Purification and Properties of Alanine Dehydrogenase from Bacillus sphaericus

1 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 species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2 The enzyme has a molecular weight of about 230000, and is composed of six identical subunits ( M r 38000). 3 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 + . 4 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. 5 Alanine dehydrogenase is inhibited significantly by HgCl 2 , p ‐chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5′‐phosphate influence the activity. 6 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 released in the order of l ‐alanine and NAD + . The Michaelis constants are as follows: NADH (10 μM), ammonia (28.2 mM), pyruvate (1.7 mM), l ‐alanine (18.9 mM) and NAD + (0.23 mM). 7 The pro‐R hydrogen at C‐4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A‐stereospecific.