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Literature summary 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

Cloned(Commentary)

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

Protein Variants

EC Number Protein Variants Comment Organism
6.3.2.2 E37Q site-directed mutagenesis, inactive mutant Synechocystis sp.
6.3.2.2 E44Q site-directed mutagenesis, inactive mutant Synechocystis sp.
6.3.2.2 H121A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme Synechocystis sp.
6.3.2.2 H121Q site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Synechocystis sp.
6.3.2.2 R167A site-directed mutagenesis, inactive mutant Synechocystis sp.
6.3.2.2 R167K site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Synechocystis sp.
6.3.2.2 R248A site-directed mutagenesis, inactive mutant Synechocystis sp.
6.3.2.2 R248K site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Synechocystis sp.
6.3.2.2 T117A site-directed mutagenesis, inactive mutant Synechocystis sp.
6.3.2.2 T117S site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Synechocystis sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
6.3.2.2 buthionine sulfoxime
-
 Synechocystis sp.
6.3.2.2 glutathione substrate inhibition Synechocystis sp.
6.3.2.3 ADP product inhibition, competitive versus ATP, noncompetitive versus gamma-L-glutamyl-L-cysteine and glycine Synechocystis sp.
6.3.2.3 glutathione product inhibition, noncompetitive versus ATP, gamma-L-glutamyl-L-cysteine, and glycine Synechocystis sp.
6.3.2.3 phosphate product inhibition, noncompetitive versus ATP, gamma-L-glutamyl-L-cysteine, and glycine Synechocystis sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.2.2 additional information
-
 additional information steady-state kinetics, overview Synechocystis sp.
6.3.2.2 0.059
-
 L-cysteine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 0.116
-
 L-cysteine pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 0.145
-
 ATP pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 0.186
-
 L-cysteine pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.279
-
 ATP pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 0.31
-
 ATP pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.352
-
 ATP pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.445
-
 ATP pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.5
-
 L-glutamate pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.707
-
 L-cysteine pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.857
-
 L-cysteine pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.953
-
 L-glutamate pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 0.969
-
 ATP pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 1.18
-
 L-glutamate pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 1.95
-
 L-cysteine pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 3.76
-
 L-glutamate pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 6.1
-
 L-glutamate pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 15.2
-
 L-glutamate pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.3 additional information
-
 additional information steady-state kinetics and kinetic mechanism, overview Synechocystis sp.
6.3.2.3 0.099
-
 gamma-L-glutamyl-L-cysteine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 0.136
-
 ATP pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 0.407
-
 glycine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.2.2 Mg2+ required Synechocystis sp.
6.3.2.3 Mg2+ required Synechocystis sp.

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
6.3.2.2 45000
-
 recombinant enzyme, gel filtration Synechocystis sp.
6.3.2.2 46000
-
 1 * 46000, recombinant enzyme, SDS-PAGE Synechocystis sp.
6.3.2.3 38000
-
 4 * 38000, recombinant enzyme, SDS-PAGE Synechocystis sp.
6.3.2.3 150000
-
 recombinant enzyme, gel filtration Synechocystis sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.3.2.2 ATP + L-glutamate + L-cysteine Synechocystis sp.
-
 ADP + phosphate + gamma-L-glutamyl-L-cysteine
-
 ?
6.3.2.3 ATP + gamma-L-glutamyl-L-cysteine + glycine Synechocystis sp.
-
 ADP + phosphate + glutathione
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.3.2.2 Synechocystis sp. P74515 gene gshA or slr0990
-
6.3.2.3 Synechocystis sp. P73493 gene gshB or slr1238
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.3.2.2 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Synechocystis sp.
6.3.2.3 recombinant His-tagged wild-type and mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Synechocystis sp.

Reaction

EC Number Reaction Comment Organism Reaction ID
6.3.2.2 ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine reaction mechanism, overview Synechocystis sp.
a
6.3.2.3 ATP + gamma-L-glutamyl-L-cysteine + glycine = ADP + phosphate + glutathione random ter-reactant reaction mechanism, overview Synechocystis sp.
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.2.2 ATP + L-glutamate + L-cysteine
-
 Synechocystis sp. ADP + phosphate + gamma-L-glutamyl-L-cysteine
-
 ?
6.3.2.3 ATP + gamma-L-glutamyl-L-cysteine + glycine
-
 Synechocystis sp. ADP + phosphate + glutathione
-
 ?

Subunits

EC Number Subunits Comment Organism
6.3.2.2 monomer 1 * 46000, recombinant enzyme, SDS-PAGE Synechocystis sp.
6.3.2.3 tetramer 4 * 38000, recombinant enzyme, SDS-PAGE Synechocystis sp.

Synonyms

EC Number Synonyms Comment Organism
6.3.2.2 GCL
-
 Synechocystis sp.
6.3.2.3 GCL
-
 Synechocystis sp.
6.3.2.3 gshB
-
 Synechocystis sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.2.2 25
-
 assay at Synechocystis sp.
6.3.2.3 25
-
 assay at Synechocystis sp.

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.3.2.2 0.008
-
 ATP pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.008
-
 L-glutamate pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.01
-
 L-cysteine pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.025
-
 L-cysteine pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.038
-
 ATP pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.045
-
 L-glutamate pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.09
-
 L-cysteine pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.098
-
 ATP pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.127
-
 L-glutamate pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.133
-
 L-cysteine pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.137
-
 ATP pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 0.137
-
 L-glutamate pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.188
-
 L-glutamate pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 0.225
-
 ATP pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.335
-
 L-cysteine pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 0.35
-
 L-glutamate pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 0.382
-
 ATP pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 0.42
-
 L-cysteine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 4.22
-
 glycine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 4.62
-
 gamma-L-glutamyl-L-cysteine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 5.4
-
 ATP pH 7.5, 25°C, wild-type enzyme Synechocystis sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3.2.2 7.5
-
 assay at Synechocystis sp.
6.3.2.3 7.5
-
 assay at Synechocystis sp.

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.2.2 ATP
-
 Synechocystis sp.
6.3.2.3 ATP
-
 Synechocystis sp.

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
6.3.2.3 1.12
-
 ADP pH 7.5, 25°C, versus ATP Synechocystis sp.
6.3.2.3 1.41
-
 ADP pH 7.5, 25°C, versus gamma-L-glutamyl-L-cysteine Synechocystis sp.
6.3.2.3 2.19
-
 ADP pH 7.5, 25°C, versus glycine Synechocystis sp.
6.3.2.3 27.3
-
 phosphate pH 7.5, 25°C, versus gamma-L-glutamyl-L-cysteine Synechocystis sp.
6.3.2.3 29.9
-
 phosphate pH 7.5, 25°C, versus ATP Synechocystis sp.
6.3.2.3 38.6
-
 phosphate pH 7.5, 25°C, versus glycine Synechocystis sp.
6.3.2.3 42.8
-
 glutathione pH 7.5, 25°C, versus gamma-L-glutamyl-L-cysteine Synechocystis sp.
6.3.2.3 54
-
 glutathione pH 7.5, 25°C, versus ATP Synechocystis sp.
6.3.2.3 68.2
-
 glutathione pH 7.5, 25°C, versus glycine Synechocystis sp.

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
6.3.2.2 7.4
-
 pH 7.5, 25°C, recombinant enzyme Synechocystis sp. glutathione
6.3.2.2 33.7
-
 pH 7.5, 25°C, recombinant enzyme Synechocystis sp. buthionine sulfoxime

General Information

EC Number General Information Comment Organism
6.3.2.2 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.
6.3.2.2 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.
6.3.2.3 evolution Synechocystis glutathione synthase shares properties with other prokaryotic enzymes Synechocystis sp.
6.3.2.3 physiological function glutathione biosynthesis catalysed by glutamate-cysteine ligase, EC 6.3.2.2, and glutathione synthetase is essential for maintaining redox homoeostasis and protection against oxidative damage in diverse eukaroytes and bacteria Synechocystis sp.

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.3.2.2 0.0009
-
 L-glutamate pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.0022
-
 L-glutamate pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.0083
-
 L-glutamate pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.0086
-
 ATP pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.0117
-
 L-cysteine pH 7.5, 25°C, mutant R248K Synechocystis sp.
6.3.2.2 0.0128
-
 L-cysteine pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.0224
-
 L-glutamate pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.0861
-
 ATP pH 7.5, 25°C, mutant R167K Synechocystis sp.
6.3.2.2 0.127
-
 L-cysteine pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.16
-
 L-glutamate pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 0.317
-
 ATP below, pH 7.5, 25°C, mutant H121A Synechocystis sp.
6.3.2.2 0.367
-
 L-glutamate pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 0.639
-
 ATP pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 0.717
-
 L-cysteine pH 7.5, 25°C, mutant T117S Synechocystis sp.
6.3.2.2 1.37
-
 ATP pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 2.89
-
 L-cysteine pH 7.5, 25°C, mutant H121Q Synechocystis sp.
6.3.2.2 3.52
-
 ATP pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.2 7.12
-
 L-cysteine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 10.36
-
 glycine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 39.71
-
 ATP pH 7.5, 25°C, wild-type enzyme Synechocystis sp.
6.3.2.3 46.63
-
 gamma-L-glutamyl-L-cysteine pH 7.5, 25°C, wild-type enzyme Synechocystis sp.