Cloned (Comment) | Organism |
---|---|
expression as C-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3) | Trypanosoma brucei |
Protein Variants | Comment | Organism |
---|---|---|
R179A | site-directed PCR-based mutagenesis, conserved residue, gamma-aminobutyrate-binding determinant, increase of Km for both L-Cys and L-Glu | Trypanosoma brucei |
R366A | site-directed PCR-based mutagenesis, mutant is active with gamma-aminobutyrate, increase of dissociation constant for L-Glu by 160fold, elimination of positive cooperativity of binding of L-Glu and ATP, interacts with the alpha-carboxylate of L-Glu, 220fold increase in Ki for GSH | Trypanosoma brucei |
R474A | site-directed PCR-based mutagenesis, R474 is an ATP binding determinant, increase of Km for ATP by 20-100fold | Trypanosoma brucei |
R487A | site-directed PCR-based mutagenesis, R487 is an ATP binding determinant, increase of Km for ATP by 20-100fold | Trypanosoma brucei |
R491A | site-directed PCR-based mutagenesis, decrease of kcat for ATP hydrolysis | Trypanosoma brucei |
T323A | site-directed PCR-based mutagenesis, T323 is an alpha-phosphate of ATP binding determinant, increase of Km for ATP by 20-100fold | Trypanosoma brucei |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
ATP | substrate inhibition of mutant R179A, when only one other substrate is saturating | Trypanosoma brucei | |
GSH | competitive | Trypanosoma brucei | |
L-alpha-aminobutyrate | substrate inhibition of mutant R179A, when only one other substrate is saturating | Trypanosoma brucei | |
L-buthionine-S-sulfoximine | irrversible inactivation, no inhibition of mutant R366A | Trypanosoma brucei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics for the wild-type and mutant enzymes | Trypanosoma brucei | |
0.071 | - |
ATP | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
0.11 | - |
ATP | mutant R491A, pH 8.0, 37°C | Trypanosoma brucei | |
0.14 | - |
ATP | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
0.24 | - |
L-glutamate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
0.61 | - |
L-glutamate | mutant R491A, pH 8.0, 37°C | Trypanosoma brucei | |
1.2 | - |
ATP | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
1.3 | - |
ATP | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
1.3 | - |
L-glutamate | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
1.6 | - |
L-glutamate | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
1.7 | - |
L-glutamate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
2.8 | - |
ATP | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
5.2 | - |
L-glutamate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
7 | - |
ATP | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
7.5 | - |
L-alpha-aminobutyrate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
10 | - |
L-alpha-aminobutyrate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
15 | - |
L-alpha-aminobutyrate | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
20 | - |
4-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
34 | - |
L-alpha-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
56 | - |
L-alpha-aminobutyrate | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
130 | - |
L-glutamate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
150 | - |
4-aminobutyrate | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
380 | - |
L-alpha-aminobutyrate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | Mg2+ or Mn2+ are required | Trypanosoma brucei | |
Mn2+ | Mg2+ or Mn2+ are required | Trypanosoma brucei |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-Glu + L-Cys | Trypanosoma brucei | first step in the de novo biosynthesis of the tripeptide glutathione | ADP + phosphate + gamma-L-Glu-L-Cys | - |
r |
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 | rapid equilibrium random ter-reactant mechanism, substrate binding modeling, active site model | Trypanosoma brucei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-Glu + gamma-aminobutyrate | mutant R366A | Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-gamma-aminobutyrate | - |
r | |
ATP + L-Glu + L-alpha-aminobutyrate | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate | - |
r | |
ATP + L-Glu + L-Cys | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
r | |
ATP + L-Glu + L-Cys | first step in the de novo biosynthesis of the tripeptide glutathione | Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
r |
Subunits | Comment | Organism |
---|---|---|
monomer | - |
Trypanosoma brucei |
Synonyms | Comment | Organism |
---|---|---|
gamma-GCS | - |
Trypanosoma brucei |
gamma-Glutamylcysteine synthetase | - |
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 |
---|---|---|---|---|---|
0.055 | - |
L-glutamate | mutant R491A, pH 8.0, 37°C | Trypanosoma brucei | |
0.14 | - |
4-aminobutyrate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
0.24 | - |
L-glutamate | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
0.32 | - |
L-glutamate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
0.59 | - |
4-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
0.61 | - |
L-glutamate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
0.79 | - |
L-glutamate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
1.2 | - |
L-glutamate | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
3.9 | - |
L-glutamate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
6.08 | - |
L-glutamate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
6.08 | - |
4-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.08 | - |
L-glutamate | mutant T323A, 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 | dependent on | Trypanosoma brucei |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
1.1 | - |
GSH | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
36 | - |
GSH | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei |