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Literature summary for 6.4.1.1 extracted from

  • Adina-Zada, A.; Jitrapakdee, S.; Surinya, K.H.; McIldowie, M.J.; Piggott, M.J.; Cleland, W.W.; Wallace, J.C.; Attwood, P.V.
    Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme (2008), Int. J. Biochem. Cell Biol., 40, 1743-1752.
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
acetyl CoA enzyme activator, major effect is to promote the carboxylation of biotin, reduces the Kms for both MgATP2- and biotin, overview Geobacillus thermodenitrificans

Cloned(Commentary)

Cloned (Comment) Organism
expression of His9-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Geobacillus thermodenitrificans

Protein Variants

Protein Variants Comment Organism
K1112A site-directed mutagenesis, the mutant lacks the biotin binding site and boud biotin, it does not catalyse the complete reaction, but catalyses ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation is not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. The mutant K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate Geobacillus thermodenitrificans

Inhibitors

Inhibitors Comment Organism Structure
MgATP2- substrate inhibition Geobacillus thermodenitrificans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information stopped flow kinetics using fluorescent ATP analogue formycin A-5'-triphosphate, presence of biotin enhanced binding, kinetics of free biotin carboxylation Geobacillus thermodenitrificans
0.6
-
ATP pH 7.8, 30°C, in presence of acetyl-CoA Geobacillus thermodenitrificans
1.8
-
ATP pH 7.8, 30°C, in absence of acetyl-CoA Geobacillus thermodenitrificans
3.3
-
biotin pH 7.8, 30°C, in presence of acetyl-CoA Geobacillus thermodenitrificans
18
-
biotin pH 7.8, 30°C, in absence of acetyl-CoA Geobacillus thermodenitrificans

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ as MgATP2- Geobacillus thermodenitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + pyruvate + HCO3- + H+ Geobacillus thermodenitrificans
-
ADP + phosphate + oxaloacetate
-
?

Organism

Organism UniProt Comment Textmining
Geobacillus thermodenitrificans
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His9-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 by affinity chromatography Geobacillus thermodenitrificans

Reaction

Reaction Comment Organism Reaction ID
ATP + pyruvate + HCO3- + H+ = ADP + phosphate + oxaloacetate reaction mechanism of ATP cleavage and biotin carboxylation, overview Geobacillus thermodenitrificans

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.004
-
free biotin carboxylation activity of the mutant K1112A in absence of acetyl-CoA at 25 mM Geobacillus thermodenitrificans
0.005
-
free biotin carboxylation activity of the mutant K1112A in presence of acetyl-CoA at 25 mM Geobacillus thermodenitrificans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + pyruvate + HCO3- ATP in form of MgATP2- Geobacillus thermodenitrificans ADP + phosphate + oxaloacetate
-
?
ATP + pyruvate + HCO3- + H+
-
Geobacillus thermodenitrificans ADP + phosphate + oxaloacetate
-
?
ATP + pyruvate + HCO3- + H+ involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation Geobacillus thermodenitrificans ADP + phosphate + oxaloacetate
-
?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at, carboxylation of free biotin, ATP cleavage, and ADP phosphorylation by carbamoyl phosphate Geobacillus thermodenitrificans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
-
assay at, carboxylation of free biotin, ATP cleavage, and ADP phosphorylation by carbamoyl phosphate Geobacillus thermodenitrificans

pH Range

pH Minimum pH Maximum Comment Organism
additional information
-
pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin Geobacillus thermodenitrificans

Cofactor

Cofactor Comment Organism Structure
ATP as MgATP2- Geobacillus thermodenitrificans
biotin covalently attached to Lys1112 Geobacillus thermodenitrificans

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.4
-
MgATP2- pH 7.8, 30°C, in absence of acetyl-CoA Geobacillus thermodenitrificans
0.5
-
MgATP2- pH 7.8, 30°C, in presence of acetyl-CoA Geobacillus thermodenitrificans