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Literature summary extracted from

  • Wolfson-Stofko, B.; Hadi, T.; Blanchard, J.S.
    Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis (2013), Arch. Biochem. Biophys., 540, 53-61 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.12 gene Rv1436, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.12 C158A site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis
1.2.1.12 C162A site-directed mutagenesis, the mutant exhibits a comparable Vmax to the wild-type enzyme and only a 2fold increased Km value for D-glyceraldehyde 3-phosphate Mycobacterium tuberculosis
1.2.1.12 H185A site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.2.1.12 arsenate linear substrate inhibition, competitive versus phosphate Mycobacterium tuberculosis
1.2.1.12 iodoacetamide an irreversible, cysteine-specific alkylator, inactivation kinetics for inactivation of mutant C162A Mycobacterium tuberculosis
1.2.1.12 NADH noncompetitive inhibition versus D-glyceraldehyde 3-phosphate, competitive inhibition versus both NAD+ and arsenate Mycobacterium tuberculosis
1.2.1.12 phosphate linear substrate inhibition, competitive versus arsenate Mycobacterium tuberculosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.12 additional information
-
 additional information kinetic isotope effects. The enzyme exhibits a kinetic mechanism in which first NAD+, then D-glyceraldehyde 3-phosphate 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 3-phospho-D-glyceroyl phosphate. Solvent and multiple kinetic isotope effects revealed that the first halfreaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation. steady-state kinetics Mycobacterium tuberculosis
1.2.1.12 6
-
 phosphate pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.1.12 D-glyceraldehyde 3-phosphate + phosphate + NAD+ Mycobacterium tuberculosis
-
 3-phospho-D-glyceroyl phosphate + NADH + H+
-
 r
1.2.1.12 D-glyceraldehyde 3-phosphate + phosphate + NAD+ Mycobacterium tuberculosis H37Rv
-
 3-phospho-D-glyceroyl phosphate + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.12 Mycobacterium tuberculosis P9WN83
-
-
1.2.1.12 Mycobacterium tuberculosis H37Rv P9WN83
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.12 recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography and dialysis to over 965% purity Mycobacterium tuberculosis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.2.1.12 D-glyceraldehyde 3-phosphate + phosphate + NAD+ = 3-phospho-D-glyceroyl phosphate + NADH + H+ the conserved residue C158 is responsible for nucleophilic catalysis and the conserved residue H185 acts as a catalytic base. The enzyme exhibits a kinetic mechanism in which first NAD+, then D-glyceraldehyde 3-phosphate 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 3-phospho-D-glyceroyl phosphate. Solvent and multiple kinetic isotope effects revealed that the first halfreaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation Mycobacterium tuberculosis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.12 D-glyceraldehyde 3-phosphate + arsenate + NAD+
-
 Mycobacterium tuberculosis 1-arseno-3-phosphoglycerate + NADH + H+
-
 r
1.2.1.12 D-glyceraldehyde 3-phosphate + arsenate + NAD+
-
 Mycobacterium tuberculosis H37Rv 1-arseno-3-phosphoglycerate + NADH + H+
-
 r
1.2.1.12 D-glyceraldehyde 3-phosphate + phosphate + NAD+
-
 Mycobacterium tuberculosis 3-phospho-D-glyceroyl phosphate + NADH + H+
-
 r
1.2.1.12 D-glyceraldehyde 3-phosphate + phosphate + NAD+
-
 Mycobacterium tuberculosis H37Rv 3-phospho-D-glyceroyl phosphate + NADH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.2.1.12 GAPDH
-
 Mycobacterium tuberculosis
1.2.1.12 glyceraldehyde 3-phosphate dehydrogenase
-
 Mycobacterium tuberculosis
1.2.1.12 Mtb-GAPDH
-
 Mycobacterium tuberculosis
1.2.1.12 Rv1436
-
 Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.12 8
-
 assay at Mycobacterium tuberculosis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.2.1.12 5.5 8.5 lack of any pH dependence of the kinetic parameters in the pH range tested, recombinant His6-tagged wild-type enzyme Mycobacterium tuberculosis

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.2.1.12 5.5 8.5 pH stability range of purified recombinant His6-tagged wild-type enzyme Mycobacterium tuberculosis

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.12 additional information no activity with NADP+ Mycobacterium tuberculosis
1.2.1.12 NAD+
-
 Mycobacterium tuberculosis
1.2.1.12 NADH
-
 Mycobacterium tuberculosis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.2.1.12 0.014
-
 NADH versus NAD+, pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis
1.2.1.12 0.024
-
 NADH versus D-glyceraldehyde 3-phosphate, pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis
1.2.1.12 0.037
-
 NADH versus arsenate, pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis
1.2.1.12 60
-
 phosphate pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis
1.2.1.12 93
-
 arsenate pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
1.2.1.12 metabolism the enzyme is involved in glycolysis Mycobacterium tuberculosis
1.2.1.12 additional information kinetic and chemical mechanism of Mtb-GAPDH, overview. C158 is the active site nucleophile reacting with the aldehyde group of D-glyceraldehyde 3-phosphate to generate the thiohemiacetal and H185 is additionally required to either stabilize thiolate anion formation or act as a catalytic acid/base group Mycobacterium tuberculosis