Title of article :
Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis
Author/Authors :
Wolfson-Stofko، نويسنده , , Brett and Hadi، نويسنده , , Timin and Blanchard، نويسنده , , John S.، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2013
Pages :
9
From page :
53
To page :
61
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.
Keywords :
glycolysis , Kinetic isotope effects , Tuberculosis , enzyme kinetics , Glyceraldehyde 3-phosphate dehydrogenase
Journal title :
Archives of Biochemistry and Biophysics
Serial Year :
2013
Journal title :
Archives of Biochemistry and Biophysics
Record number :
1603595
Link To Document :
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