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. 1989 Aug 1;261(3):853–861. doi: 10.1042/bj2610853

Purification and catalytic properties of L-valine dehydrogenase from Streptomyces cinnamonensis.

N D Priestley 1, J A Robinson 1
PMCID: PMC1138909  PMID: 2803248

Abstract

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4' of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.

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Selected References

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