightly to an enzyme than it does its substrate . e.g. penicillin is a transition state analog which binds to the
peptidoglycan transpeptidase

Suicide enzyme inhibitors ( Kcat inhibitors ) : Irreversible enzyme inhibitor which utilizes highly electrophilic species such as alpha-halogenated
9 carbonyl compounds or other strong alkylating agents which inturn irreversibly reacts with a nucleophile group on the enzyme.

Suicide enzyme inhibitors are the compounds that possess latent reactive functional groups which are unmasked by the catalytic action of the
10 enzyme. e.g. Inhibition of aldehyde dehydrogenase by cyclopropanone , a metabolite of coprine .

Active site directed irreversible inhibitors : This category of inhibitors are designed by two important aspects :a) 1st approach : A section of the reactive part of
11 the enzyme may be incorporated into the inhibitor to recognize and to direct the inhibitor molecule to the enzyme’s active site .b) 2nd approach : A strong
nucleophile is placed in the inhibitor molecule which is expected to interact with an electrophile moiety located near the active enzyme site
SUBSTRATE ANALOGUES
Substrate analogs have properties similar to those of
one of the substrates of the target enzyme
They are bound by the enzyme, but cannot be
converted further and therefore reversibly block some
of the enzyme molecules present
A higher substrate concentration is therefore needed
to achieve a half- maximum rate; the Michaelis
constant Km increases
High concentrations of the sub- strate displace the
inhibitor again. The maximum rate Vmax is therefore
not influenced by this type of inhibition
Because the substrate and the inhibitor compete with
one another for the same binding site on the enzyme,
this type of inhibition is referred to as competitive
Analogs of the transition state usually also act
competitively
SUBSTRATE ANALOGUES

• Allosteric inhibitors bind to a separate binding site outside the active center
• This results in a conformational change in the enzyme protein that indirectly
reduces its activity
• Allosteric effects practically only occur in oligomeric enzymes.
• The kinetics of this type of system can no longer be described using the simple
Michaelis–Menten model.
SUBSTRATE ANALOGUES
• Suicide substrates” are substrate analogs that also contain a
reactive group.

• Initially, they bind reversibly, and then they form a covalent


bond with the active center of the enzyme.

• Their effect is therefore also non-competitive.

• A well-known example of this is the antibiotic penicillin


Enzyme Inhibition (Mechanism)

I Competitive I Non-competitive I Uncompetitive


Substrate E
Cartoon Guide

S S E I
S

E I
S I
I
Compete for S I
Inhibitor active site Different site
E + S → ES → E + P E + S → ES → E + P E + S → ES → E + P
← ← ←
Equation and Description

+ + + +
I I I I
↓ ↓ ↓ ↓
EI EI + S →EIS EIS
↑ ↑ ↑ ↑
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S]; complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].

Juang RH (2004) BCbasics


Enzyme Inhibition (Plots)

I Competitive I Non-competitive I Uncompetitive


Vmax Vmax Vmax
vo vo
Vmax’ Vmax’
Direct Plots

I I I

Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM


Vmax unchanged Vmax decreased
Km increased Km unchanged Both Vmax & Km decreased
Double Reciprocal

1/vo 1/vo 1/vo


I I I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]

Juang RH (2004) BCbasics


ENZYME ACTIVATORS

fructose 2,6-bisphosphate, which activates phosphofructokinase 1 and increases the rate of glycolysis
in response to the hormone insulin.
ASSIGNMENT

• Difference between enzyme activators and


co-factor

• Example of each type of enzyme inhibition


THANK YOU