title: Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis.
abstract: 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, United States.
authors.forename: Brett
authors.initials: B
authors.lastname: Wolfson-Stofko
authors.forename: Timin
authors.initials: T
authors.lastname: Hadi
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D003001:Cloning, Molecular; D004789:Enzyme Activation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics
chemical_list: D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Enzyme kinetics; Glyceraldehyde 3-phosphate dehydrogenase; Glycolysis; Kinetic isotope effects; Tuberculosis