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.

1
 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.

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 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

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  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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