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Volume 272, Number 47, Issue of November 21, 1997 pp. 29643-29651
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1
RATE-LIMITING PRODUCT RELEASE ACCOUNTS FOR EFFECTS OF ISOTOPIC HYDROGEN SUBSTITUTION AND CYTOCHROME b₅ ON STEADY-STATE KINETICS

(Received for publication, June 18, 1997, and in revised form, August 13, 1997)

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

A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of ~5 on K_m (^DK = ^DK_m/^HK_m), but not on k_cat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH₂Cl₂). We observed ^DK_m values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O₂ with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k >1000 min¹) even in the absence of substrate. These results indicate that the basis for the increase in K_m is in the latter portion of the catalytic cycle. The intrinsic isotope effect (^Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min¹), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the k_cat approximates the rate of product release, and K_m is an expression in which the denominator is dominated by the rate of C-H bond breaking.

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