Nakada T, Fukui T, Saito T, Miki K, Oji C, Matsuda S, Ushijima A, Tomita K
J Biochem. 1981 Feb;89(2):625-35. doi: 10.1093/oxfordjournals.jbchem.a133239.
D(-)-beta-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(-)-beta-hydroxybutyrate and NAD in the oxidation reaction were 3.2 X 10(-4) M and 5.7 X 10(-5) M, respectively. The Km value for acetoacetate in the reduction reaction was 1.5 X 10(-4) M and that for NADH was 1.5 X 10(-5) M. Acetyl CoA, D-lactate, and 2-hydroxybutyrate were effective inhibitors for the oxidation of D(-)-beta-hydroxybutyrate. The enzyme was sensitive to the inhibitory actions of sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid) and HgCl2.
从生枝动胶菌I-16-M中纯化出D(-)-β-羟基丁酸脱氢酶，使其达到电泳纯。通过Sephadex G-200凝胶过滤法测定该酶的分子量为112,000，在十二烷基硫酸钠存在下用聚丙烯酰胺凝胶电泳法估算其单体分子量为28,000，这表明天然酶是由四个相同亚基组成的四聚体。该酶在氧化反应中的最适pH为8.0，在还原反应中的最适pH范围较宽（5.5 - 7.5）。氧化反应中D(-)-β-羟基丁酸和NAD的Km值分别为3.2×10(-4)M和5.7×10(-5)M。还原反应中乙酰乙酸和NADH的Km值分别为1.5×10(-4)M和1.5×10(-5)M。乙酰辅酶A、D-乳酸和2-羟基丁酸是D(-)-β-羟基丁酸氧化的有效抑制剂。该酶对巯基试剂如对氯汞苯甲酸、5,5'-二硫代双(2-硝基苯甲酸)和HgCl2的抑制作用敏感。
