Abstract
Human cytochrome P450 (P450) 2B6 plays an important role in the metabolism of many drugs used in the clinic, and it has been shown to be highly polymorphic and inducible by a variety of substrates. The metabolism of phencyclidine (PCP) by P450 2B6 results in mechanism-based inactivation of the enzyme. We investigated the effects of a naturally occurring mutation of P450 2B6 where a lysine 262 is changed to an arginine (K262R) on PCP metabolism and mechanism-based inactivation of 2B6 by PCP. The K262R mutant retained the 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity when it was incubated with PCP and NADPH in the reconstituted system, whereas the wild-type enzyme was readily inactivated by PCP. Spectral binding studies showed that PCP was reversibly bound in the active site of the K262R mutant with slightly higher affinity (156 μM) compared with the wild-type 2B6 (397 μM). In addition, all the metabolites of PCP (M1–M8) that were formed by the wild-type enzyme were also formed by the K262R mutant. Although the K262R mutant metabolized PCP to give similar metabolite profiles, the overall rate of metabolite formation was lower than the wild-type enzyme. A reactive intermediate of PCP was formed by wild-type P450 2B6 and trapped with glutathione (GSH). However, no GSH conjugates were detected from incubations with the K262R mutant. These data suggest that the lysine 262 residue plays an important role in the formation of a reactive intermediate of PCP that leads to the mechanism-based inactivation of P450 2B6.
Footnotes
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This work was supported in part by National Institutes of Health Grant CA-16954 (P.F.H.) and a predoctoral fellowship from Merck & Co. Inc. (M.S.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.014985.
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ABBREVIATIONS: P450, cytochrome P450; PCP, phencyclidine; 17EE, 17-α-ethynylestradiol; GSH, glutathione; TFA, trifluoroacetic acid; 7-EFC, 7-ethoxy-4-trifluoromethylcoumarin; ESI, electrospray ionization; CID, collision-induced dissociation; LC, liquid chromatography; MS/MS, tandem mass spectrometry.
- Received January 29, 2007.
- Accepted April 24, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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