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Literature summary for 1.2.4.1 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
Search for more literature for 1.2.4.1

Cloned(Commentary)

Cloned (Comment) Organism
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

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

KM Value [mM]

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

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
 Geobacillus stearothermophilus

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Geobacillus stearothermophilus
-
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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)

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

Subunits

Subunits Comment Organism
tetramer alpha2beta2 structure Geobacillus stearothermophilus

Synonyms

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

Temperature Optimum [°C]

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

Temperature Range [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

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