Abstract
Previous studies showed that natural human liver alcohol dehydrogenase gamma exhibits negative cooperativity (substrate activation) with ethanol. Studies with the recombinant gamma(2) isoenzyme now confirm that observation and show that the saturation kinetics with other alcohols are also nonhyperbolic, whereas the kinetics for reactions with NAD(+), NADH, and acetaldehyde are hyperbolic. The substrate activation with ethanol and 1-butanol are explained by an ordered mechanism with an abortive enzyme-NADH-alcohol complex that releases NADH more rapidly than does the enzyme-NADH complex. In contrast, high concentrations of cyclohexanol produce noncompetitive substrate inhibition against varied concentrations of NAD(+) and decrease the maximum velocity to 25% of the value that is observed at optimal concentrations of cyclohexanol. Transient kinetics experiments show that cyclohexanol inhibition is due to a slower rate of dissociation of NADH from the abortive enzyme-NADH-cyclohexanol complex than from the enzyme-NADH complex. Fluorescence quenching experiments confirm that the alcohols bind to the enzyme-NADH complex. The nonhyperbolic saturation kinetics for oxidation of ethanol, cyclohexanol, and 1-butanol are quantitatively explained with the abortive complex mechanism. Physiologically relevant concentrations of ethanol would be oxidized predominantly by the abortive complex pathway.
Highlights
Liver alcohol dehydrogenases (E.C. 1.1.1.1) catalyze the reversible oxidation of alcohols using NADϩ as a cofactor
Into the possible roles of alcohol dehydrogenases in alcoholism require quantitative descriptions of the kinetics of the various enzymes involved, but the properties of HsADH␥ are a challenge because both isoenzymes exhibit negative cooperativity for ethanol oxidation [6], and the mechanism has not yet been described
Negative cooperativity for ethanol oxidation was observed for a purified human liver alcohol dehydrogenase [18], which may have resulted from a mixture of isoenzymes in the preparation
Summary
HsADH and HsADH␥, human (Homo sapiens) liver alcohol dehydrogenase  and ␥ enzymes, respectively, otherwise named ADH1B and ADH1C [2]; EqADHE, horse (Equus caballus) liver alcohol dehydrogenase E (ethanol active) enzyme. Negative cooperativity for ethanol oxidation was observed for a purified human liver alcohol dehydrogenase [18], which may have resulted from a mixture of isoenzymes in the preparation. Oxidation of cyclohexanol by horse liver alcohol dehydrogenase exhibits negative cooperativity [19], and oxidation of ethanol and benzyl alcohol show substrate inhibition [21,22,23,24]. These results are explained by an ordered mechanism with alternative pathways, including abortive enzyme-NADHalcohol and binary enzyme-NADH complexes, which differ in the rate of dissociation of NADH. Substrate activation and inhibition observed for recombinant HsADH␥2 with different alcohols are explained by a similar mechanism
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