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Literature summary for 6.3.2.2 extracted from

  • Abbott, J.J.; Pei, J.; Ford, J.L.; Qi, Y.; Grishin, V.N.; Pitcher, L.A.; Phillips, M.A.; Grishin, N.V.
    Structure prediction and active site analysis of the metal binding determinants in gamma-glutamylcysteine synthetase (2001), J. Biol. Chem., 276, 42099-42107.
    View publication on PubMed
Search for more literature for 6.3.2.2

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutants in Escherichia coli BL21(DE3) as C-terminally His-tagged proteins Trypanosoma brucei

Protein Variants

Protein Variants Comment Organism
E100A site-directed mutagenesis, n1 metal binding site mutant, inactive mutant Trypanosoma brucei
E489A site-directed mutagenesis, n2 metal binding site mutant, reduced activity Trypanosoma brucei
E53A site-directed mutagenesis, n2 metal binding site mutant, reduced activity Trypanosoma brucei
E55A site-directed mutagenesis, n1 metal binding site mutant, inactive mutant Trypanosoma brucei
E93A site-directed mutagenesis, n1 metal binding site mutant, only capable of catalyzing L-Glu-dependent ATP hydrolysis and not the ligation between L-Glu and L-alpha-aminobutyrate Trypanosoma brucei
Q321A site-directed mutagenesis, n2 metal binding site mutant, reduced activity Trypanosoma brucei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics Trypanosoma brucei
0.022
-
 ATP wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
0.071
-
 ATP wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
0.1
-
 ATP mutant E489A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
0.24
-
 L-glutamate wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
0.32
-
 ATP mutant E93A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
0.89
-
 L-glutamate mutant E489A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
1
-
 L-glutamate wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
1.1
-
 L-glutamate mutant E489A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
1.1
-
 L-glutamate mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
1.2
-
 ATP mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
1.6
-
 L-glutamate mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
2
-
 ATP mutant E489A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6
-
 L-alpha-aminobutyrate wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
9.4
-
 L-alpha-aminobutyrate mutant E489A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
10
-
 L-alpha-aminobutyrate wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
11
-
 L-glutamate mutant E93A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
14
-
 L-alpha-aminobutyrate mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
15
-
 L-alpha-aminobutyrate mutant E489A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
18
-
 L-alpha-aminobutyrate mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
45
-
 ATP above, mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required, bound as MgATP2-, the metal ion specificity is determined by the second binding site n2 which also is involved in ATP binding, located in the active site and formed by 3 conserved residues Glu53, Gln321, and Glu489, Mg2+ can partly be substituted by Mn2+ Trypanosoma brucei
Mn2+ can partly substitute for Mg2+ Trypanosoma brucei
additional information the first metal binding site n1 binds free metal ions and is composed of 3 conserved residues Glu55, Glu93, and Glu100, n1 also is involved in positioning of L-glutamate for the reaction, located in the active site Trypanosoma brucei

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-Glu + L-Cys Trypanosoma brucei rate limiting and first step in glutathione biosynthesis ADP + phosphate + gamma-L-Glu-L-Cys
-
 ir

Organism

Organism UniProt Comment Textmining
Trypanosoma brucei
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli Trypanosoma brucei

Reaction

Reaction Comment Organism Reaction ID
ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine reaction mechanism via phosphorylated glutamate intermediate Trypanosoma brucei
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-Glu + L-alpha-aminobutyrate
-
 Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate
-
 ir
ATP + L-Glu + L-Cys
-
 Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-Cys
-
 ?
ATP + L-Glu + L-Cys rate limiting and first step in glutathione biosynthesis Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-Cys
-
 ir
ATP + L-glutamate + L-cysteine ATP in form of MnATP2- Trypanosoma brucei ADP + phosphate + gamma-L-glutamyl-L-cysteine
-
 ?
ATP + L-glutamate + L-cysteine ATP in form of MgATP2- Trypanosoma brucei ADP + phosphate + gamma-L-glutamyl-L-cysteine
-
 ?

Subunits

Subunits Comment Organism
More structure modeling Trypanosoma brucei

Synonyms

Synonyms Comment Organism
gamma-GCS
-
 Trypanosoma brucei
gamma-Glutamylcysteine synthetase
-
 Trypanosoma brucei
More enzyme belongs to the superfamily of carboxylate-amine/ammonia ligases, together with glutathione synthase Trypanosoma brucei

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Trypanosoma brucei

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0053
-
 ATP mutant E489A, pH 8.0, 37°C Trypanosoma brucei
0.0066
-
 Mg2+ mutant E489A, pH 8.0, 37°C Trypanosoma brucei
0.066
-
 ATP mutant E93A, pH 8.0, 37°C Trypanosoma brucei
0.078
-
 Mg2+ mutant E93A, pH 8.0, 37°C Trypanosoma brucei
0.08
-
 Mg2+ mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
0.089
-
 Mn2+ mutant E93A, pH 8.0, 37°C Trypanosoma brucei
0.24
-
 ATP above, mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
0.89
-
 ATP mutant E489A, pH 8.0, 37°C Trypanosoma brucei
0.89
-
 Mn2+ mutant E489A, pH 8.0, 37°C Trypanosoma brucei
1.5
-
 ATP mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
1.5
-
 Mn2+ mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
2.4
-
 ATP wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
2.5
-
 Mn2+ wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
3.8
-
 ATP wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
3.8
-
 Mg2+ wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.08
-
 ATP mutant E489A, pH 8.0, 37°C Trypanosoma brucei
6.08
-
 Mn2+ mutant E489A, pH 8.0, 37°C Trypanosoma brucei

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Trypanosoma brucei

Cofactor

Cofactor Comment Organism Structure
ATP associated with Mg2+ binding at the second n2 metal binding site Trypanosoma brucei