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
