ENZYME INHIBITION

 INTRODUCTION
 Inhibitors are to subtances which tend to decrease the
rate of an enzyme catalysed reaction some act on a
substrate or cofactor, we will restrict our discussion
here to those which combine directly with an enzymes.
 Enzyme inhibition is the process of decreasing the rate
of an enzyme activity.it serves as a major control
mechanism in the biological system.
ENZYME INHIBITION TYPES
 Ezymes inhibition is two types-
 Reversible inhibition
 Irreversible inhibition

REVERSIBLE INHIBITION-
It is a temparory process &
can be relieved.
 The three types of inhibition
 Competitive
 Uncompetitive
 Non-competitive
Enzyme inhibition

k3 E: Enzyme
k1 S: Substrate
E + S [ E-S ] [ E-P ] E + P P: Product
k2

Two last steps ≈irreversible, E-S to E-P rate limiting

Reaction velocity, V=k3 [E-S] Rate of form. ES:

k1[E][S]
Rate of decomp. ES: (k1 +
k3)[E][S]

Assume steady state ([E-S] doesn’t change)

k1[E][S] = (k1 + k3)[E][S]

[E] [S]
[E-S] = Michaelis const.: KM = (k2 + k3) / k1
(k2 + k3) / k1

[E] [S] [E] = [E tot] - [E-S]

[E-S] =
KM

([Etot] - [E-S] [S] [Etot] [S]

[E-S] = [E-S] =
KM [S] + KM
 k3 E: Enzyme
k1 S: Substrate
E + S [ E-S ] [ E-P ] E + P P: Product
k2

[Etot] [S]
[E-S] = V=k3 [E-S]
[S] + KM

k3[Etot] [S] Vmax: All enzyme sites occupied by S

V =
[S]
[S] + KM - 1 Vmax=k3 [Etot]
[S]>>KM,
[S] + KM

Vmax [S]
V = Michaelis Menten eq.
[S] + KM V

Vmax

Vmax : 2

KM [S]
 V

Vmax
Vmax [S]
V = Michaelis Menten eq.
[S] + KM
Vmax : 2

KM [S]

1
V

KM KM
1 1 1 Slope =
= + Lineweaver-Burk eq. Vmax
V Vmax [S] Vmax

-1
1
KM
Vmax
Measure rate at different [S]:
Determine KM and Vmax 1
[S]
 COMPETITVEINHIBITION-
Incompetitiveinhibition,thesubstrate&theexhibit
structuralsimilarityduetowhichtheycompletefortheactiveside.
 Competitiveinhibitorarealsocalledstructuralanalogs.
 IncompetitiveinhibitorVmaxremainconstantKmchange.
Competitive inhibition

Kapp
Examples of competitive
inhibitors
 Malonate vs succinate Enzyme: succinate dehydrogenase
 Krebs and his colleagues used malonate to investigate the TCA cycle.

 M etal ions also act as a competitive inhibitors.during glycolysis

Phosphoenol pyruvate +ADP pyruvate+ ATP

pyruvate kinase

Na+/Li+
Noncompetitive inhibition
Non competitive inhibitors binds to
the allosteric site(regulatory site) of
the enzyme & alters the 3-D
structure of the active site so that the
substrate can not fit the active site.
That binds to the free enzyme or to
the enzyme.
 Km is not affected

 Vmax is decreased
Noncompetitive inhibition

When Ki ≈ Ki’

 Reciprocal plot
Examples of noncompetitive inhibitors
 Heavy metals like lead, mercury (breaks disulfide bonds), chromium
will act as non-competitive inhibitor

 E – SH + Hg2+ E – S – Hg + H+
 Fluoride aldolase is enzyme that converts fructose 1-6 bisphosphate
 Fructose 1-6 bisphosphate dihydroxyacetone +
 ALDOLASE glyceraldehydes-3-
phosphates
APPLICATIONS:
To relieve cyanide poisoning.
Fluoride is used as a blood preservative for subsequent analysis of
glucose.
Uncompetitive inhibition
 Uncompetitive inhibihibition have
no affinity for substrate.it binds to
the allosteric sight of the ES
complex but not to the free
enzyme.
 Km is decreased
 Vmax is decreased
Uncompetitive inhibition
intercepts on the vertical and horizontal axis are proportionately changed
 Parallel lines in inhibited and uninhibited reactions
Vmax [S]
no EI formation  v =
Km + [S]

 [I ] 
1  ' 
 Ki 
 Reciprocal plot
 Examples of uncompetitive inhibitors

L-phenylalanine is an uncompetitive foe intestinal alkaline phosphate.

IRREVERSIBLE INHIBITION
It bind covalently with the functional gps of the active
site, thereby permanently disturbing the catalytic
activity leading to the total inactivation of the enzyme.
It is of two types
 Group specific inhibition
 Suicide inhibition
GROUP SPECIFIC INHIBITION
Group specific reagents chemical compounds that react
with specific functional grps of the active site
aminoacids.e.g. DIPF-it is a prominent component of
insecticide and nerve gas.
E-Ser-OH+DIPF____ E-Ser-O-DIP+F- + H+
 Iodoacetamide- Iodoacetamide inhibits enzyme
with active cysteine resedues by binding to the thiol
grps.
 APLICATIONS
 Thymidylate synthetase is used as an anticancer drugs
 Aspirin is used as an anti inflammatory agent.
 Penicillin is used as an anti bacterial drug due to its
ability to inhibit transpeptidase
 Xanthine oxidase is used in thetreatment of gout.
Mixed Inhibition

1. Always possible
2. Not possible with
uncompetitive inhibitors
3. Not possible with competitive
inhibitors
4. Not possible with competitive
or uncompetitive inhibitors
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
 Dose-response curves (DRC) and Dixon plots are also different for
partial inhibitors
 At high [I]  there is a residual fractional activity in DRC
 Dixon plots are not linear but hyperbolic