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Literature summary for 6.3.2.2 extracted from

  • Kumar, S.; Kasturia, N.; Sharma, A.; Datt, M.; Bachhawat, A.K.
    Redox-dependent stability of the gamma-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation (2013), Biochem. J., 449, 783-794.
    View publication on PubMed

Cloned(Commentary)

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Escherichia coli
-
gene gshA
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-glutamate + L-cysteine
-
Escherichia coli ADP + phosphate + gamma-L-glutamyl-L-cysteine
-
?

Synonyms

Synonyms Comment Organism
gamma -GCS
-
Escherichia coli
gamma-Glutamylcysteine synthetase
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Escherichia coli

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
Escherichia coli

General Information

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 [mM/s]

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