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.