Mutation of a single residue (K262R) in P450 2B6 leads to loss of mechanism-based inactivation by phencyclidine.
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 muM) compared with the wild-type 2B6 (397 muM). 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.
Shebley
Mohamad
M
Department of Pharmacology, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632, USA.
Hollenberg
Paul F
PF
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics; D003374:Coumarins / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D015786:Cytochromes b5 / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D020125:Mutation, Missense; D009249:NADP / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D010622:Phencyclidine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry
D003374:Coumarins; D011994:Recombinant Proteins; C056033:7-ethoxy-4-trifluoromethylcoumarin; D009249:NADP; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione; D010622:Phencyclidine