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Molecular Pharmacology

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Research ArticleArticle

Investigation of the Mechanisms Underlying the Differential Effects of the K262R Mutation of P450 2B6 on Catalytic Activity

Namandjé N. Bumpus and Paul F. Hollenberg
Molecular Pharmacology October 2008, 74 (4) 990-999; DOI: https://doi.org/10.1124/mol.108.048637
Namandjé N. Bumpus
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Paul F. Hollenberg
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Abstract

Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-α-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.

Footnotes

  • This study was supported in part by grant CA16954 from the National Cancer Institute, National Institutes of Health (to P.F.H.), National Institutes of Health grant T32-GM007767 and a predoctoral fellowship in pharmacology/toxicology from the PhRMA Foundation (to N.N.B.).

  • ABBREVIATIONS: P450, cytochrome P450; P450 2B6.4, P450 2B6 Lys-262-Arg mutant; BSA, bovine serum albumin; 17EE, 17-α-ethynylestradiol; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; tBHP, tert-butyl hydroperoxide; HPLC, high-performance liquid chromatography; reductase, NADPH-cytochrome P450 reductase.

    • Received May 5, 2008.
    • Accepted July 11, 2008.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 74 (4)
Molecular Pharmacology
Vol. 74, Issue 4
1 Oct 2008
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Research ArticleArticle

Investigation of the Mechanisms Underlying the Differential Effects of the K262R Mutation of P450 2B6 on Catalytic Activity

Namandjé N. Bumpus and Paul F. Hollenberg
Molecular Pharmacology October 1, 2008, 74 (4) 990-999; DOI: https://doi.org/10.1124/mol.108.048637

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Research ArticleArticle

Investigation of the Mechanisms Underlying the Differential Effects of the K262R Mutation of P450 2B6 on Catalytic Activity

Namandjé N. Bumpus and Paul F. Hollenberg
Molecular Pharmacology October 1, 2008, 74 (4) 990-999; DOI: https://doi.org/10.1124/mol.108.048637
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