Oxidation kinetics of ethanol by human cytochrome P450 2E1 - Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b(5) on steady-state kinetics

A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of similar to 5 on K-m (K-D = K-D(m)/K-H(m)), but not on k(cat), in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed K-D(m) values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O-2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k > 1000 min(-1)) 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 ((D)k) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Presteady-state studies showed a burst of product formation (k = 410 min(-1)), 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.