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Literature summary extracted from

  • Fries, M.; Chauhan, H.J.; Domingo, G.J.; Jung, H.I.; Perham, R.N.
    Site-directed mutagenesis of a loop at the active site of E1 (alpha2beta2) of the pyruvate dehydrogenase complex. A possible common sequence motif (2003), Eur. J. Biochem., 270, 861-870.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.4.1 expression of wild-type E1alpha and E1beta subunits of the pyruvate decarboxylate component and mutants in Escherichia coli strain BL21(DE3) Geobacillus stearothermophilus

Protein Variants

EC Number Protein Variants Comment Organism
1.2.4.1 D276A site-directed mutagenesis, reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 F266A site-directed mutagenesis, reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 R267A site-directed mutagenesis, highly reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 R282A site-directed mutagenesis, reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 S283C site-directed mutagenesis, reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 Y281A site-directed mutagenesis, reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 Y281A/R282A site-directed mutagenesis, highly reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 Y281A/R282A/S283A site-directed mutagenesis, highly reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus
1.2.4.1 Y281S/R282S site-directed mutagenesis, highly reduced activity with pyruvate compared to the wild-type enzyme Geobacillus stearothermophilus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.4.1 additional information
-
 additional information kinetics Geobacillus stearothermophilus
1.2.4.1 0.00056
-
 thiamine diphosphate mutant R267A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0011
-
 pyruvate wild-type enzyme, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0012
-
 pyruvate mutant F266A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0021
-
 pyruvate mutant S283C, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0027
-
 thiamine diphosphate mutant Y281S/R282S, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0029
-
 thiamine diphosphate mutant Y281A/R282A/S283A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.003
-
 thiamine diphosphate mutant Y281A/R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0052
-
 thiamine diphosphate mutant R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0055
-
 thiamine diphosphate mutant Y281A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.0088
-
 pyruvate mutant R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.011
-
 pyruvate mutant D276A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.013
-
 thiamine diphosphate mutant S283C, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.021
-
 thiamine diphosphate mutants F266A and D276A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.023
-
 thiamine diphosphate wild-type enzyme, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.049
-
 pyruvate mutant Y281A/R282A/S283A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.056
-
 pyruvate mutant Y281A/R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.063
-
 pyruvate mutant Y281A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.066
-
 pyruvate mutant Y281S/R282S, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.291
-
 pyruvate mutant R267A, pH 7.0, 25°C Geobacillus stearothermophilus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.2.4.1 Mg2+
-
 Geobacillus stearothermophilus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.4.1 pyruvate + CoA + NAD+ Geobacillus stearothermophilus first step of the overall reaction of the pyruvate dehydrogenase complex acetyl-CoA + CO2 + NADH
-
 ir

Organism

EC Number Organism UniProt Comment Textmining
1.2.4.1 Geobacillus stearothermophilus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.4.1 recombinant wild-type E1alpha and E1beta subunits of the pyruvate decarboxylate component and mutants from Escherichia coli strain BL21(DE3) Geobacillus stearothermophilus

Reaction

EC Number Reaction Comment Organism Reaction ID
1.2.4.1 pyruvate + [dihydrolipoyllysine-residue acetyltransferase] lipoyllysine = [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO2 catalytic site is formed by the conserved residues Tyr281 and Arg282 Geobacillus stearothermophilus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.4.1 acetaldehyde + benzaldehyde
-
 Geobacillus stearothermophilus (R)-phenylacetylcarbinol
-
 ?
1.2.4.1 pyruvate + CoA + NAD+
-
 Geobacillus stearothermophilus acetyl-CoA + CO2 + NADH
-
 ir
1.2.4.1 pyruvate + CoA + NAD+ first step of the overall reaction of the pyruvate dehydrogenase complex Geobacillus stearothermophilus acetyl-CoA + CO2 + NADH
-
 ir
1.2.4.1 pyruvate + CoA + oxidized 2,6-dichlorophenolindophenol artifical electron acceptor Geobacillus stearothermophilus acetyl-CoA + CO2 + reduced 2,6-dichlorophenolindophenol
-
 ir

Subunits

EC Number Subunits Comment Organism
1.2.4.1 tetramer alpha2beta2 structure Geobacillus stearothermophilus

Synonyms

EC Number Synonyms Comment Organism
1.2.4.1 More enzyme is a component of the pyruvate dehydrogenase multienzyme complex PDH Geobacillus stearothermophilus
1.2.4.1 pyruvate decarboxylase
-
 Geobacillus stearothermophilus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.4.1 65
-
 mutant 276A Geobacillus stearothermophilus
1.2.4.1 70
-
 wild-type enzyme Geobacillus stearothermophilus

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.2.4.1 additional information
-
 temperature dependence of wild-type and mutant enzymes Geobacillus stearothermophilus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.4.1 0.3
-
 pyruvate mutant F266A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.35
-
 thiamine diphosphate mutant F266A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.38
-
 pyruvate mutant S283C, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.45
-
 thiamine diphosphate mutant S283C, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.46
-
 thiamine diphosphate wild-type enzyme, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.48
-
 pyruvate wild-type enzyme, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.55
-
 pyruvate mutant R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.62
-
 thiamine diphosphate mutant R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.71
-
 pyruvate mutant D276A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.82
-
 pyruvate mutant Y281A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.91
-
 thiamine diphosphate mutant D276A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.96
-
 pyruvate mutant Y281A/R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.97
-
 thiamine diphosphate mutant Y281A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 0.98
-
 thiamine diphosphate mutant Y281A/R282A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 1.21
-
 pyruvate mutant Y281S/R282S, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 1.23
-
 pyruvate mutant Y281A/R282A/S283A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 1.25
-
 thiamine diphosphate mutant R267A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 1.3
-
 thiamine diphosphate mutant Y281S/R282S, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 1.39
-
 thiamine diphosphate mutant Y281A/R282A/S283A, pH 7.0, 25°C Geobacillus stearothermophilus
1.2.4.1 1.62
-
 pyruvate mutant R267A, pH 7.0, 25°C Geobacillus stearothermophilus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.4.1 7
-
 assay at Geobacillus stearothermophilus

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.4.1 NAD+
-
 Geobacillus stearothermophilus
1.2.4.1 thiamine diphosphate dependent on, binding site Geobacillus stearothermophilus