MECHANISM-BASED INACTIVATION OF RAT-LIVER CYTOCHROME P4502B1 BY PHENCYCLIDINE AND ITS OXIDATIVE PRODUCT, THE IMINIUM ION

Cytochrome P4502B1, the major phenobarbital-inducible isozyme in the r at liver, is inactivated by phencyclidine (PCP), Incubation of PCP wit h purified P4502B1 in the reconstituted enzyme system with NADPH-cytoc hrome P450 reductase and phospholipid resulted in a marked loss of act ivity as measured using a secondary incubation mixture for 7-ethoxycou marin O-deethylase activity, The loss of activity required NADPH and P CP, and the activity decreased in a time-dependent, pseudo-first-order process indicative of mechanism-based inactivation, The rate constant s for inactivation were dependent on the PCP concentrations and displa yed saturation kinetics, A K-l = 3.8 mu M and k(inact) = 0.12 min(-1) were determined for the inactivation by PCP, The partition ratio calcu lated from a plot of the percentage activity remaining after 45 min vs . the concentration ratios of PCP to P450 was 45, Although 90% of the catalytic activity was lost after a 45-min incubation, little loss was seen in the optical spectrum at 418 nm or in the ability of the reduc ed enzyme to bind CO, The inactivation was not inhibited by the additi on of cyanide, whereas substrates such as 7-ethoxycoumarin protected a gainst the inactivation, The iminium ion of PCP, an oxidative metaboli te, inactivated P4502B1 in the same fashion as PCP, These results demo nstrate that PCP is an efficient mechanism-based inactivator of rat li ver P4502B1 and does not inactivate by modification of the heme moiety .