Utting J M, Kohn L D
J Biol Chem. 1975 Jul 10;250(13):5233-42.
A hydroxypyruvate reductase has been induced in Pseudomonas acidovorans by growth on glyoxylate. The enzyme has been purified to homogeneity as assessed by the criteria of analytical ultracentrifugation and analytical disc gel electrophoresis. It has a molecular weight of approximately 85,000 and is composed of two identical subunits. The subunits are not interconnected by disulfide bonds although the enzyme has 4 mol of half-cystine per mol of enzyme. The enzyme catalyzes the reversible conversion of hydroxypyruvate to D(minus)-glycerate in the presence of NADH. Glyoxylate cannot replace hydroxypyruvate as a substrate and is a competitive inhibitor of hydroxypyruvate reduction. The activity of the enzyme toward hydroxypyruvate is anion-modulated; the activity of the enzyme toward D(minus)-glycerate is unaffected by anions but is increased by tris-(hydroxymethyl)aminomethane. The subunits of the induced hydroxypyruvate reductase can be renatured. After the enzyme is dissociated in solutions of 6.0 M guanidine hydrochloride containing 0.1 M 2-mercaptoethanol, optimum renaturation occurs when subunits are diluted into a renaturation solvent consisting of 0.04 M Trischloride, pH 7.4, containing 25% glycerol, 25 mM 2-mercaptoethanol, and 0.14 MM NADH. NAD is an inhibitor of renaturation and therefore cannot substitute for NADH. The optimal temperature of dilution and subsequent incubation is 15 degrees, and increases in protein concentration up to 1.2 mg/ml, the highest concentration tested, improve both the rate of renaturation and the yield of active material. The half-time of renaturation at a protein concentration of 1.2 mg/ml was 1 min. The kinetics of renaturation is second order, i.e., is compatible with a bimolecular reaction preducted by the association of two similar subunits. The physical and kinetic parameters of the renatured protein are the same as those of the native enzyme.
食酸假单胞菌在以乙醛酸为碳源生长时可诱导产生一种羟基丙酮酸还原酶。通过分析超速离心和分析型圆盘凝胶电泳的标准评估，该酶已被纯化至同质。它的分子量约为85,000，由两个相同的亚基组成。尽管每摩尔酶含有4摩尔半胱氨酸，但亚基之间不是通过二硫键相连。该酶在NADH存在的情况下催化羟基丙酮酸可逆转化为D-（-）-甘油酸。乙醛酸不能替代羟基丙酮酸作为底物，并且是羟基丙酮酸还原的竞争性抑制剂。该酶对羟基丙酮酸的活性受阴离子调节；该酶对D-（-）-甘油酸的活性不受阴离子影响，但会被三（羟甲基）氨基甲烷增强。诱导产生的羟基丙酮酸还原酶的亚基可以复性。在含有0.1 M 2-巯基乙醇的6.0 M盐酸胍溶液中使酶解离后，当将亚基稀释到由0.04 M三氯化物（pH 7.4）、25%甘油、25 mM 2-巯基乙醇和0.14 mM NADH组成的复性溶剂中时，会发生最佳复性。NAD是复性的抑制剂，因此不能替代NADH。稀释及后续孵育的最佳温度为15℃，蛋白质浓度增加至1.2 mg/ml（测试的最高浓度）可提高复性速率和活性物质的产量。蛋白质浓度为1.2 mg/ml时复性的半衰期为1分钟。复性动力学为二级反应，即与由两个相似亚基缔合预测的双分子反应相符。复性蛋白的物理和动力学参数与天然酶相同。
