Mechanism-based inactivation of rat liver cytochrome P-4502B1 by 2-methoxy-5-nitrobenzyl bromide

Mechanism-based inactivators serve as probes of enzyme mechanism, function, and structure. Koshland's Reagent II (2-methoxy-8-nitrobenzyl bromide, KR- II) is a potential mechanism-based inactivator of enzymes that perform O-de alkylations. The major phenabarbital-inducible form of cytochrome P-450 in male rat liver microsomes, CYP2B1, is capable of catalyzing O-dealkylations . The interactions of KR-II with purified CYP2B1 in the reconstituted syste m containing P-450, NADPH:P-450 oxidoreductase, and sonicated dilaurylphosp hatidyl choline were studied. The benzphetamine N-demethylase activity of C YP2B1 was inactivated by KR-II in a time- and NADPH-dependent manner, and t he loss of activity followed pseudo-first-order kinetics. The inactivation also required KR-II, and the rate of activity loss was dependent on the con centration of KR-II in a saturable fashion. The inactivator concentration r equired for the half-maximal rate of inactivation (K-1) was approximately 0 .1 mM. The inactivation was not prevented by the addition of the nucleophil es dithiothreitol and glutathione, nor was it reversed by gel filtration. T he present results demonstrate that KR-II is a mechanism-based inactivator of rat CYP2B1.