Abstract
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 (D)k(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 (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 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.
Highlights
P450 2E1 is considered to be one of the major human hepatic P450 enzymes [13]
One example is the P450 2E1-catalyzed oxidation of ethanol to acetaldehyde [10, 19]. This laboratory, as well as others, has indicated that this kinetic hydrogen isotope effect can been explained by rate-limiting product release following the isotopically sensitive and essentially irreversible C–H bondbreaking step [19, 20], and experimental evidence has been offered in support of this model [10]
KINSIM and FITSIM computer simulations are presented for ethanol oxidation by P450 2E1, and a sequential oxidation mechanism involving a rate-determining step between product formation and release is postulated
Summary
P450 2E1 is considered to be one of the major human hepatic P450 enzymes [13]. Human P450 2E1, as well as the animal orthologs, accepts a broad range of substrates, with apparent preference for small and hydrophobic molecules [4, 14, 15]. One example is the P450 2E1-catalyzed oxidation of ethanol to acetaldehyde [10, 19] This laboratory, as well as others, has indicated that this kinetic hydrogen isotope effect can been explained by rate-limiting product release following the isotopically sensitive and essentially irreversible C–H bondbreaking step [19, 20], and experimental evidence has been offered in support of this model [10]. A carbonyl product is generated in each of the oxidation reactions displaying this pattern of isotope effects [19, 21,22,23,24]. Kunitoh et al [25] and Terelius et al [26] have previously reported that acetaldehyde, generated from ethanol oxidation, serves as a substrate for P450 2E1 and is oxidized to acetic acid. KINSIM and FITSIM computer simulations are presented for ethanol oxidation by P450 2E1, and a sequential oxidation mechanism involving a rate-determining step between product formation and release is postulated
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