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into competitive inhibition, uncompetitive inhibition, and noncompetitive inhibition.
Because these types are not rigidly separated, many intermediate classes have been
described. Although enzyme kinetics of microsomal enzymes are intrinsically difficult
to elucidate because the enzymes are membrane bound and both substrate and inhibi-
tor are frequently lipophilic, methods for analysis of kinetic data are available that sim-
plify the determination of the type of inhibition. For example, see the methods used
in the study of the irreversible, mechanism-based inhibition of estradiol metabolism in
humans by chlorpyrifos (
Usmani et al., 2006
).
Competitive inhibition is usually caused by two substrates competing for the same
active site. Following classical enzyme kinetics, there should be a change in the appar-
ent
K
m
but not in
V
max
. In microsomal monooxygenase reactions, type I ligands, which
often appear to bind as substrates but do not bind to the heme iron, might be expected
to be competitive inhibitors, and this frequently appears to be the case.
Uncompetitive inhibition has seldom been reported in studies of xenobiotic
metabolism. It occurs when an inhibitor interacts with an enzyme-substrate complex
but cannot interact with free enzyme. Both
K
m
and
V
max
change by the same ratio,
giving rise to a family of parallel lines in a Lineweaver-Burke plot.
Noncompetitive inhibitors can bind to both the enzyme and the enzyme-substrate
complex to form either an enzyme-inhibitor complex or an enzyme-inhibitor-substrate
complex. The net result is a decrease in
V
max
but no change in
K
m
. Metyrapone, a well-
known inhibitor of monooxygenase reactions, can also, under some circumstances, stim-
ulate metabolism in vitro. In either case, the effect is noncompetitive, in that the
K
m
does
not change, whereas
V
max
does, decreasing in the case of inhibition and increasing in the
case of stimulation.
Irreversible inhibition, which is much more important toxicologically, can arise
from various causes. In most cases, the formation of covalent or other stable bonds or
the disruption of the enzyme structure is involved. In these cases, the effect cannot be
readily reversed in vitro by either dialysis or dilution. The formation of stable inhibi-
tory complexes may involve the prior formation of a reactive intermediate that then
interacts with the enzyme (“suicide” or mechanism-based inhibition). An excellent
example of this type of inhibition is the effect of the insecticide synergist piperonyl
butoxide on hepatic microsomal monooxygenase activity, reviewed by
Hodgson and
Levi (1998)
and
Hodgson (1999)
. This methylenedioxyphenyl compound can form
a stable inhibitory complex that blocks CO binding to CYP and also prevents sub-
strate oxidation. This complex results from the formation of a reactive intermediate,
and the type of inhibition changes from competitive to irreversible as metabolism, in
the presence of NADPH and oxygen, proceeds. It appears probable that the metabo-
lite in question is a carbene formed spontaneously by elimination of water follow-
ing hydroxylation of the methylene carbon by the cytochrome (
Dahl and Hodgson,
1979
). Piperonyl butoxide inhibits the in vitro metabolism of many substrates of
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