Literature summary for 6.3.2.3 extracted from

De Jesus, M.C.; Ingle, B.L.; Barakat, K.A.; Shrestha, B.; Slavens, K.D.; Cundari, T.R.; Anderson, M.E.
The role of strong electrostatic interactions at the dimer interface of human glutathione synthetase (2014), Protein J., 33, 403-409 .

Search for more literature for 6.3.2.3

Cloned(Commentary)

Cloned (Comment)	Organism
gene GSS, sequence comparisons, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain XL1 Blue	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
analysis of the dimeric enzyme, PDB ID 2HGS	Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D24A	site-directed mutagenesis, the mutant shows a slight increase in catalytic efficiency compared to wild-type enzyme	Homo sapiens
additional information	structure and stability comparisons of wild-type enzyme and mutant enzymes, overview	Homo sapiens
R221A	site-directed mutagenesis, the mutant shows a slight increase in catalytic efficiency compared to wild-type enzyme, the R221A mutation also has a large impact on the intrachain bonding structure	Homo sapiens
S42A	site-directed mutagenesis, the mutant shows a slight increase in catalytic efficiency compared to wild-type enzyme	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + gamma-L-glutamyl-L-cysteine + glycine	Homo sapiens	-	ADP + phosphate + glutathione	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P48637	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Homo sapiens

Storage Stability

Storage Stability	Organism
4°C, when stored in 20 mM Tris-Cl and 1 mM EDTA, pH 8.6, in sterile cryogenic tubes at 4°C, the recombinant hGS dimer interface mutant enzymes lose activity in the 30 hours after purification. Both R221A and D24A lose activity in a biphasic manner within a few hours: D24A loses 30% of activity in 4 hours, while the activity of R221A decreases by 20% in 7.5 hours	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + gamma-L-glutamyl-L-cysteine + glycine	-	Homo sapiens	ADP + phosphate + glutathione	-	?
ATP + L-gamma-glutamyl-L-alpha-aminobutyrate + glycine	gamma-GluABA, an analogue of gamma-L-glutamyl-L-cysteine (gamma-GC) with the same activity and kinetic properties as gamma-GC	Homo sapiens	ADP + L-gamma-glutamyl-L-alpha-aminobutyryl-glycine	-	?

Subunits

Subunits	Comment	Organism
homodimer	the two active sites of hGS, which are 40 A apart, display allosteric modulation by the substrate gamma-glutamylcysteine (gamma-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between residues S42, R221, and D24. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme. hGS residues S42, R221 and D24 have hydrogen bonding and ionic interactions across the dimer interface decrease activity, maintain negative cooperativity, increase L-gamma-glutamyl-L-alpha-aminobutyrate affinity, and increase catalytic efficiency when mutated to alanine	Homo sapiens

Synonyms

Synonyms	Comment	Organism
GSS	-	Homo sapiens
hGS	-	Homo sapiens

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
39.3	-	mutant D24A enzyme,unfolding or transition midpoint (Tm)	Homo sapiens
42.5	-	mutant R221A enzyme, unfolding or transition midpoint (Tm)	Homo sapiens
49.7	-	mutant S42A enzyme, unfolding or transition midpoint (Tm)	Homo sapiens
60.3	-	wild-type enzyme unfolding or transition midpoint (Tm)	Homo sapiens

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Homo sapiens

General Information

General Information	Comment	Organism
malfunction	patients with genetic deficiencies in hGS suffer from a variety of symptoms, most notably hemolytic anemia and neurological disorders. Deficiencies of GSH are associated with a variety of diseases including Parkinson's disease, Alzheimer's disease, Lou Gehrig's disease, diabetes, cystic fibrosis, and HIV/AIDS. In the R221A mutant, the interchain salt bridge and hydrogen bonds between R221 and D24 are broken, and a new salt bridge forms between R34 and D24	Homo sapiens
additional information	the obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein-protein interactions in the structural stability, activity and allostery of enzymes. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between residues S42, R221, and D24, these residues are crucial to function of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme, molecular dynamics simulations, overview. Since D24 participates in two significant interactions (a salt bridge with R221 and an ionic hydrogen bond with S42), this residue plays the largest role in hGS activity and stability. Structure and stability comparisons of wild-type enzyme and mutant enzymes, overview	Homo sapiens
physiological function	regulation of hGS plays a critical role in maintaining the cellular glutathione levels required to relieve oxidative stress	Homo sapiens

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Release 2023.1 (January 2023)