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Literature summary 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

Cloned(Commentary)

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

Protein Variants

EC Number Protein Variants Comment Organism
6.3.2.2 E100A site-directed mutagenesis, n1 metal binding site mutant, inactive mutant Trypanosoma brucei
6.3.2.2 E489A site-directed mutagenesis, n2 metal binding site mutant, reduced activity Trypanosoma brucei
6.3.2.2 E53A site-directed mutagenesis, n2 metal binding site mutant, reduced activity Trypanosoma brucei
6.3.2.2 E55A site-directed mutagenesis, n1 metal binding site mutant, inactive mutant Trypanosoma brucei
6.3.2.2 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
6.3.2.2 Q321A site-directed mutagenesis, n2 metal binding site mutant, reduced activity Trypanosoma brucei

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.2.2 additional information
-
 additional information kinetics Trypanosoma brucei
6.3.2.2 0.022
-
 ATP wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 0.071
-
 ATP wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 0.1
-
 ATP mutant E489A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 0.24
-
 L-glutamate wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 0.32
-
 ATP mutant E93A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 0.89
-
 L-glutamate mutant E489A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 1
-
 L-glutamate wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 1.1
-
 L-glutamate mutant E489A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 1.1
-
 L-glutamate mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 1.2
-
 ATP mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 1.6
-
 L-glutamate mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 2
-
 ATP mutant E489A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 6
-
 L-alpha-aminobutyrate wild-type enzyme, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 9.4
-
 L-alpha-aminobutyrate mutant E489A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 10
-
 L-alpha-aminobutyrate wild-type enzyme, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 11
-
 L-glutamate mutant E93A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 14
-
 L-alpha-aminobutyrate mutant Q321A, pH 8.0, 37°C, in presence of Mn2+ Trypanosoma brucei
6.3.2.2 15
-
 L-alpha-aminobutyrate mutant E489A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 18
-
 L-alpha-aminobutyrate mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei
6.3.2.2 45
-
 ATP above, mutant Q321A, pH 8.0, 37°C, in presence of Mg2+ Trypanosoma brucei

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.2.2 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
6.3.2.2 Mn2+ can partly substitute for Mg2+ Trypanosoma brucei
6.3.2.2 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)

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

Organism

EC Number Organism UniProt Comment Textmining
6.3.2.2 Trypanosoma brucei
-
-
-

Purification (Commentary)

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

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 via phosphorylated glutamate intermediate Trypanosoma brucei
a

Substrates and Products (Substrate)

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

Subunits

EC Number Subunits Comment Organism
6.3.2.2 More structure modeling Trypanosoma brucei

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.2.2 37
-
 assay at Trypanosoma brucei

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.3.2.2 0.0053
-
 ATP mutant E489A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.0066
-
 Mg2+ mutant E489A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.066
-
 ATP mutant E93A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.078
-
 Mg2+ mutant E93A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.08
-
 Mg2+ mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.089
-
 Mn2+ mutant E93A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.24
-
 ATP above, mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.89
-
 ATP mutant E489A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.89
-
 Mn2+ mutant E489A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.5
-
 ATP mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.5
-
 Mn2+ mutant Q321A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 2.4
-
 ATP wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 2.5
-
 Mn2+ wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 3.8
-
 ATP wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 3.8
-
 Mg2+ wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 6.08
-
 ATP mutant E489A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 6.08
-
 Mn2+ mutant E489A, pH 8.0, 37°C Trypanosoma brucei

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3.2.2 8
-
 assay at Trypanosoma brucei

Cofactor

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