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Literature summary for 6.3.2.2 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)

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

Protein Variants

Protein Variants Comment Organism
R179A site-directed PCR-based mutagenesis, conserved residue, gamma-aminobutyrate-binding determinant, increase of Km for both L-Cys and L-Glu Trypanosoma brucei
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
R474A site-directed PCR-based mutagenesis, R474 is an ATP binding determinant, increase of Km for ATP by 20-100fold Trypanosoma brucei
R487A site-directed PCR-based mutagenesis, R487 is an ATP binding determinant, increase of Km for ATP by 20-100fold Trypanosoma brucei
R491A site-directed PCR-based mutagenesis, decrease of kcat for ATP hydrolysis Trypanosoma brucei
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

Inhibitors Comment Organism Structure
ATP substrate inhibition of mutant R179A, when only one other substrate is saturating Trypanosoma brucei
GSH competitive Trypanosoma brucei
L-alpha-aminobutyrate substrate inhibition of mutant R179A, when only one other substrate is saturating Trypanosoma brucei
L-buthionine-S-sulfoximine irrversible inactivation, no inhibition of mutant R366A Trypanosoma brucei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics for the wild-type and mutant enzymes Trypanosoma brucei
0.071
-
ATP wild-type, pH 8.0, 37°C Trypanosoma brucei
0.11
-
ATP mutant R491A, pH 8.0, 37°C Trypanosoma brucei
0.14
-
ATP mutant R179A, pH 8.0, 37°C Trypanosoma brucei
0.24
-
L-glutamate wild-type, pH 8.0, 37°C Trypanosoma brucei
0.61
-
L-glutamate mutant R491A, pH 8.0, 37°C Trypanosoma brucei
1.2
-
ATP mutant R487A, pH 8.0, 37°C Trypanosoma brucei
1.3
-
ATP mutant R474A, pH 8.0, 37°C Trypanosoma brucei
1.3
-
L-glutamate mutant R487A, pH 8.0, 37°C Trypanosoma brucei
1.6
-
L-glutamate mutant R474A, pH 8.0, 37°C Trypanosoma brucei
1.7
-
L-glutamate mutant T323A, pH 8.0, 37°C Trypanosoma brucei
2.8
-
ATP mutant R366A, pH 8.0, 37°C Trypanosoma brucei
5.2
-
L-glutamate mutant R179A, pH 8.0, 37°C Trypanosoma brucei
7
-
ATP mutant T323A, pH 8.0, 37°C Trypanosoma brucei
7.5
-
L-alpha-aminobutyrate mutant T323A, pH 8.0, 37°C Trypanosoma brucei
10
-
L-alpha-aminobutyrate wild-type, pH 8.0, 37°C Trypanosoma brucei
15
-
L-alpha-aminobutyrate mutant R487A, pH 8.0, 37°C Trypanosoma brucei
20
-
4-aminobutyrate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
34
-
L-alpha-aminobutyrate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
56
-
L-alpha-aminobutyrate mutant R474A, pH 8.0, 37°C Trypanosoma brucei
130
-
L-glutamate mutant R366A, pH 8.0, 37°C Trypanosoma brucei
150
-
4-aminobutyrate wild-type enzyme, pH 8.0, 37°C Trypanosoma brucei
380
-
L-alpha-aminobutyrate mutant R179A, pH 8.0, 37°C Trypanosoma brucei

Metals/Ions

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

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 rapid equilibrium random ter-reactant mechanism, substrate binding modeling, active site model Trypanosoma brucei

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-Glu + gamma-aminobutyrate mutant R366A Trypanosoma brucei ADP + phosphate + gamma-L-Glu-gamma-aminobutyrate
-
r
ATP + L-Glu + L-alpha-aminobutyrate
-
Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate
-
r
ATP + L-Glu + L-Cys
-
Trypanosoma brucei ADP + phosphate + gamma-L-Glu-L-Cys
-
r
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

Subunits Comment Organism
monomer
-
Trypanosoma brucei

Synonyms

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

Ki Value [mM]

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