Stereopecificity and Other Properties of a Novel Secondary‐Alcohol‐Specific Alcohol Dehydrogenase

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Abstract
NAD-dependent alcohol dehydrogenase from the methanol-grown Methylcoccus sp. CRL Ml (type I membrane), Methylosinus trichosporium OB3b (type II membrane), Methylobacterium organophilum CRL 26 (type II membrane, facultative methylotroph), Pseudomonas sp. ATCC 21439 and Pichia pastoris Y-55 are secondary-alcohol-specific and that from P. pastoris Y-7556 is not. This novel secondary-alcohol-specific alcohol dehydrogenase (secondary-alcohol dehydrogenase; EC 1.1.1.1) was purified from methanol-grown Pseudomonas sp. ATCC 21439. Secondary-alcohol dehydrogenase shows a single protein band on acrylamide gel electrophoresis and has a MW of 95,000. It consists of 2 subunits of MW 48,000 daltons and 2 atoms of Zn/molecule of enzyme protein. It oxidizes secondary alcohols, notably 2-propanol and 2-butanol. Primary alcohols are not oxidized. The pH and temperature optima for secondary-alcohol dehydrogenase are 8-9 and 30-35.degree. C, respectively. The activation energy calculated is 82.8 kJ. Secondary-alcohol dehydrogenase also catalyzes the reduction of methyl ketones to their corresponding 2-alcohols in the presence of NADH (a reverse reaction). The Km values at 25.degree. C in the forward reaction for 2-butanol, (2R)-(-)-butan-2-ol and NAD, and in the reverse reaction for 2-butanone and NADH are 2.5 .times. 10-4 M, 1.6 .times. 10-4 M, 1.1 .times. 10-5 M, 1.98 .times. 10-4 M and 2.1 .times. 10-6 M, respectively. The secondary-alcohol dehydrogenase activity was inhibited by metal-chelating agents and by strong thio reagents such as p-hydroxymercuribenzoate and 5,5''-dithiobis(2-nitrobenzoic acid). The substrate specificity and mobility on gel electrophoresis of secondary-alcohol dehydrogenase and primary-alcohol dehydrogenases were compared. Secondary-alcohol dehydrogenase preferentially oxidizes the (-)-2-butanol. This is different from primary-alcohol dehydrogenase from bakers'' yeast which oxidizes only the (+)-2-butanol. This may be explained in terms of the structure of the enzymes.