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Department of Molecular and Experimental Medicine (F.J., T.H.R.,
E.F.J.), The Scripps Research Institute; and
The Agouron Institute
(J.L.R.)
The present study provides a detailed kinetic analysis of diazepam
metabolism by all four known members of the human P4502C subfamily
expressed from their cDNAs in Escherichia coli. Both P4502C18 and P4502C19 were found to be low KM
diazepam N-demethylases with apparent
KM values of 24 ± 4 µM and 21 ± 3 µM, respectively. These values closely resemble the low
KM component of diazepam N-demethylase activity exhibited by human liver microsomes.
In addition, P4502C19 also catalyzed diazepam 3-hydroxylation with a
KM value of 21 ± 9 µM. Although P4502C8
was essentially inactive in catalyzing diazepam metabolism, P4502C9
catalyzed the N-demethylation with a relatively high
KM of 80 ± 15 µM and an overall 3- to
6-fold lower catalytic efficiency, compared with P4502C18 and P4502C19, respectively. At a substrate concentration of 10 µM, diazepam N-demethylation in a panel of human liver microsomes was
inhibited 42 ± 12% (mean ± SD, N = 6) by
a polyclonal anti-CYP2C antibody. In the same experiment,
3-hydroxylation remained unaffected (<10% inhibition). 1 µM of the
CYP3A inhibitor ketoconazole inhibited 37 ± 19% of the
N-demethylation and 86 ± 5% of 3-hydroxylation. Estimates of relative contributions to diazepam
N-demethylation of P4502C9 (8 ± 4%), P4502C18
(<2%), and P4502C19 (33 ± 14%) and to diazepam 3-hydroxylation
of P4502C19 (9 ± 3%) based on the kinetic parameters of the
recombinant enzymes and on specific contents of the individual 2C P450s
determined in immunoblots are consistent with the inhibition data. In
conclusion, these data confirm that both P4502C19 and P4503A are major
contributors to human liver microsomal diazepam
N-demethylation at low substrate concentrations, whereas
P4503A is the major enzyme responsible for 3-hydroxylation.
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