Enzyme Inhibition

• Since most clinical drug therapy is based on

inhibiting the activity of enzymes, analysis of
enzyme inhibition kinetics is fundamental to the
modern design of pharmaceuticals
– Well-known examples of such therapy include the use of
methotrexate in cancer chemotherapy to semi-selectively
inhibit DNA synthesis of malignant cells
– the use of aspirin to inhibit the synthesis of
prostaglandins which are at least partly responsible for
the aches and pains of arthritis
– the use of sulfa drugs to inhibit the folic acid
synthesis that is essential for the metabolism and growth
of disease-causing bacteria
• In addition, many poisons (such as cyanide, carbon
monoxide and polychlorinated biphenols (PCBs))
produce their life- threatening effects by means of
enzyme inhibition
 Enzyme Inhibition
• Reversible inhibition: the effect of an inhibitor
can be reversed by decreasing the concentration of
inhibitor
• Irreversible inhibition: there is no reversal of
inhibition on decreasing the inhibitor
concentration: an example of enzyme inactivation
– e.g. Cyanide: by covalently binding mitochondrial
cytochrome oxidase, it inhibits all the reactions
associated with electron transport
– Penicillin for bacterial peptidase

• The distinction between reversible and irreversible

inhibition is not absolute and may be difficult to
make if the inhibitor binds very tightly to the
enzyme and is released very slowly (tight-binding
inhibitors)
Enzyme inhibition
Lineweaver-Burk plot (double-
reciprocal)
 Kinetics of competitive inhibitor

Increase [S] to
Ki = overcome
dissociation inhibition
constant for
nhibitor Vmax attainable,
Km is increased
 Competitive inhibitor
max unaltered, Km increased
 Competitive
• Competitive inhibition
inhibitors are especially attractive as
clinical modulators of enzyme activity because they
offer two routes for the reversal of enzyme inhibition
1. like all kinds of reversible inhibitors, a
decreasing concentration of the inhibitor
reverses the equilibrium
2. since substrate and competitive inhibitors
both bind at the same site, raising [S],
while holding [I] constant, provides the
second route for reversal of competitive
inhibition
Examples of competitive inhibitors
Methotrexate (A competitive inhibitor of dihydrofolate
reductase - role in purine & pyrimidine
biosynthesis-Cancer Treatment)
2,3-biphosphoglycerate
– Inhibits its own formation by inhibiting
biphosphoglycerate mutase
 Metabolic regulation by product inhibition
Examples of competitive
inhibitors

• Malonate vs
succinate Enzyme:
succinate
dehydrogenase
• Krebs and his
colleagues used
malonate to
investigate the
TCA cycle
Kinetics of non-competitive inhibitor
Increasing [S] cannot
overcome inhibition

Less E available,
Vmax is lower,
Km remains the same
for available E
 Noncompetitive inhibitor
Km unaltered, Vmax decreased
Examples of noncompetitive inhibitors

• Heavy metals like lead, mercury (breaks disulfide

bonds), chromium will act as non-competitive
inhibitors

• Mono-amine oxidase (MAO) inhibitors that are used

as anti-depressants: They covalently react with
the enzyme in the liver and effectively remove it.

– There are many potent drug interactions with MAO

inhibitors. One of these is tyramine, a compound
that is present in red wine and aged cheeses
– MAO inhibitors and tyramine (blocks neurotransmitter
reuptake in the brain)  hypertensive crisis
– Patients are still subject to hypertension for as
long as two weeks after discontinuing the drug
 Uncompetitive inhibition

• The ES complex
dissociates the
substrate with a
dissociation constant
equal to Ks, whereas the
ESI complex does not
dissociate it (i.e has a
Ks value equal to zero)
 Km is decreased

• Increasing [S] leads to increasing [ESI] (a complex

incapable of progressing to reaction products),
therefore the inhibition can not be removed
 Vmax is decreased
Uncompetitive
Inhibition

14
 Examples of uncompetitive
inhibitors
• Lithium and the phosphoinositide cycle: an
example of uncompetitive inhibition and its
pharmacological consequences
Nahorski SR, et al Trends Pharmacol Sci. 1991
Aug;12(8):297-303
– The ability of lithium to exert profound and
selective psychopharmacological effects to ameliorate
manic-depressive psychosis has been the focus of
considerable research effort.
– There is increasing evidence that lithium exerts its
therapeutic action by interfering with
polyphosphoinositide metabolism in brain and
prevention of inositol recycling by an uncompetitive
inhibition of inositol monophosphatase
 Non competitive &
Uncompetitive
– For uncompetitive inhibition: Inhibitor
binding should only occur if the active site
is occupied by substrate. But in most cases,
the inhibitor will have some affinity for
the unoccupied enzyme as well

– For non-competitive inhibition: the

inhibitor affinity should be unchanged
regardless of whether substrate is bound or
not. The affinity for the inhibitor usually
changes when substrate is bound in reality
 Partial inhibitors
• In some situations, the enzyme can still work
with the inhibitor bound, but at a reduced rate
 Activity of the enzyme can not be driven to
zero...

• To be sure if the compound is a partial

inhibitor or not, one should pay attention to
experimental conditions

• Lineweaver-Burk plot would be linear, BUT

secondary plots of intercept or slope vs [I]
will not be linear
 Enzyme inhibition by DIPF
p - specific reagents react with R groups of amino acids

diisopropylphosphofluoridate

(nerve gas) reacts with Ser in acetylcholinesterase

Affinity inhibitor: covalent modification
Affinity inhibitor, bromoacetol
phosphate

Active site modified

 Mechanism-based enzyme
inactivators (Suicide Inhibitors)
• Active-site directed reagent (unreactive)
binds to the enzyme active site 
transformed to a reactive form. Once
activated, a covalent bond between the
inhibitor and the enzyme forms

• This approach minimizes side reactions (non

specific covalent bond formation) which may
occur with an affinity reagent

• 1) inhibitor binds to active site

2) converted to reactive compound via
enzyme's catalytic capabilities
3) covalently reacts with the enzyme