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CHMI 2227E
Biochemistry I
Enzymes:
-
Inhibition
CHMI 2227 - E.R. Gauthier, Ph.D.
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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;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Enzyme inhibition
Example 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!
VEGF-R
VEGF
Endothelial cell
growth/migration
ZD6474
Endothelial cell
CHMI 2227 - E.R. Gauthier,
Ph.D. of Cancer (2005) 92(Suppl 1), S6 – S1
British Journal
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Int. J. Impot. Res. (2004) 16, S11–S14
Enzyme inhibition
Example 2 – Sildenafil:
Sildenafil:

cGMP-Phosphodiesterase
inhibitor;

Initially tested as an antihypertension drug;
Acetylcholine
Endothelial cell
Vascular smooth
muscle cell
GMP
PDE
Nitric Oxide
Synthase (NOS)

Arginine
Muscle
relaxation
Sildenafil
cGMP
NO
NO
Guanylate
cyclase
CHMI 2227 - E.R. Gauthier, Ph.D.
Blood vessel
Dilation
GTP
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Enzyme inhibition
Example 3 – Acetaminophen (tylenol):
CHMI 2227 - E.R. Gauthier, Ph.D.
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Reversible Enzyme inhibition
1- 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:


P
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.
Trypsin inhibitor
CHMI 2227 - E.R. Gauthier, Ph.D.
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Reversible Enzyme inhibition
1- 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.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Reversible Enzyme inhibition
2- 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.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Reversible Enzyme inhibition
2- Uncompetitive inhibition

Vmaxapp= Vmax / (1 + [I]/Ki)

Kmapp= Km / (1 + [I]/Ki)
CHMI 2227 - E.R. Gauthier, Ph.D.
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Reversible Enzyme inhibition
3- 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).
CHMI 2227 - E.R. Gauthier, Ph.D.
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Reversible Enzyme inhibition
3- Noncompetitive inhibition

Vmaxapp= Vmax / (1 + [I]/Ki)
CHMI 2227 - E.R. Gauthier, Ph.D.
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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
CHMI 2227 - E.R. Gauthier, Ph.D.
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Irreversible enzyme inhibition
1. 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)
CHMI 2227 - E.R. Gauthier, Ph.D.
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Irreversible enzyme inhibition
2. 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;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Irreversible enzyme inhibition
3. 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;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Irreversible enzyme inhibition
3. Suicide inhibitors - penicillin

Tetrapeptide
Sugars
Interfere with the synthesis of the
bacterial cell wall

Makes bacteria much less
resistant to stress;
pentaGly
bridges
Pen

Cell wall:


Peptidoglycan
Penicillin blocks the formation of
the link between the tetrapeptide
and the pentaGly bridge;
Structure of the bacterial
cell wall
CHMI 2227 - E.R. Gauthier, Ph.D.
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Irreversible enzyme inhibition
3. Suicide inhibitors - penicillin
Glycopeptide
transpeptidase
Glycopeptide
transpeptidase
pentaGly
bridge
Penicillin
Tetrapeptide
Glycopeptide
transpeptidase
CHMI 2227 - E.R. Gauthier, Ph.D.
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Irreversible enzyme inhibition
3. Suicide inhibitors - penicillin
CHMI 2227 - E.R. Gauthier, Ph.D.
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