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

NAD‐ dependent alcohol dehydrogenase from the methanol‐grown Methylcoccus sp. CRL MI (type I membrane). Methylosinus trichosporium OB3b (type II membrane), Methylobacterium organophillum 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) has been purified from methanol‐grown Pseudomonas sp. ATCC 21439. Secondary‐alcohol dehydrogenase shows a single protein band on acrylamide gel electrophoresis and has a molecular weigth of 95000. It consists of two subunits of M r 48000 daltons and two atoms of zinc per 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 °C, respectively. The activation enrgy calculated is 82.8kJ.Secondary‐alcohol dehydrogenase also catalyzes the reduction of methyl ketones to their corresponding 2‐alcohols in the presence of NADH (a reverse reaction). The K m values at 25 °C in the forward reaction for 2‐butanol, (2 R )‐(−)‐butan‐2‐ol, and NAD, and in the reverse reaction for 2‐butanone and NADH are 2.5×10 −4 M, 1.1×10 −5 M, 1.98×10 −4 M, and 2.1×10 −6 M, respectively. The secondary‐alcohol dehydrogenase activity was inhibited by metal‐chelating agents and by strong thio regents such as p ‐hydroxymercuribenzoate and 5,5′‐dithiobis(2‐nitrobenzoic acid).The substrate specificity, and mobility on gel electrophoresis of secondary‐alcohol dehydrogenase oxidizes preferentially the (−)‐2‐butanol. This different from primary‐aclcohol dehydrogenase from bakers' yeast which oxidizes only the(+)‐2‐butanol. This may be explained in terms of the structure of the enzymes.