Mechanism of inactivation of human cytochrome P450 2B6 by phencyclidine.

The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 microM PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.

Selective pathways for the metabolism of phencyclidine by cytochrome p450 2b enzymes: identification of electrophilic metabolites, glutathione, and N-acetyl cysteine adducts.

The metabolism of phencyclidine (PCP) has been studied previously in cytochrome P450 (P450)-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH4 resulted in the disappearance of the signal at m/z 240, consistent with reduction of a 2,3-dihydropyridinium species. Furthermore, the reactive metabolite trapped by GSH resulted in an adduct exhibiting an m/z of 547, consistent with the mass of the 2,3-dihydropyridinium species of PCP (m/z 240), that has reacted with a molecule of GSH (m/z 308). However, P450 2B6 formed a different reactive intermediate of PCP that was isolated as a GSH adduct exhibiting an m/z of 581 and an NAC adduct with an m/z of 437. Liquid chromatography-tandem mass spectrometry analysis of these adducts suggested that a di-oxygenated iminium metabolite of PCP could be the reactive intermediate formed by P450 2B6 but not by the other 2B isoforms. These data suggest that P450 2B6 favors oxidation pathways for PCP metabolism that are different from those of P450s 2B1 and 2B4.

Catalytic turnover of pyrene by CYP3A4: evidence that cytochrome b5 directly induces positive cooperativity.

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b5 (b5) on turnover kinetics. In the absence of b5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate-velocity curve where K(m1) and V(max1) were 1.3 microM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a pi-pi stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V(max) was 15.7 pmol/min/pmol P450, the K(m) was 7.5 microM, and the Hill coefficient was greater than two. This demonstrated that b5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.

The mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine.

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. The K(I) was 10 microM, k(inact) was 0.01 min(-1), which corresponds to a t(1/2) 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 of (3)H-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 using (3)H-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.