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Vol. 300, Issue 2, 549-558, February 2002
-Ethynylestradiol on Activities of Cytochrome
P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s
2B1 and 2B6 and Identification of Metabolites
Department of Pharmacology, University of Michigan, Ann Arbor,
Michigan (U.M.K., D.E.M., P.F.H.); and Department of Chemistry, Tulane
University, New Orleans, Louisiana (R.V.R., W.L.A.)
17-
-Ethynylestradiol (17EE) inactivated purified, reconstituted rat
hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a
mechanism-based manner. Little or no inactivation was observed when
P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s
2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed
pseudo-first order kinetics. The maximal rate constants for the
inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min
1, respectively. For P450s 2B1 and 2B6 the apparent
KI was 11 and 0.8 µM, respectively.
Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a
75% loss in enzymatic activity and a concurrent 20 to 25% loss of the
enzyme's ability to form a reduced CO complex. With P450 2B6, an 83%
loss in enzymatic activity and a 5 to 10% loss in the CO reduced
spectrum were observed. The extrapolated partition ratios for 17EE with
P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation
of an alternate substrate together with 17EE protected both enzymes
from inactivation. A 1.3:1 stoichiometry of labeling for binding of the
radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate
that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the
apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes
that become inactivated differ primarily by their ability to generate
two metabolites that were not produced by P450s 2B2 or 2B4.
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