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Research ArticleArticle

The Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Monica I. Jushchyshyn, Ute M. Kent and Paul F. Hollenberg
Drug Metabolism and Disposition January 2003, 31 (1) 46-52; DOI: https://doi.org/10.1124/dmd.31.1.46
Monica I. Jushchyshyn
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Ute M. Kent
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Paul F. Hollenberg
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Abstract

Phencyclidine (PCP) was analyzed for its ability to inactivate human cytochrome P450 (P450) 2B6. PCP inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 in a concentration-, time-, and NADPH-dependent manner and exhibited pseudo-first order kinetics. TheKI was 10 μM,kinact was 0.01 min−1, which corresponds to a t1/2 of 31 min. The partition ratio was approximately 45. Spectral analysis of the heme moiety demonstrated that the heme was not modified during inactivation. Extensive dialysis of the PCP-inactivated P450 2B6 did not cause a return in catalytic activity demonstrating PCP inactivation was irreversible. Including 7-ethoxycoumarin, an alternate substrate, protected 2B6 from inactivation by PCP indicating competition of the two substrates for the active site. Exogenous nucleophiles such as glutathione (GSH) and cyanide could not protect P450 2B6 from PCP inactivation demonstrating that the reactive intermediate remained within the P450 active site. High performance liquid chromatography analysis of P450 2B6 inactivated in the presence of3H-labeled PCP showed that PCP binding was specific for the P450 and not to other proteins in the reaction mixture. The stoichiometry of binding of PCP to P450 2B6 was demonstrated using3H-labeled PCP. In the absence of GSH, the stoichiometry was 5.5:1 (PCP/P450). In the presence of GSH, the stoichiometry was 1:1. This stoichiometry was further supported using electrospray ionization-liquid chromatography-mass spectrometry to analyze PCP-inactivated P450 2B1, 2B4, and 2B6.

Footnotes

  • This work was supported in part by National Institutes of Health Grant CA 16954.

  • Abbreviations used are::
    P450
    cytochrome P450
    PCP
    phencyclidine
    ESI-LC-MS
    electrospray ionization-liquid chromatography-mass spectrometry
    DLPC
    dilauroyl-l-α-phosphatidylcholine
    GSH
    glutathione
    DMSO
    dimethyl sulfoxide
    HPLC
    high performance liquid chromatography
    TFA
    trifluoroacetic acid
    7-EFC
    7-ethoxy-4-(trifluoromethyl)coumarin
    HFC
    7-hydroxy-4-(trifluoromethyl) coumarin
    EC
    ethoxycoumarin
    KCN
    potassium cyanide
    CN
    cyanide
    7-EC
    7-ethoxycoumarin
    reductase
    NAPDH-cytochrome P450 reductase
    • Received July 8, 2002.
    • Accepted September 25, 2002.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 31 (1)
Drug Metabolism and Disposition
Vol. 31, Issue 1
1 Jan 2003
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Research ArticleArticle

The Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Monica I. Jushchyshyn, Ute M. Kent and Paul F. Hollenberg
Drug Metabolism and Disposition January 1, 2003, 31 (1) 46-52; DOI: https://doi.org/10.1124/dmd.31.1.46

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Research ArticleArticle

The Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Monica I. Jushchyshyn, Ute M. Kent and Paul F. Hollenberg
Drug Metabolism and Disposition January 1, 2003, 31 (1) 46-52; DOI: https://doi.org/10.1124/dmd.31.1.46
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