Rate-determining steps in phenacetin oxidations by human cytochrome P450 1A2 and selected mutants

Mutants with altered activities were obtained from random libraries of huma n cytochrome P450 (P450) 1A2 with the putative substrate recognition sequen ces (SRS) mutated [Parikh, A., Josephy, P. D., and Guengerich, F, P. (1999) Biochemistry 38, 5283-5289]. Six mutants from SRS 2 (E225I, E225N, F226I, and F226Y) and 4 (D320A and V322A) regions were expressed as oligohistidine -tagged proteins, purified to homogeneity, and used to analyze kinetics of individual steps in the catalytic cycle, to determine which reaction steps have been altered. When the wild-type, E225I, E225N, F226I, F226Y, D320A, a nd V322A proteins were reconstituted with NADPH-P450 reductase, rates of 7- ethoxyresorufin O-deethylation and phenacetin O-deethylation were in accord with those expected from membrane preparations. Within each assay, the val ues of k(cat)/K-m varied by 2-3 orders of magnitude, and in the case of E22 5I and E225N, these parameters were 7-8-fold higher than for the wild-type enzyme. The coupling efficiency obtained from the rates of product formatio n and NADPH oxidation was low (<20%) in all enzymes. No correlation was fou nd between activities and several individual steps in the catalytic cycle e xamined, including substrate binding, reduction kinetics, NADPH oxidation, and H2O2 formation. Quench reactions did not show a burst for either phenac etin O-deethylation or formation of the acetol, a minor product, indicating that rate-determining steps occur prior to product formation. Inter- and i ntramolecular kinetic deuterium isotope effects for phenacetin O-deethylati on were 2-3. In the case of phenacetin acetyl hydroxylation (acetol formati on), large isotope effects [(D)k(cat) or (D)(k(cat)/K-m) > 10] were observe d, providing evidence for rate-limiting C-H bond cleavage. We suggest that the very high isotope effect for acetol formation reflects rate-limiting hy drogen atom abstraction; the lower isotope effect for O-deethylation may be a consequence of a 1-electron transfer pathway resulting from the low oxid ation potential of the substrate phenacetin. These pre-steady-state, steady -state, and kinetic hydrogen isotope effect studies indicate that the rate- limiting steps are relatively unchanged over an 800-fold range of catalytic activity. We hypothesize that these SRS mutations alter steps leading to t he formation of the activated Michaelis complex following the introduction of the first electron.