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 rat liver, is inactivated by phencyclidine (PCP). Incubation of PCP with purified P4502B1 in the reconstituted enzyme system with NADPH-cytochrome P450 reductase and phospholipid resulted in a marked loss of activity as measured using a secondary incubation mixture for 7-ethoxycoumarin O-deethylase activity. The loss of activity required NADPH and PCP, and the activity decreased in a time-dependent, pseudo-first-order process indicative of mechanism-based inactivation. The rate constants for inactivation were dependent on the PCP concentrations and displayed saturation kinetics. A KI = 3.8 microM and kinact = 0.12 min-1 were determined for the inactivation by PCP. The partition ratio calculated 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 reduced enzyme to bind CO. The inactivation was not inhibited by the addition of cyanide, whereas substrates such as 7-ethoxycoumarin protected against the inactivation. The iminium ion of PCP, an oxidative metabolite, inactivated P4502B1 in the same fashion as PCP. These results demonstrate that PCP is an efficient mechanism-based inactivator of rat liver P4502B1 and does not inactivate by modification of the heme moiety.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002374:Catalase / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007097:Imines / Q000494:pharmacology*; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010622:Phencyclidine / Q000378:metabolism / Q000494:pharmacology*; D011190:Potassium Cyanide / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors*
D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007097:Imines; C017299:7-ethoxycoumarin; D009249:NADP; D002374:Catalase; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D010622:Phencyclidine; D011190:Potassium Cyanide