Information on EC 6.3.2.3 - glutathione synthase and Organism(s) Homo sapiens

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria


The taxonomic range for the selected organisms is: Homo sapiens

EC NUMBER
COMMENTARY hide
6.3.2.3
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RECOMMENDED NAME
GeneOntology No.
glutathione synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + gamma-L-glutamyl-L-cysteine + glycine = ADP + phosphate + glutathione
show the reaction diagram
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
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxamide formation
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carboxylic acid-amide formation
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
glutathione biosynthesis
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glutathione metabolism
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Cysteine and methionine metabolism
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Glutathione metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming)
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CAS REGISTRY NUMBER
COMMENTARY hide
9023-62-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop, Asp458 is important for cooperativity and active site structure, it impacts the allostery of the enzyme. Asp458 is important for loop closure and is a critical residue within the enzyme's A-loop. Enzyme structure-function modeling, molecular dynamics, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + gamma-Glu-L-Cys + Gly
?
show the reaction diagram
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ATP + gamma-Glu-L-Cys + Gly
ADP + phosphate + glutathione
show the reaction diagram
ATP + gamma-glutamyl-alpha-aminobutyrate
?
show the reaction diagram
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?
ATP + gamma-L-Glu-aminobutanoate + Gly
ADP + phosphate + ophthalmic acid
show the reaction diagram
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?
ATP + gamma-L-Glu-L-Cys + Gly
ADP + phosphate + glutathione
show the reaction diagram
ATP + gamma-L-glutamyl-L-cysteine + glycine
ADP + phosphate + glutathione
show the reaction diagram
glycine + ATP + gamma-L-glutamyl-L-cysteine
ADP + phosphate + glutathione
show the reaction diagram
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assay at pH 8.2
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?
additional information
?
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dysregulation of GSH synthesis occurs in aging and is increasingly being recognized as contributing to the pathogenesis of many pathological conditions including diabetes mellitus, pulmonary fibrosis, cholestatic liver injury, endotoxemia and drug-resistant tumor cells, detailed overview. Decrease in the activity of GS alone without a change in glutathione cysteine ligase, GCL EC 6.3.2.2, and a fall in muscle GSH levels occur after surgical trauma in human skeletal muscle
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + gamma-Glu-L-Cys + Gly
?
show the reaction diagram
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ATP + gamma-Glu-L-Cys + Gly
ADP + phosphate + glutathione
show the reaction diagram
ATP + gamma-L-Glu-L-Cys + Gly
ADP + phosphate + glutathione
show the reaction diagram
ATP + gamma-L-glutamyl-L-cysteine + glycine
ADP + phosphate + glutathione
show the reaction diagram
additional information
?
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dysregulation of GSH synthesis occurs in aging and is increasingly being recognized as contributing to the pathogenesis of many pathological conditions including diabetes mellitus, pulmonary fibrosis, cholestatic liver injury, endotoxemia and drug-resistant tumor cells, detailed overview. Decrease in the activity of GS alone without a change in glutathione cysteine ligase, GCL EC 6.3.2.2, and a fall in muscle GSH levels occur after surgical trauma in human skeletal muscle
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoate)
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iodoacetate
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NEM
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PCMB
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additional information
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decrease of enzyme activity in hypoxia: 20% after 6 h, 17% after 12 h, 23% after 24 h, hypoxia-induced decrease in enzyme activity may be prevented by MAPK inhibition and catalase
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Thioacetamide
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increases the enzyme expression and activity in Chang cells by 50% at 6.66 mM
additional information
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oxidative stress induces the enzyme, key transcription factors include Nrf2/Nrf1 via the antioxidant response element, ARE, activator protein-1, AP-1, and nuclear factor kappa B, NFkappaB. Nrf1 and Nrf2 overexpression induces the human GS promoter activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 2.66
ATP
0.5
gamma-Glu-2-aminobutyrate
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ATP
0.2
gamma-Glu-Cys
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0.063
gamma-glutamyl-alpha-aminobutyrate
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pH 8.2, 37C
0.1 - 1.6
gamma-L-Glu-aminobutanoate
0.34 - 0.66
gamma-L-Glu-L-Cys
0.1 - 2
gamma-L-glutamyl-L-cysteine
0.36 - 2.52
Gly
0.25 - 51
glycine
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
13.6 - 21.7
gamma-L-Glu-aminobutanoate
0.003 - 6.5
gamma-L-Glu-L-Cys
1.17 - 15.68
gamma-L-glutamyl-L-cysteine
0.052 - 18.8
Gly
1.17 - 15.68
glycine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.5
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7.3
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assay at
7.4 - 8
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52352
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2 * 52352, calculation from nucleotide sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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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
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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+
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
41.3
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melting temperature, mutant V45W
42.8
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melting temperature, mutant V44A/V45A
47.8
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melting temperature, mutant V45A
51.1
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melting temperature, mutant V44A
57
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melting temperature, mutant V44W
60.4
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melting temperature, wild-type
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme expressed in Escherichia coli, to homogeneity
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recombinant His-tagged wild-type enzyme from Escherichia coli, to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the enzyme promoter and molecular mechanisms of GS transcriptional regulation, overview. Nrf1 and Nrf2 overexpression induces the human GS promoter activity. Human GS promoter contains two regions with homology to the nuclear factor erythroid 2, NFE2, motif that are required for basal activity as mutation of these sites reduces the human GS promoter activity by 66%
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expression in Escherichia coli
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expression in Escherichia coli BL21(DE3)
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expression of N-terminally His-tagged wild-type enzyme in Escherichia coli BL21(DE3)
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gene gshB, expression of His-tagged wild-type and mutants
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seven naturally occurring missense mutations (L188P, D219A, D219G, Y270C, Y270H, R283C and P314L) are expressed using a His-tagged, Escherichia coli-based expression system
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
novel alternative splicing variant
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C294A
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mutant enzymes Cys294Ala and Cys409Ala retain significant residual activity. Substantial decreases in activity are detected with mutant Cys522Ala and Cys-free mutant Cys294/Cys409/Cys422 to Ala294/Ala409/Ala422
C409A
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mutant enzymes Cys294Ala and Cys409Ala retain significant residual activity. Substantial decreases in activity are detected with mutant Cys522Ala and Cys-free mutant Cys294/Cys409/Cys422 to Ala294/Ala409/Ala422
C522A
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mutant enzymes Cys294Ala and Cys409Ala retain significant residual activity. Substantial decreases in activity are detected with mutant Cys522Ala and Cys-free mutant Cys294/Cys409/Cys422 to Ala294/Ala409/Ala422
D219A
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naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 4% of the wild-type activity
D219G
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naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 27% of the wild-type activity. Negative cooperativity for L-gamma-glutamyl-L-alpha-aminobutyric acid is changed to positive
D458A
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site-directed mutagenesis, the mutant shows 10% activity compared to the wild-type enzyme
D458N
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site-directed mutagenesis, the mutant shows 15% activity compared to the wild-type enzyme
D458R
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site-directed mutagenesis, the mutant shows 7% activity compared to the wild-type enzyme
E144A
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site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure
E144K
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site-directed mutagenesis, inactive mutant, unaltered tertiary structure
G369V
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mutation in G-loop glycine triad, about 0.7% of wild-type activity. Mutation decreases ligand binding and prevent active site closure and protection
G370V
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mutation in G-loop glycine triad, about 0.3% of wild-type activity. Mutation decreases ligand binding and prevent active site closure and protection
G371V
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mutation in G-loop glycine triad, about 13% of wild-type activity
K305A
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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
K305E
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site-directed mutagenesis, 5% activity compared to the wild-type enzyme, loss of negative cooperativity, 40fold increased Km for ATP, unaltered tertiary structure
K364A
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site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure
K364E
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site-directed mutagenesis, 0.2% activity compared to the wild-type enzyme, unaltered tertiary structure
L188P
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naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 9% of the wild-type activity
N146A
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site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure
N146D
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site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure
N146K
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site-directed mutagenesis, inactive mutant, unaltered tertiary structure
P314L
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naturally occurring neutral mutation
R283C
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naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 13% of the wild-type activity
V44A
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decrease in melting temperature, slight decrease in activity
V44A/V45A
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decrease in melting temperature, initial activity similar to wild-type
V44W
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decrease in melting temperature, 16% decrease in activity
V45A
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decrease in melting temperature, slight decrease in activity
V45W
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decrease in melting temperature, 30% decrease in activity
Y208C
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naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 2% of the wild-type activity
Y270H
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naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 6% of the wild-type activity
additional information
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all Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type enzyme, differential scanning calorimetry
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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assay of glutathione synthetase in erythrocytes by HPLC with fluorimetric detection, useful for rapid screening of erythrocytes glutathione synthetase activity in various pathological conditions
medicine
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the enzyme is a target for therapy of disease like diabetes mellitus, pulmonary fibrosis, cholestatic liver injury, endotoxemia and drug-resistant tumor cells, since manipulation of the GSH synthetic capacity is beneficial in treatment of many of these disorders