EC Number | Cloned (Comment) | Organism |
---|---|---|
6.3.2.2 | expression as C-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3) | Trypanosoma brucei |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
6.3.2.2 | R179A | site-directed PCR-based mutagenesis, conserved residue, gamma-aminobutyrate-binding determinant, increase of Km for both L-Cys and L-Glu | Trypanosoma brucei |
6.3.2.2 | 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 |
6.3.2.2 | R474A | site-directed PCR-based mutagenesis, R474 is an ATP binding determinant, increase of Km for ATP by 20-100fold | Trypanosoma brucei |
6.3.2.2 | R487A | site-directed PCR-based mutagenesis, R487 is an ATP binding determinant, increase of Km for ATP by 20-100fold | Trypanosoma brucei |
6.3.2.2 | R491A | site-directed PCR-based mutagenesis, decrease of kcat for ATP hydrolysis | Trypanosoma brucei |
6.3.2.2 | 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 |
EC Number | Inhibitors | Comment | Organism | Structure |
---|---|---|---|---|
6.3.2.2 | ATP | substrate inhibition of mutant R179A, when only one other substrate is saturating | Trypanosoma brucei | |
6.3.2.2 | GSH | competitive | Trypanosoma brucei | |
6.3.2.2 | L-alpha-aminobutyrate | substrate inhibition of mutant R179A, when only one other substrate is saturating | Trypanosoma brucei | |
6.3.2.2 | L-buthionine-S-sulfoximine | irrversible inactivation, no inhibition of mutant R366A | Trypanosoma brucei |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
6.3.2.2 | additional information | - |
additional information | kinetics for the wild-type and mutant enzymes | Trypanosoma brucei | |
6.3.2.2 | 0.071 | - |
ATP | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.11 | - |
ATP | mutant R491A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.14 | - |
ATP | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.24 | - |
L-glutamate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.61 | - |
L-glutamate | mutant R491A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 1.2 | - |
ATP | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 1.3 | - |
ATP | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 1.3 | - |
L-glutamate | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 1.6 | - |
L-glutamate | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 1.7 | - |
L-glutamate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 2.8 | - |
ATP | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 5.2 | - |
L-glutamate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 7 | - |
ATP | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 7.5 | - |
L-alpha-aminobutyrate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 10 | - |
L-alpha-aminobutyrate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 15 | - |
L-alpha-aminobutyrate | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 20 | - |
4-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 34 | - |
L-alpha-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 56 | - |
L-alpha-aminobutyrate | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 130 | - |
L-glutamate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 150 | - |
4-aminobutyrate | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 380 | - |
L-alpha-aminobutyrate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
6.3.2.2 | Mg2+ | Mg2+ or Mn2+ are required | Trypanosoma brucei | |
6.3.2.2 | Mn2+ | Mg2+ or Mn2+ are required | Trypanosoma brucei |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
6.3.2.2 | 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 |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
6.3.2.2 | Trypanosoma brucei | - |
- |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
6.3.2.2 | recombinant His-tagged wild-type and mutant enzymes from Escherichia coli | Trypanosoma brucei |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
6.3.2.2 | 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 |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
6.3.2.2 | ATP + L-Glu + gamma-aminobutyrate | mutant R366A | Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-gamma-aminobutyrate | - |
r | |
6.3.2.2 | ATP + L-Glu + L-alpha-aminobutyrate | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate | - |
r | |
6.3.2.2 | ATP + L-Glu + L-Cys | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
r | |
6.3.2.2 | 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 |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
6.3.2.2 | monomer | - |
Trypanosoma brucei |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
6.3.2.2 | gamma-GCS | - |
Trypanosoma brucei |
6.3.2.2 | gamma-Glutamylcysteine synthetase | - |
Trypanosoma brucei |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
6.3.2.2 | 37 | - |
assay at | Trypanosoma brucei |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
6.3.2.2 | 0.055 | - |
L-glutamate | mutant R491A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.14 | - |
4-aminobutyrate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.24 | - |
L-glutamate | mutant R474A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.32 | - |
L-glutamate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.59 | - |
4-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.61 | - |
L-glutamate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 0.79 | - |
L-glutamate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 1.2 | - |
L-glutamate | mutant R487A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 3.9 | - |
L-glutamate | wild-type, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 6.08 | - |
L-glutamate | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 6.08 | - |
4-aminobutyrate | mutant R366A, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 6.08 | - |
L-glutamate | mutant T323A, pH 8.0, 37°C | Trypanosoma brucei |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
6.3.2.2 | 8 | - |
assay at | Trypanosoma brucei |
EC Number | Cofactor | Comment | Organism | Structure |
---|---|---|---|---|
6.3.2.2 | ATP | dependent on | Trypanosoma brucei |
EC Number | Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|---|
6.3.2.2 | 1.1 | - |
GSH | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
6.3.2.2 | 36 | - |
GSH | mutant R179A, pH 8.0, 37°C | Trypanosoma brucei |