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 (Comment) | Organism |
---|---|
expression of His9-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 | Geobacillus thermodenitrificans |
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 | Comment | Organism | Structure |
---|---|---|---|
MgATP2- | substrate inhibition | Geobacillus thermodenitrificans |
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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | as MgATP2- | Geobacillus thermodenitrificans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + pyruvate + HCO3- + H+ | Geobacillus thermodenitrificans | - |
ADP + phosphate + oxaloacetate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Geobacillus thermodenitrificans | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His9-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 by affinity chromatography | Geobacillus thermodenitrificans |
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 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at, carboxylation of free biotin, ATP cleavage, and ADP phosphorylation by carbamoyl phosphate | Geobacillus thermodenitrificans |
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 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 | Comment | Organism | Structure |
---|---|---|---|
ATP | as MgATP2- | Geobacillus thermodenitrificans | |
biotin | covalently attached to Lys1112 | Geobacillus thermodenitrificans |
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 |