1887

Abstract

SUMMARY: Two pseudomonad strains grown on -aminopropan-2-o1 as sole source of carbon + nitrogen provided rich sources of (+)--aminopropan-2-o1: NAD oxidoreductase. The activity of this enzyme in crude extracts of sp. strain 8858 was about 400 mμmoles aminoacetone formed/mg. protein/min., under optimum conditions. Growth on media containing other carbon sources commonly repressed enzyme formation. Enzyme formation was induced by incubating succinate-grown bacteria in media containing -aminopropan-2-ol, aminoacetone or -threonine; but induction was inhibited by -2-hydroxy-2-phenylethylamine or -2-phenylserine. The enzyme was purified twenty-fold by treatment with protamine sulphate and ammonium sulphate followed by gel-filtration. The molecular weight of the enzyme was estimated to be 70 to 80 thousand. The partly purified enzyme was optimally active at pH 9.5. The Km values for -aminopropan-2-ol and NAD were about 0.1 and 0.3 m, respectively. Activity with NADP as the coenzyme was about the same as that with NAD, the Km being about 0.2 m. The enzyme was inactive with α-NAD. Activity was unaffected by a variety of thiols, thiol-alkylating and metal-chelating agents. The enzyme was virtually inactive with twenty-one potential substrates but oxidized -aminobutan-2-ol, -2-hydroxy-2-phenylethylamine and -phenylserine at significant rates and ,3-diaminopropan-2-ol and -3-hydroxybutyrate slowly. Enzyme activity towards -amino-propan-2-ol was stereospecific for the (+)-isomer, which was oxidized about six times more rapidly than the racemic substrate. The (-)-enantiomorph was a competitive inhibitor of the reaction, Ki = about 0.3 m, and no dehydrogenase activity towards this isomer was observed with either purified enzyme preparations or crude cell-free extracts. Aminoacetone-dependent oxidation of NADH occurred optimally at pH 5 in acetate buffer; a second peak of activity was found at pH 8.4 in diethanolamine + HCl buffers. Several buffers appeared to inhibit activity at pH 7.5. 2′-Aminoacetophenone was active as a substrate, but 5-aminolaevulate had little activity. NADPH was also active as coenzyme.

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/content/journal/micro/10.1099/00221287-54-1-115
1968-11-01
2019-12-05
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