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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on August 9, 2004; DOI: 10.1124/jpet.104.071670

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Received for publication June 1, 2004.
Revised August 9, 2004.
Accepted for publication August 9, 2004.

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

Hsia-lien Lin 1, Ute M. Kent 1, Haoming Zhang 2, Lucy Waskell 3, Paul F. Hollenberg 4*

1 University of Michigan 2 Veteran Affairs Health Services 3 Veterna Affairs Health Services 4 University of Michigan School of Medicine

* Address correspondence to: E-mail: phollen{at}umich.edu

Abstract

We have previously demonstrated that substituting Val for Thr-205 abolishes the 16{beta}-hydroxylation of testosterone and markedly decreases the ability of 2-ethnylnaphthalene (2EN) and 17{alpha}-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 were not significantly altered compared to WT. In contrast, the efficiency of the inactivation of the Val-mutant decreased by ~6-fold and ~30-fold for 2EN and 17EE, respectively. 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- and Ala-mutants. 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.


Key words: F helix structure-function studies, P450 2B1, T205 mutagenesis, ethynylestradiole, ethynylnaphtalene, mechanism-based inactivation




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