Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction.
The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D013379:Substrate Specificity
D009243:NAD; D000431:Ethanol; C005941:3-acetylpyridine adenine dinucleotide; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase