Grape Alcohol Dehydrogenase. II. Kinetic Studies: Mechanism, Substrate, and Coenzyme Specificity

Abstract

Kinetic studies of grape alcohol dehydrogenase (GADH) were carried out at pH 5.8 and 9.4 for the forward and backward reactions, respectively. Primary double reciprocal plots for several constant concentrations of the associate substrate are indicative of a bi-bi sequential mechanism. Exploitation of secondary plots gives the following values for Michaelis constants: acetaldehyde = 0.48 mM, ethanol = 13.6 mM, NADH = 0.013 mM, NAD⁺ = 0.012 mM. For inhibition kinetic constants the values given are: acetaldehyde = 0.59 mM, ethanol = 8.33 mM, NADH = 0.016 mM, NAD⁺ = 0.07 mM. The results obtained in inhibition studies agree with an ordered bi-bi mechanism, the coenzyme being the first substrate bound. Values obtained for kinetic constants and maximum velocity for ethanol to acetaldehyde interconversion, as for equilibrium constant, show that the reaction tends towards aldehyde reduction. This fact was confirmed by substrate specificity studies, aldehydes and particularly six-carbon aldehydes being generally better substrates than the corresponding alcohols as indicated by the highest values of the catalytic efficiency (V'_max/K'_M). Kinetic studies suggested that GADH possesses a single catalytic site which accepts NADH or NADPH, the catalytic efficiency being more important in the presence of NADH. No NADP⁺-linked activity was detected with purified enzyme.