ENZYME INHIBITION
Rate of reaction of an enzyme can be affected
specifically by:
Activator: to enhance/increase the rate of
reaction
Inhibitor: to reduce the rate of reaction
The importance of inhibition and activation
studies:
i. Inhibition and activation are a discreet
metabolic regulation in biological system
ii. External interference on metabolism, either
by drugs, pesticides or any toxic agents
usually by enzyme inhibition.
iii. Inhibition is an important method in the
study of the mechanism of enzymatic
reaction.
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INHIBITION
Two considerations :
Specificity
The degree of reversibility
Irreversible Inhibitor
Usually involved the action non-specific
agents for example, acid, alkali, urea,
detergent, heavy metals, reducing agents and
protease, or high temperature which destroy
(denature) the structure of the protein. Thus
the enzyme is deactivated (inactive- the
enzyme losing activity).
Reversible Inhibitor
Usually involved the action of selective or
specific agents eg, protein inhibitors,
metabolite inhibitors, drugs etc. interact
with the enzyme at specific sites without
destroying /denaturing the 3-dimentional
structure of the enzyme but inhibiting the
enzyme activity.
2
Examples of Inhibition :
Example 1
- Inhibition of glyseraldehyde 3-phoshate
dehydrogenase by iodoacetate
E-SH + ICH2COOH E-SCH2COOH + HI
- Irreversible inhibition
- The inhibition could be reverted or
retrieved by dialysis or competitively by
addition of thiol groups.
Example 2
- Inhibition by DTNB or Ellman’s reagent
( 5,5’-dithiobis-(2-nitrobenzoate) )
- the binding (covalent) of
thionitrobenzoate to the enzyme
- the modification is stable
- irreversible inhibition
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Example 3
- Inhibition of glutamate dehydrogenase by
pyridoxal phosphate
bes Schiff
atau imin
- formation of covalent bond
- could be reverted by dialysis or addition
of amino compounds
- Irreversible inhibition
Example 4
- Inhibition of succinate dehydrogenase by
malonate
succinate + FAD fumarate + FADH2
- Reversible inhibition
- Covalent bond not involved
- Could be reverted by dialysis or dilution
4
Competitive inhibitor:
Compound which closely resembles the
chemical structure and molecular geometry
of the substrate.
- The inhibitor competes for the same active
site as the substrate molecule
- The inhibitor may interact with the enzyme at
the active site, but no reaction takes place.
- The inhibitor ‘stuck’ on the enzyme and
prevents any substrate molecules from
reacting with the enzyme
However: Competitive inhibition is usually
reversible; if sufficient substrate molecules
are available it could displace the inhibitor
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Methanol poisoning: Occurs because
methanol is oxidized to formaldehyde and
formic acid which attack the optic nerve
causing blindness.
Ethanol is given as an antidote for
methanol poisoning because ethanol
competitively inhibits the oxidation of
methanol.
Ethanol is oxidized in preference to
methanol and consequently the oxidation of
methanol is slowed down
The toxic by-products do not have a chance
to accumulate.
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Reversible inhibition
Could be classified base on the pattern of its
reaction
In a simple uni-substrate kinetics an
inhibitor could bind with :
- E but not with ES
(competitive inhibition)
- ES but not with E
(uncompetitive inhibition)
- Both E and ES
(noncompetitive inhibition)
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Competitive inhibition
- E could bind with:
- S to form ES
or
- I to form EI
- In this case two incidences could happen:
i. Inhibitor could bind to the active site
ii. inhibitor could bind to other site and
cause the active site unavailable
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k1 k3
E + S ES E + P
+ k2
I
Ki
EI
[E][I] = Ki or [EI] = [E][I]
[EI] Ki
Ki = dissociation constant for complex EI
Km = [E] [S] or [E] = [ES] Km
[ES] [S]
- conservation enzyme equation :
e = [ES] + [E]
- will be :
e = [ES] + [E] + [EI]
- substituting the value of [EI] :
e = [ES] + [E] + [E][I]
Ki
e = [ES] + [E] (1 + [I])
Ki
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- substituting the value of [E] :
e = [ES] + [ES] Km (1 + [I])
[S] Ki
e = [ES][1 + Km (1 + [I])]
[S] Ki
- as in the absence of inhibitor
v = k3 [ES]
- substituting the value of [ES] :
v = k3e
1 + Km (1 + [I])
[S] Ki
v = k3e[S]
[S] + Km(1 + [I])
Ki
- substituting the value of Vm :
v = Vm[S]
[S] + Km(1 + [I])
Ki
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- Lineweaver-Burk plot for competitive
inhibition could be derived as :
1 = Km(1 + [I]/Ki) 1 + 1
v Vm [S] Vm
- 1/v against 1/[S] plot will give a straight
line with :
- intercept at x-axis = -1
Km (1 + [I]/Ki)
- intercept at y-axis = 1/Vm
- gradient of the graph = Km (1 + [I]/Ki)
Vm
Kawalan
Vm
- Vm value remains unchanged
- Km value increases with a factor of
(1 + [I]/Ki)
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Determination of Ki value
- For competitive inhibition Ki value could
be determined by :
i. Lineweaver-Burk plot
- Gradient and intercept values at x-axis
is different by a factor of (1 + [I]/Ki)
ii. LB gradient against [I] plot
LB gradient = Km (1 + [I]/Ki)
Vm
= Km .[I] + Km
Vm Ki Vm
- LB gradient against [I] will give a
straight line with an intercept at
x-axis = - Ki
Kecerunan
plot LB
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iii. Dixon plot
1 = Km (1 + [I]/Ki) 1 + 1
v Vm [S] Vm
= Km + Km [I] + 1
Vm [S] Vm [S] Ki Vm
= Km [I] + 1 (1 + Km/[S])
Vm [S] Ki Vm
- Graph of 1/v against [I] will give a
straight line
Km (1 + [I]/Ki) 1 + 1 = Km (1 + [I]/Ki) 1 + 1
Vm [S]1 Vm Vm [S]2 Vm
(1 + [I]/Ki) 1 = (1 + [I]/Ki) 1
[S]1 [S]2
It could only happen at [I] = - Ki
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Also at [I] = - KI , the value of
1 = 1
v Vm
- 1/v is independent of [S]
- Dixon plot at different values of [S]
will intercept at [I] = - Ki
Uncompetitive inhibition
- The binding of [I] only to ES but not to E
k1 k3
E + S ES E + P
k2 +
I
Ki
EIS
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[ES][I] = Ki or [EIS] = [ES][I]
[EIS] Ki
Km = [S][E] or [E] = Km[ES]
[ES] [S]
- Conservation of enzyme equation :
e = [ES] + [E] + [EIS]
- Substituting [E] and [EIS] values :
e = [ES] + Km[ES] + [ES][I]
[S] Ki
e = [ES] (1 + Km + [I] )
[S] Ki
- As in the absence of inhibitor :
v = k3 [ES]
- Substituting the value of [ES]
v = k3e
1 + Km + [I]
[S] Ki
= k3e
(1 + [I]) + Km
Ki [S]
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= k3e[S]
[S](1 + [I]) + Km
Ki
- Substituting the value of Vm
v = Vm[S]
[S](1 + [I]) + Km
Ki
v = Vm[S] / (1 + [I]/Ki )
[S] + Km / (1 + [I]/Ki )
- Both Vm and Km reduce by a factor of
(1 + [I]/Ki )
- LB plot for uncompetitive could be
derived as :
1 = Km 1 + (1 + [I]/Ki)
v Vm [S] Vm
- Graph of 1/v against 1/[S] gives a straight
line with :
- Intercept at x-axis = - (1 + [I]/Ki)
Km
- Intercept at y-axis = (1 + [I]/Ki)
Vm
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- graph gradient = Km/Vm
Kawalan
- intercept values for both axis increase by
the same factor that resulting parallel
straight lines
Noncompetitive inhibition
- Could bind to both E and ES to form EI
and EIS
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k1 k3
E + S ES E + P
+ k2 +
I I
KiE KiES
EI EIS
[E] [I] = Ki = [ES] [I]
[EI] [EIS]
[EI] = [E] [I] and [EIS] = [ES] [I]
Ki Ki
Km = [E] [S] or [E] = Km [ES]
[ES] [S]
- Conservation of enzyme equation :
- e = [ES] + [E] + [EI] + [EIS]
- substituting the values of [E], [EI] and
[EIS]
e = [ES] + Km [ES] + [E] [I] + [ES] [I]
[S] Ki Ki
= [ES] + Km[ES] + Km[ES][I] + [ES][I]
[S] [S] Ki Ki
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= [ES] (1 + Km + Km [I] + [I] )
[S] [S] Ki Ki
- as in absence of inhibitor :
v = k3 [ES]
- substituting the value of [ES] :
v = k3e
1 + Km + Km [I] + [I]
[S] [S] Ki Ki
= k3e [S]
[S] (1 + [I] ) + Km (1 + [I] )
Ki Ki
- Substituting the value of Vm :
v = Vm[S]
[S] (1 + [I] ) + Km (1 + [I] )
Ki Ki
= Vm[S] / (1 + [I]/Ki )
[S] + Km
- Vm value reduces by a factor of
(1 + [I]/Ki)
- Km value is unchanged
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- LB plot for noncompetitive inhibition
could be derived as :
1 = Km (1 + [I]/Ki ) 1 + (1 + [I]/Ki )
v Vm [S] Vm
- graph 1/v against 1/[S] will give a straight
line with :
- intercept at x-axis = - 1/Km
- intercept at y-axis = (1 + [I]/Ki)
Vm
Kawalan
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Summary of characteristics of different
types of reversible inhibitors
Inhibition Binding of I Vm Km
Competitive E =
Uncompetitive ES
Noncompetitive E and ES =
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Competitive Inhibition :
vo = Vm [ S ]
[ S ] + Km ( 1 + [ I ] )
Ki
1 = Km ( 1 + [ I ] ) 1 + 1
v Vm ( Ki ) [ S ] Vm
Vm I2
1/v
v I1
MM
[S]
1/[ S ]
Ki is the concentration of [ I ] which increases the
gradient of Lineweaver-Burk plot 2 x or increases
Km by 2 x at M-M
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Non-competitive Inhibition :
vo = Vm [ S ]
(1+[I])
Ki
[ S ] + Km
1 = Km ( 1 + [ I ] ) + ( 1 + [ I ] )
v Ki Ki
Vm [ S ] Vm
Vm I3
1/v I2
I1
Vm I0
I
[S] 1/[ S ]
Ki is the concentration of [ I ] which inhibits 50% Vm
at M-M .
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Un-competitive Inhibition :
vo = Vm [ S ]
(1+[I])
Ki
[ S ] + Km
(1+[I])
Ki
1 = Km + ( 1 + [ I ] / Ki )
v Vm [ S ] Vm
Vm
1/v I2
v I I1
[S]
1/[ S ]
Ki is the concentration of [ I ] which increases
intercept at Y by 2 x ie. 1/Vm by 2 x
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