Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
Biochemistry. 2011 Aug 16;50(32):6879-87. doi: 10.1021/bi2007299. Epub 2011 Jul 18.
Malate synthase catalyzes the Claisen-like condensation of acetyl-coenzyme A (AcCoA) and glyoxylate in the glyoxylate shunt of the citric acid cycle. The Mycobacterium tuberculosis malate synthase G gene, glcB, was cloned, and the N-terminal His(6)-tagged 80 kDa protein was expressed in soluble form and purified by metal affinity chromatography. A chromogenic 4,4'-dithiodipyridine assay did not yield linear kinetics, but the generation of an active site-directed mutant, C619S, gave an active enzyme and linear kinetics. The resulting mutant exhibited kinetics comparable to those of the wild type and was used for the full kinetic analysis. Initial velocity studies were intersecting, suggesting a sequential mechanism, which was confirmed by product and dead-end inhibition. The inhibition studies delineated the ordered binding of glyoxylate followed by AcCoA and the ordered release of CoA followed by malate. The pH dependencies of k(cat) and k(cat)/K(gly) are both bell-shaped, and catalysis depends on a general base (pK = 5.3) and a general acid (pK = 9.2). Primary kinetic isotope effects determined using [C(2)H(3)-methyl]acetyl-CoA suggested that proton removal and carbon-carbon bond formation were partially rate-limiting. Solvent kinetic isotope effects on k(cat) suggested the hydrolysis of the malyl-CoA intermediate was also partially rate-limiting. Multiple kinetic isotope effects, utilizing D(2)O and [C(2)H(3)-methyl]acetyl-CoA, confirmed a stepwise mechanism in which the step exhibiting primary kinetic isotope effects precedes the step exhibiting the solvent isotope effects. We combined the kinetic data and the pH dependence of the kinetic parameters with existing structural and mutagenesis data to propose a chemical mechanism for malate synthase from M. tuberculosis.
苹果酸合酶催化柠檬酸循环的乙醛酸支路中乙酰辅酶 A（AcCoA）和草酰乙酸的Claisen 样缩合。结核分枝杆菌苹果酸合酶 G 基因 glcB 被克隆，N 端 His(6)-标签 80 kDa 蛋白以可溶形式表达，并通过金属亲和层析纯化。显色 4,4'-二硫代二吡啶测定法没有得到线性动力学，但生成活性位点定向突变体 C619S，得到了活性酶和线性动力学。所得突变体表现出与野生型相当的动力学特性，并用于全动力学分析。初始速度研究相交，表明存在顺序机制，这通过产物和无终产物抑制得到证实。抑制研究描绘了草酰乙酸的有序结合，然后是 AcCoA，以及 CoA 的有序释放，然后是苹果酸。k(cat)和 k(cat)/K(gly)的 pH 依赖性均呈钟形，催化依赖于一个广义碱（pK = 5.3）和一个广义酸（pK = 9.2）。使用 [C(2)H(3)-甲基]乙酰辅酶 A 测定的一级动力学同位素效应表明，质子的去除和碳-碳键的形成部分受到限速。对 k(cat)的溶剂动力学同位素效应表明，马来酰辅酶 A 中间体的水解也是部分限速的。利用 D(2)O 和 [C(2)H(3)-甲基]乙酰辅酶 A 的多个动力学同位素效应证实了逐步机制，其中表现出一级动力学同位素效应的步骤先于表现出溶剂同位素效应的步骤。我们将动力学数据和动力学参数的 pH 依赖性与现有的结构和突变数据相结合，提出了结核分枝杆菌苹果酸合酶的化学机制。
