Purification and Properties of Alanine Dehydrogenase from Bacillus sphaericus

Abstract

The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus sp. The enzyme was purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. The enzyme has a MW of about 230,000, and is composed of 6 identical subunits (MW 38,000). The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also aminated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. The enzyme is stable over a wide pH range (pH 6.0-10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5''-phosphate influence the activity. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are relased in the order of L-alanine and NAD+. The Km are as follows: NADH (10 .mu.M), NH3 (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). The pro-R H at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.