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Literature summary extracted from

  • Abbott, J.J.; Ford, J.L.; Phillips, M.A.
    Substrate binding determinants of Trypanosoma brucei gamma-glutamylcysteine synthetase (2002), Biochemistry, 41, 2741-2750.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.3.2.2 expression as C-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3) Trypanosoma brucei

Protein Variants

EC Number Protein Variants Comment Organism
6.3.2.2 R179A site-directed PCR-based mutagenesis, conserved residue, gamma-aminobutyrate-binding determinant, increase of Km for both L-Cys and L-Glu Trypanosoma brucei
6.3.2.2 R366A site-directed PCR-based mutagenesis, mutant is active with gamma-aminobutyrate, increase of dissociation constant for L-Glu by 160fold, elimination of positive cooperativity of binding of L-Glu and ATP, interacts with the alpha-carboxylate of L-Glu, 220fold increase in Ki for GSH Trypanosoma brucei
6.3.2.2 R474A site-directed PCR-based mutagenesis, R474 is an ATP binding determinant, increase of Km for ATP by 20-100fold Trypanosoma brucei
6.3.2.2 R487A site-directed PCR-based mutagenesis, R487 is an ATP binding determinant, increase of Km for ATP by 20-100fold Trypanosoma brucei
6.3.2.2 R491A site-directed PCR-based mutagenesis, decrease of kcat for ATP hydrolysis Trypanosoma brucei
6.3.2.2 T323A site-directed PCR-based mutagenesis, T323 is an alpha-phosphate of ATP binding determinant, increase of Km for ATP by 20-100fold Trypanosoma brucei

Inhibitors

EC Number Inhibitors Comment Organism Structure
6.3.2.2 ATP substrate inhibition of mutant R179A, when only one other substrate is saturating Trypanosoma brucei
6.3.2.2 GSH competitive Trypanosoma brucei
6.3.2.2 L-alpha-aminobutyrate substrate inhibition of mutant R179A, when only one other substrate is saturating Trypanosoma brucei
6.3.2.2 L-buthionine-S-sulfoximine irrversible inactivation, no inhibition of mutant R366A 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 for the wild-type and mutant enzymes Trypanosoma brucei
6.3.2.2 0.071
-
 ATP wild-type, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.11
-
 ATP mutant R491A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.14
-
 ATP mutant R179A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.24
-
 L-glutamate wild-type, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.61
-
 L-glutamate mutant R491A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.2
-
 ATP mutant R487A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.3
-
 ATP mutant R474A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.3
-
 L-glutamate mutant R487A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.6
-
 L-glutamate mutant R474A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.7
-
 L-glutamate mutant T323A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 2.8
-
 ATP mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 5.2
-
 L-glutamate mutant R179A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 7
-
 ATP mutant T323A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 7.5
-
 L-alpha-aminobutyrate mutant T323A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 10
-
 L-alpha-aminobutyrate wild-type, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 15
-
 L-alpha-aminobutyrate mutant R487A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 20
-
 4-aminobutyrate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 34
-
 L-alpha-aminobutyrate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 56
-
 L-alpha-aminobutyrate mutant R474A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 130
-
 L-glutamate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 150
-
 4-aminobutyrate wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 380
-
 L-alpha-aminobutyrate mutant R179A, pH 8.0, 37°C Trypanosoma brucei

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.2.2 Mg2+ Mg2+ or Mn2+ are required Trypanosoma brucei
6.3.2.2 Mn2+ Mg2+ or Mn2+ are required 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 first step in the de novo biosynthesis of the tripeptide glutathione ADP + phosphate + gamma-L-Glu-L-Cys
-
 r

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 rapid equilibrium random ter-reactant mechanism, substrate binding modeling, active site model 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 + gamma-aminobutyrate mutant R366A Trypanosoma brucei ADP + phosphate + gamma-L-Glu-gamma-aminobutyrate
-
 r
6.3.2.2 ATP + L-Glu + L-alpha-aminobutyrate
-
 Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate
-
 r
6.3.2.2 ATP + L-Glu + L-Cys
-
 Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-Cys
-
 r
6.3.2.2 ATP + L-Glu + L-Cys first step in the de novo biosynthesis of the tripeptide glutathione Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-Cys
-
 r

Subunits

EC Number Subunits Comment Organism
6.3.2.2 monomer
-
 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

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 0.055
-
 L-glutamate mutant R491A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.14
-
 4-aminobutyrate wild-type, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.24
-
 L-glutamate mutant R474A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.32
-
 L-glutamate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.59
-
 4-aminobutyrate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.61
-
 L-glutamate mutant R179A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 0.79
-
 L-glutamate mutant T323A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 1.2
-
 L-glutamate mutant R487A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 3.9
-
 L-glutamate wild-type, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 6.08
-
 L-glutamate mutant R179A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 6.08
-
 4-aminobutyrate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 6.08
-
 L-glutamate mutant T323A, 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 dependent on Trypanosoma brucei

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
6.3.2.2 1.1
-
 GSH wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
6.3.2.2 36
-
 GSH mutant R179A, pH 8.0, 37°C Trypanosoma brucei