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Enzyme Inhibition

Bryant Miles
Reversible versus Irreversible Inhibitiors.
There are many types of enzyme inhibitors. Inhibitors interact with enzymes in one of
two ways, reversibly and irreversibly. Reversible inhibitors interact with the enzyme
through noncovalent interactions and associate and dissociate with the enzyme.
Irreversible inhibitors form stable covalent bonds with the enzyme which block the active
site from binding substrate. The consequence of irreversible enzyme inhibition is a
decrease in the concentration of enzyme active sites.
I. Reversible Inhibitors.
Three categories of reversible enzyme inhibitors are competitive, noncompetitive and
mixed inhibitors.
Competitive inhibitors compete with the substrate for the same binding site on the
enzyme. The inhibitor is prevented from binding to the active site if the active site is
already occupied by a substrate molecule. Thus, increasing the substrate concentration
favors the binding of substrate to the enzyme over the inhibitor, I.
E + S
+
I
Ki
EI

k1
k2

ES

k3

E +P

Ki =

[ E ][ I ]
[ E ][ I ]
; [ EI ]K i = [ E ][ I ];[ EI ] =
Ki
[ EI ]

d [ E ] d [ ES ] d [ EI ]
=
=
=0
dt
dt
dt
d [ ES ]
= k1[ E ][ S ] k 2 [ ES ] k 3 [ ES ] = 0
dt
k1[ E ][ S ] = k 2 [ ES ] + k 3 [ ES ]
[E] =

(k 2 + k 3 )
[ ES ]
k1[ S ]

[ E0 ] = [ E ] + [ ES ] + [ EI ]
[ E0 ] = [ E ] + [ ES ] +
[ E0 ] =

[ E ][ I ]
Ki

[ I ](k 2 + k 3 )
(k 2 + k 3 )
[ ES ]
[ ES ] + [ ES ] +
k1[ S ]
K i k1[ S ]

(k + k 3 ) [ I ](k 2 + k 3 )
[ ES ]
[ E0 ] = 1 + 2
+
k
S
K
k
S
[
]
[
]
i 1
1

K k [ S ] + K i (k 2 + k 3 ) + [ I ](k 2 + k 3 )
[ ES ]
[ E0 ] = i 1
K i k1[ S ]

K i k1[ S ][ E0 ]
= [ ES ]
K i k1[ S ] + K i (k 2 + k 3 ) + [ I ](k 2 + k 3 )
[ ES ] =

v=

[ S ][ E0 ]
(k + k 3 ) [ I ](k 2 + k 3 )
[S ] + 2
+
k1
K i k1

dP
= k 3 [ ES ] =
dt

k 3 [ S ][ E0 ]
(k + k 3 ) [ I ](k 2 + k 3 )
[S ] + 2
+
k1
K i k1

Vmax = k 3 [ E0 ]; Km =
v=

v=

(k 2 + k 3 )
k1

Vmax [ S ]
Vmax [ S ]
=
[ I ]K m
[I ]
[S ] + K m +

[ S ] + K m 1 +
Ki
Ki
Vmax [ S ]
[I ]

[ S ] + K m 1 +
Ki

[I ]

[ S ] + K m 1 +
K i
Vmax [ S ]
K m [I ]
1
1

1 +
v=
; =
=
+
Vmax [ S ]
Vmax Vmax [ S ] K i
[I ] v

[ S ] + K m 1 +
Ki

The Lineweaver-Burk Plot is useful for


evaluating competitive inhibition. The
derivation for the double reciprocal plot is
shown above. A Lineweaver-plot of
competitive inhibition is shown to the
right.
Each of the plots at various inhibitor
concentrations pass through the same
intercept corresponding to 1/Vmax. This is
because the effect of the inhibitor
disappears as the substrate concentration
becomes high.
The slope of the plots depend on the
product
K m [I ]
compared to Km/Vmax for the uninhibited reaction. By measuring the
1 +
Vmax K i
slope of the plots as a function of the inhibitor concentration we can determine Ki.

The specificity pocket of trypsin can accommodate an


arginine side chain of a polypeptide substrate, or a
benzamidine ion which competes with the substrate for
the binding site and acts as a competitive inhibitor.

Noncompetitive inhibitors
Noncompetitive is when the binding of the inhibitor has no effect on the binding of
substrate. That is I and S bind to different sites on the enzyme such that the binding of I
has no effect of the binding of S.
Ki'
Ki
E+I
EI
ES + I
ESI
For pure noncompetitive inhibition Ki=Ki.
The characteristic of a noncompetitive
inhibitor is that it decreases the maximum rate
of the reaction without affecting the Km. The
inhibitor inhibits a fraction of the enzyme
regardless of the substrate concentration.
Lineweaver-Burk plots of noncompetitive
inhibitors all intersect the x-axis at the same
point which corresponds to -1/Km.

Mixed Noncompetitive Inhibition


It is rare due to the allosteric nature of ligand binding, that the substrate does not affect
the binding of an inhibitor. Even when the inhibitor binds to a separate site on the
enzyme, the binding of substrate is communicated to that site by conformational changes.
Similarly the binding of an inhibitor at a separate site is communicated to the substrate
binding site. In these situations, Ki does not equal Ki. The result is both the Km and the
Vmax are affected by inhibitor binding. This is a very common inhibition pattern.

Shown above are two Lineweaver-Burk plots of mixed noncompetitive inhibition. Note
that both the slopes and the intercepts change in the presence of I. The first curve on the
left is the situation where Ki < Ki. The graph on the right corresponds to Ki >Ki.

Uncompetitive Inhibition
There some cases where the inhibitor will only bind to the enzyme-substrate complex and
not to the free enzyme. These types of inhibitors are called uncompetitive inhibitors.
These inhibitors are common for enzymes that bind multiple substrates.
k1
k3
E + S
ES
E +P
k2
+
I
Ki
ESI
The Lineweaver-Burk Plots of these inhibitors are a series of parallel lines.

II. Irreversible Inhibition


Irreversible inhibitors covalently modify the enzyme which destroys catalytic activity.
The Lineweaver-Burk plot for irreversible inhibitors cannot be distinguished from the
noncovalent pattern, because of the net loss of active enzyme. Irreversible inhibition can
be distinguished from noncompetitive inhibition by measuring the time dependent loss of
enzyme activity. Also dialysis does not restore activity, as it would for a noncovalent
inhibitor.
Suicide Substrates.
Suicide substrates is the popular name for mechanism based inhibitors. These inhibitors
are designed to mimic substrate, and use knowledge of the mechanism of the enzymes
catalytic action to covalently and irreversibly inactivate the enzyme. These Trojan horse
substrates are a special type of affinity label. They bind at the active site of the enzyme
where the activated the activity of the enzyme and become covalently bound.

An example of a suicide substrate is Penicillin. The cell walls of bacteria are formed by
macromolecules called peptidoglycan which consists of linear polysaccharide chains
(NAG-NAM)x that are cross linked by short peptides. The cross-linking reaction is
catalyzed by an enzyme called glycoprotein peptidase. The enzyme catalyzed crosslinking reaction involves the formation of an acyl enzyme intermediate.

The antibiotic penicillin covalently reacts with an active site serine if glycoprotein
peptidase. Penicillin effectively blocks cell wall synthesis and makes the cells
susceptible to osmotic lysis.
Penicillin is an effective inhibitor of glycoprotein peptidase
because it resembles the transition state of the normal
substrate RDAlaDAla. (A) is the crystal structure of
penicillin. (B) is the proposed transition state of the
transpeptidase reaction.

Penicillin is an irreversible inhibitor of


glycopeptide transpeptidase which
catalyzes an essential step in bacterial
cell wall synthesis. Penicillin consists
of a thiozolidine ring fused to a lactam ring. A serine residue reacts
with the reactive peptide bond of the
lactam ring and becomes covalently
attached to the enzyme.