Tomato alcohol dehydrogenase: Purification and substrate specificity

Abstract

Alcohol dehydrogenase of tomato ( Lycopersicon esculentum) has been purified to homogeneity, using affinity chromatography on Cibacron F3GA-agarose. The enzyme is a dimer, M r 90,000–100,000. The coenzyme is NAD +; no NADP +-dependent activity was detected even in crude extracts. Among saturated substrates, ethanol and acetaldehyde show the lowest apparent K m values (2.67 and 0.174 m m, respectively) and highest V values, supporting a primary role in acetaldehyde metabolism, with action also on “flavor aldehydes”; 2-unsaturated alcohols show still lower K m values, probably due to a more favorable K eq. This enzyme and other plant alcohol dehydrogenases form a definite class, intermediate in specificity between liver and yeast alcohol dehydrogenases: they differ from the former in being essentially inactive on secondary and aromatic substrates, from the latter in showing only a mild decrease in V with increasing chain length of alkyl substrates, and from both in showing the lowest K m as well as highest V on ethanol and acetaldehyde. The tomato enzyme differs from other reported plant enzymes in showing substantial activity on geraniol. Kinetic studies are in agreement with an ordered sequential mechanism. The enzyme is inhibited slowly by iodoacetamide, and reversibly by acetamide and zinc-chelating compounds.