Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism.

摘要: Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had molecular mass of about 230 kDa consisted six subunits with an identical 38 kDa. much more thermostable than that mesophile, B. sphaericus, retained its full activity upon heating at 75 degrees C for least 60 min incubation in pH 5.5-9.5 10 min. can be stored without loss frozen state (-20 C, 7.2) over 5 months. optimum the L-alanine deamination pyruvate amination were around 10.5 8.2, respectively. exclusively catalyzed oxidative presence NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate 3-fluoropyruvate are also aminated as well NADH ammonia. Initial velocity product inhibition studies reductive proceeded through sequential mechanism containing partially random binding. binds first enzyme, then ammonia bind fashion. products sequentially released order NAD+. A dead-end formation abortive ternary complex which consists included reaction. possible role is prevent functioning synthesis. Michaelis constants substrates follows: NADH, 0.10 mM; pyruvate, 0.50 ammonia, 38.0 L-alanine, mM 0.26 mM.