SPECIFICITY AND OTHER PROPERTIES OF AN ALCOHOL-DEHYDROGENASE PURIFIED FROM COMAMONAS-TERRIGENA - AN ENZYME EXHIBITING PREFERENCE FOR L-STEREOISOMERS OF SECONDARY ALCOHOLS

An NAD-dependent alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) active towards L-octam-2-ol but not towards the corresponding D-isomer was purified to homogeneity from the soil bacterium C. terrigena. The enzyme is a tetramer (MW 125,000-141,000) and is most active at pH 8.5-9.9. Preferred alcohol substrates are L-alkan-2-ols, activity towards which was inhibited by EDTA, 1,10-phenanthroline and 2,2''-dipyridine. The enzyme exhibits much weaker activity towards primary alcohols, symmetrical secondary alcohols and asymmetric secondary alcohols in which the hydroxyl moiety is located at positions other than C-2, and little or no activity towards D-alkan-2-ols. For L-alkan-2-ols, symmetrical secondary alcohols and primary alcohols, log Km values decrease linearly with increase in the number of C atoms in the alkyl chain. A plot of standard free-energy of binding (.DELTA. G0'') of substrates against the number of C atoms in the alkyl chain (primary alcohols) or the longer of the 2 portions of the alkyl chain (secondary alcohols) gives a single straight-line relationship, suggesting that hydrophobic interactions make an important contribution to substrate binding. The observed specificity was interpreted in terms of a model in which secondary alcohols interact with the enzyme through the H and OH group that participate in NAD+ reduction, and 1 of the 2 alkyl segments. The size of the unbound alkyl segment markedly affects V, the optimum being a single methyl unit. This specificity was correlated with that of the CS2 secondary alkylsulfohydrolase that catalyzes the production of L-alkan-2-ols from D-alkan-2-yl sulfate surfactants.