Cloned (Comment) | Organism |
---|---|
gene gshA, phylogenetic tree analysis of GCS lineage I, expression of C-terminally His-tagged wild-type and mutants in Escherichia coli strain BL21(DE3) or Origami from pTEF416, subcloning in Escherichia coli strain DH5alpha, complementation of Saccharomyces cerevisiae gsh deficient strain ABC1195 | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
C 164S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S | site-directed mutagenesis, inactive mutant | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395Y | site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q | site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195, the mutant enzyme lacking cysteine residues shows a decreased in vivo half-life | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395W | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372F/C395S | site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372F/S395C | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372W/S395C | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/V375F | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C372S/C433S/C439S | site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C357S/C433S/C439S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C106S/C164S/C205S/C223S/C433S/C439S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C205S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C223S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
C433S/C439S | site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195 | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0001 | - |
L-cysteine | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
0.0001 | - |
ATP | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
0.0001 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
0.00011 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
0.00011 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
0.00014 | - |
ATP | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
0.00014 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
0.00014 | - |
L-cysteine | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
0.00016 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
0.00019 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
0.00024 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
0.00025 | - |
ATP | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
0.00046 | - |
ATP | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
0.00053 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
0.0032 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
0.0039 | - |
L-glutamate | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
0.004 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
0.004 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
0.0041 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
0.0051 | - |
L-glutamate | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
0.0053 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + L-cysteine | Escherichia coli | - |
ADP + phosphate + gamma-L-glutamyl-L-cysteine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
gene gshA | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + L-cysteine | - |
Escherichia coli | ADP + phosphate + gamma-L-glutamyl-L-cysteine | - |
? |
Synonyms | Comment | Organism |
---|---|---|
gamma -GCS | - |
Escherichia coli |
gamma-Glutamylcysteine synthetase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
21.9 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
22.1 | - |
L-glutamate | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
22.2 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
22.7 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
23.8 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
24.2 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
24.2 | - |
L-cysteine | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
27.2 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
28.7 | - |
ATP | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
29.2 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
30.1 | - |
ATP | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
30.4 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
30.5 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
31.2 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
31.4 | - |
L-glutamate | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
31.8 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
34 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
39.7 | - |
ATP | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
39.7 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
46.6 | - |
L-cysteine | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
46.9 | - |
ATP | pH 8.0, 25°C, wild-type enzyme | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | in eukaryotes gamma -glutamylcysteine synthetase and glutathione synthetase, EC 6.3.2.3, activities are encoded by two distinct enzymes In some prokaryotes, such as Escherichia coli and Vibrio cholerae, separate enzymes exist for these two reactions. However, in some prokaryotes, such as Streptococcus agalactiae, Pasteurella multicoda and Listeria monocytogenes, both of these activities are encoded by a single bifunctional enzyme, GshF. Evolution of gamma-GCS has occurred by convergent evolution in three different lineages with no significant sequence similarities between the lineages, the Escherichia coli enzyme belongs to lineage I | Escherichia coli |
metabolism | biosynthesis of GSH occurs by two sequential ATP-dependent enzymatic steps. The first enzyme, gamma -glutamylcysteine synthetase ligates glutamate and cysteine to yield gamma -glutamylcysteine. Glutathione synthetase, EC 6.3.2.3, the second enzyme, then catalyses the addition of glycine to yield glutathione | Escherichia coli |
physiological function | gamma-GCS is rate-limiting catalyzing the regulated step of GSH biosynthesis, being both transcriptionally and post-translationally regulated, post-translational regulation of the gamma-GCS enzyme by the redox environment | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
4.3 | - |
L-glutamate | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
4.5 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
6.9 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
7.4 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
7.4 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
8.1 | - |
L-glutamate | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
8.5 | - |
L-glutamate | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
46.6 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
62.4 | - |
ATP | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
120.4 | - |
ATP | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
127.1 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
142.9 | - |
ATP | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
172.9 | - |
L-cysteine | pH 8.0, 25°C, wild-type enzyme, in presence of DTT | Escherichia coli | |
198.8 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W | Escherichia coli | |
199.1 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y | Escherichia coli | |
222 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S, in presence of DTT | Escherichia coli | |
288.6 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F | Escherichia coli | |
335 | - |
ATP | pH 8.0, 25°C, wild-type enzyme | Escherichia coli | |
396.9 | - |
L-cysteine | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
397 | - |
ATP | pH 8.0, 25°C, mutant C106S,C164S,C205S,C223S,C357S,C433S,C439S | Escherichia coli | |
466 | - |
L-cysteine | pH 8.0, 25°C, wild-type enzyme | Escherichia coli |