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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

The Functional Role of Threonine-205 in the Mechanism-Based Inactivation of P450 2B1 by Two Ethynyl Substrates: The Importance of the F Helix in Catalysis

Departments of Pharmacology (H.-L.L., U.M.K., P.F.H.) and Anesthesiology (H.Z., L.W.), University of Michigan, Ann Arbor, Michigan; and Veterans Affairs Health Service (H.Z., L.W.), Ann Arbor, Michigan.

We have previously demonstrated that substituting Val for Thr-205 in P450 2B1 abolishes the 16-hydroxylation of testosterone and markedly decreases the ability of 2-ethnylnaphthalene (2EN) and 17-ethynylestradiol (17EE) to inactivate P450 2B1. The role of Thr-205 has been further investigated by measuring the kinetics of the mechanism-based inactivation of the 7-ethoxy-(trifluoromethyl)coumarin deethylation activity of 2B1 by 2EN and 17EE in wild-type (WT) and mutant P450s. In general, the kinetics of the inactivation of the Ser and Ala mutants was not significantly altered compared with WT. In contrast, the efficiency of the inactivation of the Val mutant decreased by 6- and 30-fold for 2EN and 17EE, respectively. High-pressure liquid chromatography (HPLC) analysis and SDS gel electrophoresis demonstrated the covalent binding of radiolabeled 2EN- and 17EE-reactive intermediates to the WT apoprotein, but not the Val mutant. The Val mutant was able to metabolize 2EN to 2-naphthylacetic acid, except the initial rate was slower than the WT. HPLC analysis of the 17EE incubation mixtures revealed three major metabolites and showed a correlation between the efficiency of inactivation and the generation of one of the major metabolites (C). Metabolite C was generated by the WT, Ser mutant, and Ala mutant. Metabolite C may be formed by the oxidation of the ethynyl group, and this reactive intermediate contributes to the inactivation of P450 2B1 by 17EE. The site-specific mutation of one residue, Thr-205 to Val, is sufficient to alter the profile of products formed during 17EE metabolism, such that very low levels of metabolite C are formed and inactivation is essentially abolished.

Address correspondence to: Paul F. Hollenberg, Department of Pharmacology, 2301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632. E-mail: phollen{at}umich.edu