Kinetic cooperativity of human liver alcohol dehydrogenase gamma(2)

Previous studies showed that natural human liver alcohol dehydrogenase gamm a exhibits negative cooperativity (substrate activation) with ethanol. Stud ies with the recombinant gamma(2) isoenzyme now confirm that observation an d show that the saturation kinetics with other alcohols are also nonhyperbo lic, whereas the kinetics for reactions with NAD(+), NADH, and acetaldehyde are hyperbolic. The substrate activation with ethanol and 1-butanol are ex plained by an ordered mechanism with an abortive enzyme-NADH-alcohol comple x that releases NADH more rapidly than does the enzyme-NADH complex, In con trast, 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 inhibit ion is due to a slower rate of dissociation of NADH from the abortive enzym e-NADH-cyclohexanol complex than from the enzyme-NADH complex. Fluorescence quenching experiments confirm that the alcohols bind to the enzyme-NADH co mplex. The nonhyperbolic saturation kinetics for oxidation of ethanol, cycl ohexanol, and 1-butanol are quantitatively explained with the abortive comp lex mechanism. Physiologically relevant concentrations of ethanol would be oxidized predominantly by the abortive complex pathway.