Cloned (Comment) | Organism |
---|---|
expression of N-terminally His-tagged wild-type enzyme in Escherichia coli BL21(DE3) | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
computational structure modeling of wild-type and mutant enzymes using crystal structure data at 2.1 A resolution, interactions of actie site residues Glu144, Asn146, Lys305, and Lys364, and ligands ADP2-, SO42-, and Mg2+ | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
E144A | site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure | Homo sapiens |
E144K | site-directed mutagenesis, inactive mutant, unaltered tertiary structure | Homo sapiens |
K305A | site-directed mutagenesis, 6.5% activity compared to the wild-type enzyme, 7fold increased Km for glycine, loss of negative cooperativity, 105fold increased Km for ATP, unaltered tertiary structure | Homo sapiens |
K305E | site-directed mutagenesis, 5% activity compared to the wild-type enzyme, loss of negative cooperativity, 40fold increased Km for ATP, unaltered tertiary structure | Homo sapiens |
K364A | site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure | Homo sapiens |
K364E | site-directed mutagenesis, 0.2% activity compared to the wild-type enzyme, unaltered tertiary structure | Homo sapiens |
N146A | site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure | Homo sapiens |
N146D | site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure | Homo sapiens |
N146K | site-directed mutagenesis, inactive mutant, unaltered tertiary structure | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics, substrate and ligand binding, wild-type enzyme and mutants | Homo sapiens | |
0.07 | - |
ATP | wild-type enzyme, pH 8.2, 37°C | Homo sapiens | |
0.25 | - |
glycine | mutant K305A, pH 8.2, 37°C | Homo sapiens | |
0.34 | - |
gamma-L-Glu-L-Cys | mutant K305A, pH 8.2, 37°C | Homo sapiens | |
0.4 | - |
gamma-L-Glu-L-Cys | mutant K305E, pH 8.2, 37°C | Homo sapiens | |
0.66 | - |
gamma-L-Glu-L-Cys | wild-type enzyme, pH 8.2, 37°C | Homo sapiens | |
0.83 | - |
ATP | mutant K305A, pH 8.2, 37°C | Homo sapiens | |
1.75 | - |
glycine | wild-type enzyme, pH 8.2, 37°C | Homo sapiens | |
1.96 | - |
glycine | mutant K305E, pH 8.2, 37°C | Homo sapiens | |
2.66 | - |
ATP | mutant K305E, pH 8.2, 37°C | Homo sapiens |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | 2 ions bound per enzyme molecule, binding structure and kinetics | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + gamma-L-Glu-L-Cys + Gly | Homo sapiens | second step in the biosynthesis of glutahione | ADP + phosphate + glutathione | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P48637 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type enzyme from Escherichia coli, to homogeneity | Homo sapiens |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + gamma-L-glutamyl-L-cysteine + glycine = ADP + phosphate + glutathione | mechanism, active site residues are Glu144, Asn146, Lys305, and Lys364, interaction of these residues with the ligands is essential for enzyme activity, especially Glu144 seems to be very important for stabilization of the reaction intermediate, but the active site residues are not essential for the overall enzyme structure | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + gamma-L-Glu-L-Cys + Gly | wild-type enzyme shows negative cooperativity | Homo sapiens | ADP + phosphate + glutathione | - |
? | |
ATP + gamma-L-Glu-L-Cys + Gly | second step in the biosynthesis of glutahione | Homo sapiens | ADP + phosphate + glutathione | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | active site residues are Glu144, Asn146, Lys305, and Lys364, interaction of these residues with the ligands is essential for enzyme activity, especially with Glu144, but they are not essential for the overall enzyme structure | Homo sapiens |
Synonyms | Comment | Organism |
---|---|---|
Glutathione synthetase | - |
Homo sapiens |
GS | - |
Homo sapiens |
More | the enzyme belongs to the glutathione synthetase ATP-binding domain-like superfamily | Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Homo sapiens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.003 | - |
gamma-L-Glu-L-Cys | mutant E144K and mutant N146D, pH 8.2, 37°C | Homo sapiens | |
0.007 | - |
gamma-L-Glu-L-Cys | mutant N146A, pH 8.2, 37°C | Homo sapiens | |
0.008 | - |
gamma-L-Glu-L-Cys | mutant K364A, pH 8.2, 37°C | Homo sapiens | |
0.015 | - |
gamma-L-Glu-L-Cys | mutant K364E, pH 8.2, 37°C | Homo sapiens | |
0.336 | - |
gamma-L-Glu-L-Cys | mutant K305E, pH 8.2, 37°C | Homo sapiens | |
0.423 | - |
gamma-L-Glu-L-Cys | mutant K305A, pH 8.2, 37°C | Homo sapiens | |
6.5 | - |
gamma-L-Glu-L-Cys | wild-type enzyme, pH 8.2, 37°C | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.2 | - |
assay at | Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | dependent on | Homo sapiens |