Kinetic and chemical mechanism of malate synthase from mycobacterium tuberculosis

Christine E. Quartararo, John S. Blanchard

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 4,4-2-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 2H 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 2O and [C 2H 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.

Original languageEnglish (US)
Pages (from-to)6879-6887
Number of pages9
JournalBiochemistry
Volume50
Issue number32
DOIs
StatePublished - Aug 16 2011

Fingerprint

Malate Synthase
Mycobacterium tuberculosis
Kinetics
Acetyl Coenzyme A
Isotopes
His-His-His-His-His-His
Cats
Carbon
Chromogenics
Affinity chromatography
Mutagenesis
Dilatation and Curettage
Citric Acid Cycle
Coenzyme A
Kinetic parameters
Catalysis
Affinity Chromatography
Byproducts
Protons
Condensation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and chemical mechanism of malate synthase from mycobacterium tuberculosis. / Quartararo, Christine E.; Blanchard, John S.

In: Biochemistry, Vol. 50, No. 32, 16.08.2011, p. 6879-6887.

Research output: Contribution to journalArticle

@article{c944dba6b9db43e1a575b8f1df7cc18d,
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 4,4-2-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 2H 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 2O and [C 2H 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.",
author = "Quartararo, {Christine E.} and Blanchard, {John S.}",
year = "2011",
month = "8",
day = "16",
doi = "10.1021/bi2007299",
language = "English (US)",
volume = "50",
pages = "6879--6887",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "32",

}

TY - JOUR

T1 - Kinetic and chemical mechanism of malate synthase from mycobacterium tuberculosis

AU - Quartararo, Christine E.

AU - Blanchard, John S.

PY - 2011/8/16

Y1 - 2011/8/16

N2 - 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 4,4-2-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 2H 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 2O and [C 2H 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.

AB - 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 4,4-2-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 2H 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 2O and [C 2H 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.

UR - http://www.scopus.com/inward/record.url?scp=80051505605&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80051505605&partnerID=8YFLogxK

U2 - 10.1021/bi2007299

DO - 10.1021/bi2007299

M3 - Article

VL - 50

SP - 6879

EP - 6887

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 32

ER -