Cloned (Comment) | Organism |
---|---|
gene gshA, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Synechocystis sp. |
Protein Variants | Comment | Organism |
---|---|---|
E37Q | site-directed mutagenesis, inactive mutant | Synechocystis sp. |
E44Q | site-directed mutagenesis, inactive mutant | Synechocystis sp. |
H121A | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Synechocystis sp. |
H121Q | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme | Synechocystis sp. |
R167A | site-directed mutagenesis, inactive mutant | Synechocystis sp. |
R167K | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme | Synechocystis sp. |
R248A | site-directed mutagenesis, inactive mutant | Synechocystis sp. |
R248K | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme | Synechocystis sp. |
T117A | site-directed mutagenesis, inactive mutant | Synechocystis sp. |
T117S | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme | Synechocystis sp. |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
buthionine sulfoxime | - |
Synechocystis sp. | |
glutathione | substrate inhibition | Synechocystis sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics, overview | Synechocystis sp. | |
0.059 | - |
L-cysteine | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
0.116 | - |
L-cysteine | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
0.145 | - |
ATP | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
0.186 | - |
L-cysteine | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.279 | - |
ATP | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
0.31 | - |
ATP | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.352 | - |
ATP | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.445 | - |
ATP | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.5 | - |
L-glutamate | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.707 | - |
L-cysteine | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.857 | - |
L-cysteine | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.953 | - |
L-glutamate | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
0.969 | - |
ATP | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
1.18 | - |
L-glutamate | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
1.95 | - |
L-cysteine | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
3.76 | - |
L-glutamate | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
6.1 | - |
L-glutamate | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
15.2 | - |
L-glutamate | pH 7.5, 25°C, mutant H121A | Synechocystis sp. |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Synechocystis sp. |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
45000 | - |
recombinant enzyme, gel filtration | Synechocystis sp. |
46000 | - |
1 * 46000, recombinant enzyme, SDS-PAGE | Synechocystis sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + L-cysteine | Synechocystis sp. | - |
ADP + phosphate + gamma-L-glutamyl-L-cysteine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Synechocystis sp. | P74515 | gene gshA or slr0990 | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Synechocystis sp. |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine | reaction mechanism, overview | Synechocystis sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + L-cysteine | - |
Synechocystis sp. | ADP + phosphate + gamma-L-glutamyl-L-cysteine | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | 1 * 46000, recombinant enzyme, SDS-PAGE | Synechocystis sp. |
Synonyms | Comment | Organism |
---|---|---|
GCL | - |
Synechocystis sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Synechocystis sp. |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.008 | - |
ATP | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.008 | - |
L-glutamate | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.01 | - |
L-cysteine | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.025 | - |
L-cysteine | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.038 | - |
ATP | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.045 | - |
L-glutamate | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.09 | - |
L-cysteine | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.098 | - |
ATP | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.127 | - |
L-glutamate | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.133 | - |
L-cysteine | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.137 | - |
ATP | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
0.137 | - |
L-glutamate | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.188 | - |
L-glutamate | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
0.225 | - |
ATP | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.335 | - |
L-cysteine | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
0.35 | - |
L-glutamate | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
0.382 | - |
ATP | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
0.42 | - |
L-cysteine | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Synechocystis sp. |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Synechocystis sp. |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
7.4 | - |
pH 7.5, 25°C, recombinant enzyme | Synechocystis sp. | glutathione | |
33.7 | - |
pH 7.5, 25°C, recombinant enzyme | Synechocystis sp. | buthionine sulfoxime |
General Information | Comment | Organism |
---|---|---|
evolution | Synechocystis GCL is part of the plant-like GCL family, the Synechocystis enzyme lacks the redox regulation associated with the plant enzymes and functions as a monomeric protein, indicating that evolution of redox regulation occurs later in the green lineage | Synechocystis sp. |
physiological function | glutathione biosynthesis catalysed by glutamate-cysteine ligase and glutathione synthetase, EC 6.3.2.3, is essential for maintaining redox homoeostasis and protection against oxidative damage in diverse eukaroytes and bacteria | Synechocystis sp. |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0009 | - |
L-glutamate | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.0022 | - |
L-glutamate | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.0083 | - |
L-glutamate | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.0086 | - |
ATP | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.0117 | - |
L-cysteine | pH 7.5, 25°C, mutant R248K | Synechocystis sp. | |
0.0128 | - |
L-cysteine | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.0224 | - |
L-glutamate | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.0861 | - |
ATP | pH 7.5, 25°C, mutant R167K | Synechocystis sp. | |
0.127 | - |
L-cysteine | pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.16 | - |
L-glutamate | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
0.317 | - |
ATP | below, pH 7.5, 25°C, mutant H121A | Synechocystis sp. | |
0.367 | - |
L-glutamate | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
0.639 | - |
ATP | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
0.717 | - |
L-cysteine | pH 7.5, 25°C, mutant T117S | Synechocystis sp. | |
1.37 | - |
ATP | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
2.89 | - |
L-cysteine | pH 7.5, 25°C, mutant H121Q | Synechocystis sp. | |
3.52 | - |
ATP | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. | |
7.12 | - |
L-cysteine | pH 7.5, 25°C, wild-type enzyme | Synechocystis sp. |