Properties of crystalline leucine dehydrogenase from Bacillus sphaericus

The distribution of Bacterial leucine dehydrogenase (L-leucine:NAD+ oxidoreductase, deaminating, EC 1.4.1.9) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the Enzyme. Leucine dehydrogenase, which was purified to homogeneity and crystallized from B. sphaericus, has a molecular weight of about 245,000 and consists of six identical subunits (Mr = 41,000). The Enzyme catalyzes the oxidative deamination of L-leucine, L-valine, L-isoleucine, L-norvaline, L-alpha-aminobutyrate, and L-norleucine, and the reductive amination of their keto analogues. The Enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. D-Enantiomers of the substrate Amino acids inhibit competitively the oxidation of L-leucine. The Enzyme activity is significantly reduced by both sulfhydryl reagents and pyridoxal 5'-phosphate. Purine and pyrimidine bases, nucleosides and nucleotides have no effect on the Enzyme activity. Initial velocity and product inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the Enzyme followed by alpha-ketoisocaproate and ammonia, and the products are released in the order of L-leucine and NAD+. The Michaelis constants are as follows: L-leucine (1 mM), NAD+ (0.39 mM), NADH (35 micrometer), alpha-ketoisocaproate (0.31 mM), and ammonia (0.2 M). The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH is exclusively transferred to the substrate; the Enzyme is B-stereospecific.