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Research ArticleMetabolism, Transport, and Pharmacogenomics

Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex

Haoming Zhang, Chitra Sridar, Cesar Kenaan, Hemali Amunugama, David P. Ballou and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics September 2011, 338 (3) 803-809; DOI: https://doi.org/10.1124/jpet.111.183111
Haoming Zhang
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Chitra Sridar
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Cesar Kenaan
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Hemali Amunugama
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David P. Ballou
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Paul F. Hollenberg
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Abstract

In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311:34–43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the Km values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the charge-reversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype–phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.

Footnotes

  • This work was supported in part by the National Institutes of Health National Cancer Institute [Grant CA16945] (to P.F.H.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.111.183111.

  • ABBREVIATIONS:

    7-EFC
    7-ethoxy-4-trifluoromethylcoumarin
    7-HFC
    7-hydroxy-4-trifluoromethylcoumarin
    P450
    cytochrome P450
    P420
    cytochrome P420
    CPR
    NADPH-dependent cytochrome P450 reductase
    tBHP
    tert-butyl hydroperoxide
    WT
    wild type
    TFA
    trifluoroacetic acid.

  • Received April 19, 2011.
  • Accepted June 8, 2011.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 338 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 338, Issue 3
1 Sep 2011
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Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex
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Research ArticleMetabolism, Transport, and Pharmacogenomics

Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex

Haoming Zhang, Chitra Sridar, Cesar Kenaan, Hemali Amunugama, David P. Ballou and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics September 1, 2011, 338 (3) 803-809; DOI: https://doi.org/10.1124/jpet.111.183111

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Research ArticleMetabolism, Transport, and Pharmacogenomics

Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex

Haoming Zhang, Chitra Sridar, Cesar Kenaan, Hemali Amunugama, David P. Ballou and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics September 1, 2011, 338 (3) 803-809; DOI: https://doi.org/10.1124/jpet.111.183111
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