Cloned (Comment) | Organism |
---|---|
expression of the C-terminally His8-tagged wild-type enzyme in Escherichia coli BL21(DE3), expression of the mutant C319A in Escherichia coli BO265 | Trypanosoma brucei |
expression of wild-type and mutant C319A enzymes in Escherichia coli BL21(DE3) as His-tagged proteins | Trypanosoma brucei |
Protein Variants | Comment | Organism |
---|---|---|
C319A | mutant is insensitive to cystamine, but the catalytic efficiency, the kinetic mechanism, or the substrate affinities remain unaltered | Trypanosoma brucei |
C319A | site-directed mutagenesis, loss of sensitivity against cysteamine inactivation | Trypanosoma brucei |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
cystamine | irreversible inactivation of the wild-type enzyme, loss of 75% activity within 10 min at 0.01 mM, binds to active site Cys319 and in this way blocks the binding of substrate to the enzyme, no inhibition of the mutant C319A enzyme | Trypanosoma brucei | |
cysteamine | wild-type enzyme is inhibited by 75% at 0.01 mM after 10 min, complete irreversible inhibition at 10 mM, the mutant C319Ais completely resistant to cysteamine | Trypanosoma brucei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics | Trypanosoma brucei | |
additional information | - |
additional information | kinetics, wild-type enzyme and mutant C319A | Trypanosoma brucei | |
1.4 | - |
ATP | wild-type enzyme and mutant C319A, pH 8.0, 37°C | Trypanosoma brucei | |
2.6 | - |
L-Glu | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
5.4 | - |
L-2-aminobutyric acid | wild-type enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
5.9 | - |
L-Glu | mutant C319A, pH 8.0, 37°C | Trypanosoma brucei | |
6.1 | - |
L-2-aminobutyric acid | mutant C319A, pH 8.0, 37°C | Trypanosoma brucei |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
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 biosynthesis of trypanothione via GSH | ADP + phosphate + gamma-L-Glu-L-Cys | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trypanosoma brucei | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant C319A enzymes from Escherichia coli | Trypanosoma brucei |
recombinant mutant C319A and recombinant His8-tagged wild-type 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, Cys319 is an active site cysteine not essential for activity | Trypanosoma brucei | |
ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine | reaction and kinetic mechanism, modeling | Trypanosoma brucei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-Glu + L-2-aminobutyrate | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-2-aminobutyrate | - |
? | |
ATP + L-Glu + L-alpha-aminobutyrate | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate | - |
? | |
ATP + L-Glu + L-Cys | - |
Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
? | |
ATP + L-Glu + L-Cys | first step in the biosynthesis of trypanothione via GSH | Trypanosoma brucei | ADP + phosphate + gamma-L-Glu-L-Cys | - |
? | |
additional information | binding of ATP to the enzyme increases the binding affinity for L-Glu by 18fold, while binding of L-Glu or L-alpha-aminobutyrate decreases the affinity for binding of the other by 6fold | 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 |
---|---|---|---|---|---|
23 | - |
ATP | wild-type and mutant enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
23 | - |
L-Glu | wild-type and mutant enzyme, pH 8.0, 37°C | Trypanosoma brucei | |
23 | - |
L-alpha-aminobutyrate | wild-type and mutant enzyme, 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 | - |
Trypanosoma brucei | |
ATP | binding of L-Glu to the enzyme increases the binding affinity for ATP by 18fold | Trypanosoma brucei |