title: Kinetic and chemical mechanism of malate synthase from Mycobacterium tuberculosis.
abstract: 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.
authors: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
authors: Christine E
authors: CE
authors: Quartararo
authors: John S
authors: JS
authors: Blanchard
mesh_terms: D002384:Catalysis; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008292:Malate Synthase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D016133:Polymerase Chain Reaction
chemical_list: D008292:Malate Synthase