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Literature summary for 1.2.1.12 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)

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

Protein Variants

Protein Variants Comment Organism
C158A site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis
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
H185A site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis

Inhibitors

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

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
6
-
phosphate pH 8.0, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis P9WN83
-
-
Mycobacterium tuberculosis H37Rv P9WN83
-
-

Purification (Commentary)

Purification (Comment) Organism
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

Reaction Comment Organism Reaction ID
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

Substrates and Products (Substrate)

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

Synonyms

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

pH Optimum

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

pH Range

pH Minimum pH Maximum Comment Organism
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

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

Cofactor

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

Ki Value [mM]

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

General Information

General Information Comment Organism
metabolism the enzyme is involved in glycolysis Mycobacterium tuberculosis
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