CHMI 2227 - E.R. Gauthier, Ph.D. 1

CHMI 2227EBiochemistry I

Enzymes:- Inhibition

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Enzyme inhibition

Enzyme inhibitors inactivate the enzyme;

Two main types of inhibition exist:

Reversible enzyme inhibition: enzyme activity can be recovered by removing the inhibitor (e.g. dialysis, gel filtration);

Irreversible enzyme inhibition: inhibitor binds covalently to enzyme, which is then irreversibly inactivated.

The inhibition (i.e. inactivation) of an enzyme can tell us a lot about the way it works;

Enzyme inhibitors are frequently used to define biological phenomena;

Enzyme inhibitors are also sought by the big pharma to block enzymes involved in diseases;

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Enzyme inhibitionExample 1 - VEGF Receptor inhibitors: VEGF (Vascular Endothelial Growth Factor):

Produced in embryos and tumours;

Acts via a cell surface receptor to trigger the growth of blood vessels;

Why inhibit VEGF-R: Blocking the action of VEGF (an enzyme) will block the

growth of blood vessels and starve tumours to death!

British Journal of Cancer (2005) 92(Suppl 1), S6 – S1Endothelial cell

VEGF

VEGF-R

Endothelial cell growth/migration

ZD6474

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Enzyme inhibitionExample 2 – Sildenafil:

Int.

J. Im

pot.

Res

. (20

04)

16, S

11–S

14

Endothelial cell Vascular smoothmuscle cell

Acetylcholine

Nit

ric

Ox

ide

S

yn

tha

se

(N

OS

)

NO

Arginine

NO Guanylate cyclase

GTP

cGMP

GMPMusclerelaxation Blood vessel

Dilation

PDE

Sildenafil

Sildenafil: cGMP-Phosphodiesterase

inhibitor;

Initially tested as an anti-hypertension drug;

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Enzyme inhibitionExample 3 – Acetaminophen (tylenol):

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Trypsin inhibitor

Reversible Enzyme inhibition1- Competitive inhibition

Most frequently encountered inhibitors;

I is very similar to S (i.e. it is a structural analog)

I and S compete for the same binding site on the enzyme: the active site;

Vmax stays the same: At high enough [S], S will outcompete I

Km is increased (Kmapp): Because I can bind E, the amount of S

required to reach ½ Vmax will be increased.

P

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Reversible Enzyme inhibition1- Competitive inhibition

The value of Kmapp can be used to obtain Km and Ki (the dissociation constant for the inhibitor): Kmapp = Km (1 + [I]/Ki)

Ki = [E][I]/[EI]

Ki is a measure of the affinity of I for E: the smaller Ki, the more potent the inhibition.

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Reversible Enzyme inhibition2- Uncompetitive inhibition

I only bind to ES, not the free enzyme;

Example: glycophosphate (Round-up herbicide)

Vmax is decreased: Some of the E is converted into an inactive ESI

complex.

Km is decreased: I reduces the amount of E that can participate in

the reaction;

ESI shifts the E + S ES to the right, leading to an apparent decrease in Km.

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Reversible Enzyme inhibition2- Uncompetitive inhibition

Vmaxapp= Vmax / (1 + [I]/Ki)

Kmapp= Km / (1 + [I]/Ki)

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Reversible Enzyme inhibition3- Noncompetitive inhibition I and S bind to different sites on E;

Binding of I on E doesn’t affect the binding of S on E (and vice versa);

So: Km is unchanged, but Vmax is decreased (I reduces the [E] that can generate P);

E.g. deoxycyclin (an antibiotic), which inhibits collagenase (a proteolytic enzyme involved in periodontal diseases).

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Reversible Enzyme inhibition3- Noncompetitive inhibition

Vmaxapp= Vmax / (1 + [I]/Ki)

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Irreversible enzyme inhibition

Irreversible inhibitors bind covalently to the enzyme and permanently inhibit it.

Very useful to identify the amino acids involved in catalysis

Three types: Group-specific Active site-directed reagents (aka Affinity labels) Suicide inhibitors

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Irreversible enzyme inhibition1. Group-specific inhibitors

React with amino acid side chains;

Lead to inhibition by interfering with the catalysis (e.g. by reacting with side-chains important for the catalysis);

E.g. diisopropyl fluorophosphate (DFP); Nerve gas Inhibits acetylcholine esterase

(and many other proteases with Ser at the active site)

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Irreversible enzyme inhibition2. Affinity labels

Inhibitor is structurally similar to S;

Reacts with active site residues;

I reacts with E to form a covalent bond that cannot be hydrolysed;

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Irreversible enzyme inhibition3. Suicide inhibitors

Modified substrates;

Initially processed by E as if it were the normal S;

However, an reaction intermediate covalently and irreversibly binds the E, leading to its inhibition;

Example 1: monoamine oxidase (MAO) inhibitors (MAO – breaks down certain neurotransmitters, e.g. serotonine, adrenaline) high MAO activity = depression;

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Irreversible enzyme inhibition3. Suicide inhibitors - penicillin Interfere with the synthesis of the

bacterial cell wall Makes bacteria much less

resistant to stress;

Cell wall: Peptidoglycan Penicillin blocks the formation of

the link between the tetrapeptide and the pentaGly bridge;

Structure of the bacterial cell wall

SugarsTetrapeptide

pentaGly bridges

Pen

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Irreversible enzyme inhibition3. Suicide inhibitors - penicillin

Tetrapeptide

pentaGly bridge

Glycopeptide transpeptidase

Glycopeptide transpeptidase

Glycopeptide transpeptidase

Penicillin

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Irreversible enzyme inhibition3. Suicide inhibitors - penicillin