Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis

Brett Wolfson-Stofko, Timin Hadi, John S. Blanchard

Research output: Contribution to journalArticle

7 Scopus citations


Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD+, then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD+ on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.

Original languageEnglish (US)
Pages (from-to)53-61
Number of pages9
JournalArchives of Biochemistry and Biophysics
Issue number1-2
Publication statusPublished - Nov 12 2013



  • Enzyme kinetics
  • Glyceraldehyde 3-phosphate dehydrogenase
  • Glycolysis
  • Kinetic isotope effects
  • Tuberculosis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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