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Literature summary for 6.3.2.2 extracted from

  • Musgrave, W.B.; Yi, H.; Kline, D.; Cameron, J.C.; Wignes, J.; Dey, S.; Pakrasi, H.B.; Jez, J.M.
    Probing the origins of glutathione biosynthesis through biochemical analysis of glutamate-cysteine ligase and glutathione synthetase from a model photosynthetic prokaryote (2013), Biochem. J., 450, 63-72.
    View publication on PubMed
Search for more literature for 6.3.2.2

Cloned(Commentary)

Cloned (Comment) Organism
gene gshA, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Synechocystis sp.

Protein Variants

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

Inhibitors Comment Organism Structure
buthionine sulfoxime
-
 Synechocystis sp.
glutathione substrate inhibition Synechocystis sp.

KM Value [mM]

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

Metals/Ions Comment Organism Structure
Mg2+ required Synechocystis sp.

Molecular Weight [Da]

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Synechocystis sp. P74515 gene gshA or slr0990
-

Purification (Commentary)

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

Reaction Comment Organism Reaction ID
ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine reaction mechanism, overview Synechocystis sp.
a

Substrates and Products (Substrate)

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

Subunits

Subunits Comment Organism
monomer 1 * 46000, recombinant enzyme, SDS-PAGE Synechocystis sp.

Synonyms

Synonyms Comment Organism
GCL
-
 Synechocystis sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Synechocystis sp.

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Synechocystis sp.

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Synechocystis sp.

IC50 Value

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

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 [mM/s]

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.