Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2:Structure-activity relationships and simulation of rate constants of individual steps in catalysis

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many impor tant drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effor t to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in t he O-alkyl and N-acyl substituents altered the rates of the two oxidation r eactions and the ratio of acetol/phenol products. Moving one methylene grou p of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations similar to5-fold and improved the coupling efficiency ( oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive ki netic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope ef fect for E225I > wild-type, mutant D320A was observed for O-demethylation o f p-methoxyacetanilide, which follows the trend for k(cat). The set of O-de alkylation and acetol formation results for wild-type P450 1A2 and the E225 I mutant with several of the protiated and deuterated substrates were fit t o a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k( cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of forma tion of the oxygenating species, the rate of substrate oxidation by the oxy genating species, and the rates of generation of reduced oxygen species (H2 O2, H2O). This analysis of the effects of the individual rate constants pro vides a framework for consideration of other P450 reactions and rate-limiti ng steps.