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

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. 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.