98
chapter6
Enzymes
1:
General Properties, Kinetics, and Inhibition
of (1 + [I] /
Ki
) (Table 6-2). Uncompetitive inhibition
is rarely observed in single-substrate reactions. A
noteworthy example in clinical enzymology is the
inhibition of intestinal alkaline phosphatase by
L-phenylalanine. Uncompetitive inhibition is more
common in two-substrate reactions with a
double-displacement reaction mechanism.
Irreversible Inhibition
Irreversible inhibition occurs when the inhibitor reacts at
or near the active site of the enzyme with covalent mod-
ification of the active site or when the inhibitor binds so
tightly that, for practical purposes, there is no dissocia-
tion of enzyme and inhibitor. The latter situation occurs
in the case of proteinase inhibitors (see below). Thus,
physical separative processes are ineffective in removing
the irreversible inhibitor from the enzyme. Irreversible in-
hibitor reaction is written
E + I -»• El (inactive enzyme)
Examples of irreversible inhibitors of enzymes are:
1. Enzymes that contain free sulfhydryl groups at the
active site (e.g., glyceraldehyde-3-phosphate
dehydrogenase; see Chapter 13) react with an
alkylating reagent, iodoacetic acid, resulting in
inactivation of the enzyme.
Enzyme-SH + ICH2COOH -*
Iodoacetic acid
enzyme-S —
CH2COOH + HI
Inactive covalent derivative of enzyme
The imidazole ring of histidine also undergoes
alkylation on reaction with iodoacetate. In
ribonucléase, two residues (His 12 and His 119) are
alkylated with loss of activity when the enzyme is
treated with iodoacetate at pH 5.5.
2. Enzymes with seryl hydroxyl groups at the active
sites can be inactivated by organophosphorous
compounds. Thus, diisopropylphosphofluoridate
(DPF) inactivates serine hydrolases by
phosphorylation at the active site:
A specific example is inactivation of
acetylcholinesterase (Table 6-1), which catalyzes
hydrolysis of acetylcholine to acetate and choline.
Acetylcholine is a
neurotransmitter,
a chemical
mediator of a nerve impulse at a junction—known as
a
synapse
—between two neurons or between a
neuron and a muscle fiber. On arrival of a nerve
impulse at the ending of the neuron, acetylcholine
(which is stored in the vesicles of the presynaptic
nerve terminal) is released. The released
H3C
CH3
w
c
I
0
1
Enzyme-CH2-OH + F— P = 0
Active seryl
I
residue
0
i
C
/ H
\
H3C
CH3
Diisopropylphosphofluoridate
(DPF)
^ H F
H3C
CH3
\ H /
c
I
0
1
Enzyme— CH2— O— P = 0
I
0
1
c
/
h\
H3C
CH3
acetylcholine acts on the postsynaptic membrane to
increase the permeability of Na+ entry across the
membrane. Depolarization results in the inside of the
membrane becoming more positive than the outside;
normally, the inside of the membrane is more negative
than the outside. This process may propagate an
action potential along a nerve fiber, or it may lead to
contraction of a muscle (Chapter 21). Acetylcholine is
quickly destroyed by acetylcholinesterase present in
the basal lamina of the neuromuscular junction
(Figure
6
-
8
). If, however, acetylcholine is not
destroyed, as in the case of inactivation of
acetylcholinesterase by DPF, its continued presence
causes extended transmission of impulses. In muscle
fibers, continuous depolarization leads to paralysis.
The cause of death in DPF intoxication is respiratory
failure due to paralysis of the respiratory muscles
(including the diaphragm and abdominal muscles).
Several organophosphorous compounds are used as
agricultural insecticides, improper exposure to which
can result in toxic manifestations and death.
Knowledge of the mechanisms of action of acetyl-
cholinesterase and of the reaction of organophosphorous
compounds with esterases led to the development of drugs
useful in the treatment of this kind of intoxication. The ac-
tive site of acetylcholinesterase consists of two subsites:
