Purification and Properties of D-β-Hydroxybutyrate Dehydrogenase from Zoogloea ramigera I-16-M1

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Abstract
D(−)-β-Hydroxybutyrate dehydrogenase was purified from Zoogloea ramigera I-16-M to electrophoretic homogeneity. The molecular weight of the enzyme as determined by Sephadex G-200 gel filtration was 112,000, and the monomer molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 28,000, indicating that the native enzyme is a tetramer with four identical subunits. The enzyme showed a pH optimum at 8.0 in the oxidation reaction, and a broad pH optimum (5.5–7.5) in the reduction reaction. The Km values for D(−)-β-hydroxybutyrate and NAD in the oxidation reaction were 3.2×10 −4 M and 5.7 × 10 −6 M, respectively. The K m value for acetoacetate in the reduction reaction was 1.5 × 10 −4 M and that for NADH was 1.5 × 10 −5 M. Acetyl CoA, D-lactate, and 2-hydroxybutyrate were effective inhibitors for the oxidation of D(−)-β-hydroxy-butyrate. The enzyme was sensitive to the inhibitory actions of sulfhydryl reagents such as p -chloromercuribenzoic acid, 5, 5'-dithiobis(2-nitrobenzoic acid) and HgCl 2 .