Kinetics of the hydroperoxide-dependent dealkylation reactions catalyzed by rabbit liver microsomal cytochrome P-450.
Rabbit liver microsomal cytochrome P-450 catalyzes the dealkylation of a variety of substrates when organic hydroperoxides, peracids, or peroxyesters are substituted for NADPH and O2. The peroxide-supported demethylation of p-nitroanisole by rabbit liver microsomes exhibited normal Michaelis-Menten kinetics with respect to both substrates. The Vmax values for the hydroperoxide-dependent reactions were dependent on the identity of the hydroperoxide and with several of the peroxides they were significantly greater than those obtained with NADPH and O2. The pH profiles for the hydroperoxide-supported demethylations were similar to the profile for the NADPH-supported reaction and exhibited optima from 7.0 to 7.6, depending on the identity of the organic oxidant. The kinetic mechanism of the t-butyl hydroperoxide-supported demethylation of p-nitroanisole was determined. Plots of reciprocal velocity versus the reciprocal concentration of either substrate at several different fixed concentrations of the other substrate converged to common points of intersection on the negative side of the ordinate and above the abscissa, suggesting a sequential mechanism involving the formation of a ternary complex between cytochrome P-450, p-nitroanisole, and t-butyl hydroperoxide followed by one or more reactions and the subsequent release of the products. Potassium cyanide was a competitive inhibitor with respect to t-butyl hydroperoxide and uncompetitive with respect to p-nitroanisole. t-Butyl alcohol, a product of the t-butyl hydroperoxide-supported reaction, was a noncompetitive inhibitor with respect to both substrates. These results, which indicate that the reaction proceeds via an Ordered Bi Bi mechanism in which p-nitroanisole binds to the enzyme prior to the binding of t-butyl hydroperoxide, are discussed in relationship to the peroxidase-type mechanism which has been suggested for the action of cytochrome P-450.
D000818:Animals; D000440:Butanols / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010545:Peroxides; D011817:Rabbits; D013379:Substrate Specificity
D000440:Butanols; D003486:Cyanides; D010545:Peroxides; D003577:Cytochrome P-450 Enzyme System; D010089:Oxidoreductases, N-Demethylating