Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutants in Escherichia coli BL21(DE3) as C-terminally His-tagged proteins | Trypanosoma brucei |
Protein Variants | Comment | Organism |
---|---|---|
E100A | site-directed mutagenesis, n1 metal binding site mutant, inactive mutant | Trypanosoma brucei |
E489A | site-directed mutagenesis, n2 metal binding site mutant, reduced activity | Trypanosoma brucei |
E53A | site-directed mutagenesis, n2 metal binding site mutant, reduced activity | Trypanosoma brucei |
E55A | site-directed mutagenesis, n1 metal binding site mutant, inactive mutant | Trypanosoma brucei |
E93A | site-directed mutagenesis, n1 metal binding site mutant, only capable of catalyzing L-Glu-dependent ATP hydrolysis and not the ligation between L-Glu and L-alpha-aminobutyrate | Trypanosoma brucei |
Q321A | site-directed mutagenesis, n2 metal binding site mutant, reduced activity | Trypanosoma brucei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics | Trypanosoma brucei | |
0.022 | - |
ATP | wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
0.071 | - |
ATP | wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
0.1 | - |
ATP | mutant E489A, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
0.24 | - |
L-glutamate | wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
0.32 | - |
ATP | mutant E93A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
0.89 | - |
L-glutamate | mutant E489A, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
1 | - |
L-glutamate | wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
1.1 | - |
L-glutamate | mutant E489A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
1.1 | - |
L-glutamate | mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
1.2 | - |
ATP | mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
1.6 | - |
L-glutamate | mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
2 | - |
ATP | mutant E489A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
6 | - |
L-alpha-aminobutyrate | wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
9.4 | - |
L-alpha-aminobutyrate | mutant E489A, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
10 | - |
L-alpha-aminobutyrate | wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
11 | - |
L-glutamate | mutant E93A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
14 | - |
L-alpha-aminobutyrate | mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ | Trypanosoma brucei | |
15 | - |
L-alpha-aminobutyrate | mutant E489A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
18 | - |
L-alpha-aminobutyrate | mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei | |
45 | - |
ATP | above, mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ | Trypanosoma brucei |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, bound as MgATP2-, the metal ion specificity is determined by the second binding site n2 which also is involved in ATP binding, located in the active site and formed by 3 conserved residues Glu53, Gln321, and Glu489, Mg2+ can partly be substituted by Mn2+ | Trypanosoma brucei | |
Mn2+ | can partly substitute for Mg2+ | Trypanosoma brucei | |
additional information | the first metal binding site n1 binds free metal ions and is composed of 3 conserved residues Glu55, Glu93, and Glu100, n1 also is involved in positioning of L-glutamate for the reaction, located in the active site | Trypanosoma brucei |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-Glu + L-Cys | Trypanosoma brucei | rate limiting and first step in glutathione biosynthesis | ADP + phosphate + gamma-L-Glu-L-Cys | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trypanosoma brucei | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli | Trypanosoma brucei |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine | reaction mechanism via phosphorylated glutamate intermediate | Trypanosoma brucei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-Glu + L-alpha-aminobutyrate | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate | - |
ir | |
ATP + L-Glu + L-Cys | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
? | |
ATP + L-Glu + L-Cys | rate limiting and first step in glutathione biosynthesis | Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
ir | |
ATP + L-glutamate + L-cysteine | ATP in form of MnATP2- | Trypanosoma brucei | ADP + phosphate + gamma-L-glutamyl-L-cysteine | - |
? | |
ATP + L-glutamate + L-cysteine | ATP in form of MgATP2- | Trypanosoma brucei | ADP + phosphate + gamma-L-glutamyl-L-cysteine | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | structure modeling | Trypanosoma brucei |
Synonyms | Comment | Organism |
---|---|---|
gamma-GCS | - |
Trypanosoma brucei |
gamma-Glutamylcysteine synthetase | - |
Trypanosoma brucei |
More | enzyme belongs to the superfamily of carboxylate-amine/ammonia ligases, together with glutathione synthase | Trypanosoma brucei |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Trypanosoma brucei |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0053 | - |
ATP | mutant E489A, pH 8.0, 37°C | Trypanosoma brucei | |
0.0066 | - |
Mg2+ | mutant E489A, pH 8.0, 37°C | Trypanosoma brucei | |
0.066 | - |
ATP | mutant E93A, pH 8.0, 37°C | Trypanosoma brucei | |
0.078 | - |
Mg2+ | mutant E93A, pH 8.0, 37°C | Trypanosoma brucei | |
0.08 | - |
Mg2+ | mutant Q321A, pH 8.0, 37°C | Trypanosoma brucei | |
0.089 | - |
Mn2+ | mutant E93A, pH 8.0, 37°C | Trypanosoma brucei | |
0.24 | - |
ATP | above, mutant Q321A, pH 8.0, 37°C | Trypanosoma brucei | |
0.89 | - |
ATP | mutant E489A, pH 8.0, 37°C | Trypanosoma brucei | |
0.89 | - |
Mn2+ | mutant E489A, pH 8.0, 37°C | Trypanosoma brucei | |
1.5 | - |
ATP | mutant Q321A, pH 8.0, 37°C | Trypanosoma brucei | |
1.5 | - |
Mn2+ | mutant Q321A, pH 8.0, 37°C | Trypanosoma brucei | |
2.4 | - |
ATP | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
2.5 | - |
Mn2+ | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
3.8 | - |
ATP | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
3.8 | - |
Mg2+ | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
6.08 | - |
ATP | mutant E489A, pH 8.0, 37°C | Trypanosoma brucei | |
6.08 | - |
Mn2+ | mutant E489A, pH 8.0, 37°C | Trypanosoma brucei |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Trypanosoma brucei |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | associated with Mg2+ binding at the second n2 metal binding site | Trypanosoma brucei |