Mechanism-based Inactivation of P450 2B Enzymes: Effectws of 2-phenyl-2piperidino Propane on the Activities of Rat Isoform 2B1, Rat 2B2, Rabbit 2B4, and Human2B6
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Mammalian P450s serve to metabolize endogenous substrates and to facilitate excretion or detoxification of exogenous compounds, yet high-resolution crystal structures are difficult to obtain because mammalian P450s are membrane-bound. Mechanism-based inactivators have been used to label critical and subsequently identify residues in the P450 active site that are involved in substrate binding and catalysis. Previous studies demonstrated that 2-phenyl-2-piperidino propane (PPP), a derivative of phencyclidine (PCP), inactivated rat P450 2B1 and human 2B6 in a mechanism-based manner due to the binding of a PPP-reactive intermediate to the apoprotein (Chun et al., 2000). The effects of PPP on rabbit P450 2B4 and rat P450 2B2 had not been previously investigated. In this study, P450 2B4 in a reconstituted system was inactivated by PPP in a mechanism-based manner with a Ki that was approximately 100-fold higher than that previously observed for P450s 2B1 and 2B6. Liquid chromatography-mass spectral (LCMS) analysis and Western blotting with polyclonal anti-PCP antibody (PCHAP) recognition indicated that a PPP-reactive intermediate bound to the apoprotein of P450 2B4. A decrease in enzymatic activity correlated with an increase in antibody binding to the PPP-modified P450 2B6, as shown by PCHAP recognition. Binding of the PCHAP antibody did not decrease nor was inactivation of P450 2B6 abolished when samples were incubated with glutathione. Preliminary studies using LC-MS analysis failed to show a mass adduct to the apoprotein of P450 2B6. Initial studies with pH effects and enzymatic or chemical digestions have paved the way for future work designed to isolate and identify PPP-labeled peptides of P450s 2B4 and 2B6.Department of Pharmacology. University of Michigan. Ann Arbor, Michigan.