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Enzyme Inhibition

Enzyme inhibitors are molecules that interact in some way with the enzyme to
prevent it from working in the normal manner; thus they decrease the rate of
an enzyme-catalyzed reaction.

There are a variety of types of inhibitors including: nonspecific, irreversible,


reversible competitive, uncompetitive and noncompetitive (mixed). Poisons
and drugs are examples of enzyme inhibitors.
Nonspecific Inhibitors

A nonspecific inhibition effects all enzymes in the same way. Non-


specific methods of inhibition include any physical or chemical changes
which ultimately denatures the protein portion of the enzyme and are
therefore irreversible.
Nonspecific Inhibitors

Temperature: Usually, the reaction rate increases with temperature,


but with enzyme reactions, a point is reached when the reaction rate
decreases with increasing temperature. At high temperatures the
protein part of the enzyme begins to denature, thus inhibiting the
reaction.
Nonspecific Inhibitors

Acids and Bases: Enzyme activity is also controlled by pH. As the pH is


decreased or increased, the nature of the various acid and amine
groups on side chains is altered with resulting changes in the overall
shape structure of the enzyme.
Irreversible Inhibition

MichaelisMenten kinetics cannot be applied to irreversible inhibition.


The inhibitor forms a covalent linkage with the enzyme molecule and
cannot be removed. The effectiveness of an irreversible inhibitor is
determined not by the equilibrium constant but by the rate at which
the binding takes place.
Irreversible Inhibition

Iodoacetamides and maleimides act as irreversible inhibitors to the


sulfhydryl groups:
Irreversible Inhibition

Another example is the action of diisopropyl phosphofluoridate (a


nerve gas) on the enzyme acetylcholinesterase.

The nerve gas diisopropyl phosphorofluoridate forms a strong covalent bond with
the hydroxyl group of the serine residue at the active site of acetylcholinesterase.
Irreversible Inhibition
When a nerve makes a muscle cell contract, it gives the cell a tiny squirt of acetylcholine
molecules. Acetylcholine is called a neurotransmitter because it acts as a messenger
between the nerve and the final destination (in this case, the muscle cell).

Once they have performed the proper function, the acetylcholine molecules must be
destroyed; otherwise, the resulting excess of this substance will hyperstimulate glands and
muscle, producing convulsions, choking, and other distressing symptoms. Many victims of
exposure to this nerve gas suffer paralysis or even death.
Irreversible Inhibition

The effective removal of excess acetylcholine is by means of a


hydrolysis reaction.

The catalyst for this reaction is acetylcholinesterase.


Irreversible Inhibition

The irreversible inhibition of this enzyme takes place via the formation
of a covalent bond between the phosphorus atom and the hydroxyl
oxygen of the serine residue in the enzyme.

The complex formed is so stable that for practical purposes the


restoration of normal nerve function must await the formation of new
enzyme molecules by the exposed persons body.
Reversible Inhibition

There are three important types of reversible inhibition: competitive


inhibition, uncompetitive inhibition, and noncompetitive (mixed)
inhibition. We shall discuss each type in turn.
Competitive Inhibition
A competitive inhibitor is any 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 is "stuck" on the enzyme and prevents any substrate


molecules from reacting with the enzyme.
Competitive Inhibition

Competitive inhibition. Both the substrate and the inhibitor


compete for the same active site. Only the ES complex leads to
product formation.
Competitive Inhibition
The reactions are

where
Competitive Inhibition
Note that the complex EI does not react with S to form products. Applying the
steady-state approximation for ES, we obtain:

[]
= [I]
(10)
1+ +[]
I

Which has the same form as the MichaelisMenten expression (Eq. 6), except that
the KM term has been modified by 1 + [I] I , thereby reducing .
Competitive Inhibition
The LineweaverBurk equation is given by

1 [I] 1 1
= 1+ + (11)
I []

Thus, a straight line results when 1/r is plotted versus 1/[S] at constant [I]. The
difference between this Equation and the rearranged Michaelis-Menten expression
(Eq. 9) is that in the former, the slope is enhanced by the factor 1 + [I] I .
Competitive Inhibition
The intercept on the 1/r axis is the same for because rmax does not
change.
Competitive Inhibition
Dividing the MichaelisMenten expression (Eq. 6) by Equation (10), we
obtain

To overcome competitive inhibition, we need to increase the substrate


concentration relative to that of the inhibitor; that is, at high substrate
concentrations, []I I, so that
Competitive Inhibition
A well-known example of a competitive inhibitor is malonic acid, CH2(COOH)2,
which competes with succinic acid in the dehydrogenation reaction catalyzed by
succinic dehydrogenase:

Because malonic acid resembles succinic acid in structure, it can combine with the
enzyme, although no product is formed in this reaction.
Uncompetitive Inhibition
An uncompetitive inhibitor does not bind to the free enzyme; instead,
it binds reversibly to the enzymesubstrate complex to yield an inactive
ESI complex.

Uncompetitive inhibition. The inhibitor binds only to the ES


complex. The ESI complex does not lead to product formation.
Uncompetitive Inhibition
The reactions are

where
Uncompetitive Inhibition

The ESI complex does not form a product. Again, because I does not
interfere with the formation of ES, uncompetitive inhibition cannot be
reversed by increasing the substrate concentration.
Uncompetitive Inhibition
The initial rate is given by

(12)

Comparison of Equation (12) with the MichaelisMenten expression (Eq. 6),


shows that both rmax and KM have been reduced by the factor 1 + [I] I .
Uncompetitive Inhibition
The LineweaverBurk equation is given by

(13)

Thus, a straight line is obtained by plotting 1/r versus 1/[S] at constant [I]. The
difference between this Equation and the rearranged Michaelis-Menten expression
(Eq. 9) is that the intercept on the 1/r axis is altered by the factor 1 + [I] I , but
the slope remains the same.
Uncompetitive Inhibition
Uncompetitive Inhibition
Dividing the MichaelisMenten expression (Eq. 6) by Equation (12), we get

If conditions are such that [] , then the equation above becomes

We see that increasing the substrate concentration cannot overcome the


effect of I in uncompetitive inhibition.
Uncompetitive Inhibition

Uncompetitive inhibition is rarely observed in one-substrate systems.


Multisubstrate enzymes, however, often give parallel line plots with
inhibitors.
Noncompetitive (Mixed) Inhibition
A noncompetitive inhibitor binds to the enzyme at a site that is distinct
from the substrate binding site; therefore, it can bind to both the free
enzyme and the enzymesubstrate complex

Noncompetitive inhibition.
The inhibitor binds to a
site other than the active
site.
The ESI complex does not
lead to product formation.
Noncompetitive (Mixed) Inhibition
The binding of the inhibitor has no effect on the substrate binding, and vice versa.
The reactions are

Neither EI nor ESI forms products. Because I does not interfere with the formation
of ES, noncompetitive inhibition cannot be reversed by increasing the substrate
concentration.
Noncompetitive (Mixed) Inhibition
The initial rate is given by

(14)

Comparing Equation (14) with the MichaelisMenten expression (Eq. 6), we see
that rmax has been reduced by the factor 1 + [I] I but KM is unchanged.
Noncompetitive (Mixed) Inhibition
The LineweaverBurk equation is given by

(15)

We see that a plot of 1/r versus 1/[S] gives a straight line with an
increase in slope and intercept on the 1/r axis.
Noncompetitive Inhibition
Noncompetitive Inhibition
Dividing the MichaelisMenten expression (Eq. 6) by Equation (14), we get

the extent of noncompetitive inhibition is independent of [S] and depends


only on [I] and I .
Noncompetitive (Mixed) Inhibition
Noncompetitive inhibition is very common with multisubstrate
enzymes. Other examples are the reversible reactions between the
sulfhydryl groups of cysteine residues on enzymes with heavy metal
ions: