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Literature summary for 6.3.2.3 extracted from

  • Dinescu, A.; Cundari, T.R.; Bhansali, V.S.; Luo, J.L.; Anderson, M.E.
    Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes (2004), J. Biol. Chem., 279, 22412-22421.
    View publication on PubMed
Search for more literature for 6.3.2.3

Cloned(Commentary)

Cloned (Comment) Organism
expression of N-terminally His-tagged wild-type enzyme in Escherichia coli BL21(DE3) Homo sapiens

Crystallization (Commentary)

Crystallization (Comment) Organism
computational structure modeling of wild-type and mutant enzymes using crystal structure data at 2.1 A resolution, interactions of actie site residues Glu144, Asn146, Lys305, and Lys364, and ligands ADP2-, SO42-, and Mg2+ Homo sapiens

Protein Variants

Protein Variants Comment Organism
E144A site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure Homo sapiens
E144K site-directed mutagenesis, inactive mutant, unaltered tertiary structure Homo sapiens
K305A site-directed mutagenesis, 6.5% activity compared to the wild-type enzyme, 7fold increased Km for glycine, loss of negative cooperativity, 105fold increased Km for ATP, unaltered tertiary structure Homo sapiens
K305E site-directed mutagenesis, 5% activity compared to the wild-type enzyme, loss of negative cooperativity, 40fold increased Km for ATP, unaltered tertiary structure Homo sapiens
K364A site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure Homo sapiens
K364E site-directed mutagenesis, 0.2% activity compared to the wild-type enzyme, unaltered tertiary structure Homo sapiens
N146A site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure Homo sapiens
N146D site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure Homo sapiens
N146K site-directed mutagenesis, inactive mutant, unaltered tertiary structure Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics, substrate and ligand binding, wild-type enzyme and mutants Homo sapiens
0.07
-
 ATP wild-type enzyme, pH 8.2, 37°C Homo sapiens
0.25
-
 glycine mutant K305A, pH 8.2, 37°C Homo sapiens
0.34
-
 gamma-L-Glu-L-Cys mutant K305A, pH 8.2, 37°C Homo sapiens
0.4
-
 gamma-L-Glu-L-Cys mutant K305E, pH 8.2, 37°C Homo sapiens
0.66
-
 gamma-L-Glu-L-Cys wild-type enzyme, pH 8.2, 37°C Homo sapiens
0.83
-
 ATP mutant K305A, pH 8.2, 37°C Homo sapiens
1.75
-
 glycine wild-type enzyme, pH 8.2, 37°C Homo sapiens
1.96
-
 glycine mutant K305E, pH 8.2, 37°C Homo sapiens
2.66
-
 ATP mutant K305E, pH 8.2, 37°C Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ 2 ions bound per enzyme molecule, binding structure and kinetics Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + gamma-L-Glu-L-Cys + Gly Homo sapiens second step in the biosynthesis of glutahione ADP + phosphate + glutathione
-
 ?

Organism

Organism UniProt Comment Textmining
Homo sapiens P48637
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type enzyme from Escherichia coli, to homogeneity Homo sapiens

Reaction

Reaction Comment Organism Reaction ID
ATP + gamma-L-glutamyl-L-cysteine + glycine = ADP + phosphate + glutathione mechanism, active site residues are Glu144, Asn146, Lys305, and Lys364, interaction of these residues with the ligands is essential for enzyme activity, especially Glu144 seems to be very important for stabilization of the reaction intermediate, but the active site residues are not essential for the overall enzyme structure Homo sapiens
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + gamma-L-Glu-L-Cys + Gly wild-type enzyme shows negative cooperativity Homo sapiens ADP + phosphate + glutathione
-
 ?
ATP + gamma-L-Glu-L-Cys + Gly second step in the biosynthesis of glutahione Homo sapiens ADP + phosphate + glutathione
-
 ?

Subunits

Subunits Comment Organism
More active site residues are Glu144, Asn146, Lys305, and Lys364, interaction of these residues with the ligands is essential for enzyme activity, especially with Glu144, but they are not essential for the overall enzyme structure Homo sapiens

Synonyms

Synonyms Comment Organism
Glutathione synthetase
-
 Homo sapiens
GS
-
 Homo sapiens
More the enzyme belongs to the glutathione synthetase ATP-binding domain-like superfamily Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.003
-
 gamma-L-Glu-L-Cys mutant E144K and mutant N146D, pH 8.2, 37°C Homo sapiens
0.007
-
 gamma-L-Glu-L-Cys mutant N146A, pH 8.2, 37°C Homo sapiens
0.008
-
 gamma-L-Glu-L-Cys mutant K364A, pH 8.2, 37°C Homo sapiens
0.015
-
 gamma-L-Glu-L-Cys mutant K364E, pH 8.2, 37°C Homo sapiens
0.336
-
 gamma-L-Glu-L-Cys mutant K305E, pH 8.2, 37°C Homo sapiens
0.423
-
 gamma-L-Glu-L-Cys mutant K305A, pH 8.2, 37°C Homo sapiens
6.5
-
 gamma-L-Glu-L-Cys wild-type enzyme, pH 8.2, 37°C Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.2
-
 assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
ATP dependent on Homo sapiens