Received for publication January 30, 2007.
Revised April 19, 2007.
Accepted for publication April 24, 2007.

Mutation of a Single Residue (K262R) in P450 2B6 Leads to Loss of Mechanism-based Inactivation by Phencyclidine

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 (7-EFC) O-deethylation activity when it was incubated with PCP and NADPH in the reconstituted system, while the wild type enzyme was readily inactivated by PCP. Spectral binding studies demonstrated that PCP was reversibly bound in the active site of the K262R mutant with slightly higher affinity (156 µM) as compared to the wild type 2B6 (397 µM). In addition, all of 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.

Key words: CYP inhibition, CYP2B, cytochrome P450 catalyzed oxidations, cytochrome P450 structure, genetic polymorphism, glutathione conjugates, human CYP enzymes, mechanism-based inhibition, reactive intermediate, suicide inhibition