Human liver alcohol dehydrogenase isozymes: reduction of aldehydes and ketones

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Abstract
The class I (.alpha., .beta.1, .gamma.1 and .gamma.2), II (.pi.) and III (.chi.) isozymes of human liver alcohol dehydrogenase (ADH) were isolated as electrophoretically homogeneous preparations to examine their kinetics of aldehyde and ketone reduction. While the oxidation of a wide variety of alcohols by ADH has been investigated extensively, the reduction of aldehydes and ketones has received much less attention even though the equilibrium favors the latter process. For each isozyme, the Km and kcat values were measured at pH 7.0 with acetaldehyde, pentanal, octanal, benzaldehyde and cyclohexanone as substrates. Activity could not be detected with succinic semialdehyde and betaine aldehyde for any of the isozymes. The nonenzymatic hydration, oxidation and aldol condensation of aldehydes in aqueous solutions present serious experimental obstacles in determining the isozymes'' kinetic constants. The effects of these reactions on the enzymatic parameters were studied and compensated for. Km for all class I and II isozymes vary by more than 8000-fold, from less than 1 .mu.M for .beta.1.gamma.1 and .beta.1.beta.1 with octanal to 8.3 mM for .pi.-ADH for acetaldehyde. However, with any given aldehyde, these values vary by less than 40-fold, and the constants are approximately equal to Km values reported previously for the corresponding alcohols. In contrast, Km values for .chi.-ADH are extremely high and could be determined accurately only for octanal (75 .mu.M). The kcat values for class I and II isozymes range from 21 min-1 for .beta.1.beta.1 with cyclohexanone to 3400 min-1 for .beta.1.gamma.2 with octanal and are generally an order of magnitude greater than those for the alcohols. However, the .beta.1.beta.1 isozyme catalyzes both the reduction of aldehydes and the oxidation of alcohols at turnover rates significantly lower than those of other class I forms. Hence, in principle, poor oxidase activity does not necessarily imply good reductase activity.