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J Biol Chem, Vol. 274, Issue 34, 23833-23840, August 20, 1999

Kinetics of Cytochrome P450 2E1-Catalyzed Oxidation of Ethanol to Acetic Acid via Acetaldehyde

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K_m with no effect on k_cat, and rate-limiting product release has been proposed to account for the lack of an isotope effect on k_cat (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k_cat/K_m for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid (^H(k_cat/K_m)/^D(k_cat/K_m) = 4.5, and ^Dk_cat = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k_cat is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (~450 min¹) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, ~90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K_d values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.

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