Rate-Determining Steps in Phenacetin Oxidations by Human Cytochrome P450 1A2 and Selected Mutants

Abstract

Mutants with altered activities were obtained from random libraries of human cytochrome P450 (P450) 1A2 with the putative substrate recognition sequences (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, and 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 values of k(cat)/K(m) varied by 2-3 orders of magnitude, and in the case of E225I and E225N, these parameters were 7-8-fold higher than for the wild-type enzyme. The coupling efficiency obtained from the rates of product formation and NADPH oxidation was low (<20%) in all enzymes. No correlation was found between activities and several individual steps in the catalytic cycle examined, including substrate binding, reduction kinetics, NADPH oxidation, and H(2)O(2) formation. Quench reactions did not show a burst for either phenacetin O-deethylation or formation of the acetol, a minor product, indicating that rate-determining steps occur prior to product formation. Inter- and intramolecular kinetic deuterium isotope effects for phenacetin O-deethylation were 2-3. In the case of phenacetin acetyl hydroxylation (acetol formation), large isotope effects [(D)k(cat) or (D)(k(cat)/K(m)) > 10] were observed, providing evidence for rate-limiting C-H bond cleavage. We suggest that the very high isotope effect for acetol formation reflects rate-limiting hydrogen atom abstraction; the lower isotope effect for O-deethylation may be a consequence of a 1-electron transfer pathway resulting from the low oxidation 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 the formation of the activated Michaelis complex following the introduction of the first electron.

Preparation of human drug metabolites using fungal peroxygenases

“…A value of 3 is considerably smaller than the intrinsic isotope effect near 10 expected for a hydrogen abstraction mechanism, and may indicate that phenacetin oxidation by APOs proceeds instead via electron transfer, as proposed earlier for the P450-catalyzed reaction [40]. By contrast, the O-dealkylation of 1,4-dimethoxybenzene-d 3 by AaeAPO probably does proceed via hydrogen abstraction, because it exhibits a much higher (k H /k D ) obs near 12 [18,40].…”

Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

“…The C-H bond-breaking step appears to be less rate-limiting in P450 3A4 reactions than with several reactions catalyzed by other mammalian P450s, e.g. P450s 1A2 (42,44), 2A6 (45), 2E1 (41,103), and 2D6 (43). These results, coupled with the very selective stereochemistry of hydrogen abstraction (Fig.…”

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

“…The expression cultures were grown in 1 liter Fernbach flasks at 37 °C for 3 h and then at 28 °C with shaking at 200 rpm for 24 h. Bacterial inner membrane fractions containing CYP52A21 were isolated and prepared from 1 liter TB (with ampicillin, 100 μg/ml) expression cultures of E. coli DH5α. Purification of the CYP52A21 protein using a Ni 2+ -nitrilotriacetate column was carried out as previously described for a different protein [20,21]. Briefly, the membrane was solubilized at 4 °C overnight in 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.5 M NaCl, 10 mM β-mercaptoethanol, and 1.5% CHAPS (w/v).…”