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Literature summary extracted from

  • Fraser, J.A.; Kansagra, P.; Kotecki, C.; Saunders, R.D.C.; McLellan, L.I.
    The modifier subunit of Drosophila glutamate-cysteine ligase regulates catalytic activity by covalent and noncovalent interactions and influences glutathione homeostasis in vivo (2003), J. Biol. Chem., 278, 46369-46377.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.3.2.2 expresion of His-tagged wild-type subunits GCLM and GCLC and expression of His-tagged mutants in Escherichia coli BL21(DE3) Drosophila melanogaster

Protein Variants

EC Number Protein Variants Comment Organism
6.3.2.2 C139S/C267S sequential site-directed mutagenesis, exchange of cysteine residues in the modifier subunit, the cysteine residues seem not to be involved in intersubunit disulfide bonding Drosophila melanogaster
6.3.2.2 C213S/C214S/C267S sequential site-directed mutagenesis, exchange of cysteine residues in the modifier subunit, which can still associate with the catalytic subunit, but no longer form intersubunit disulfides, the enzyme activity is reduced but still higher than the activity of the catalytic subunit alone, the mutant is more sensitive to inhibition by GSH, human modifier subunit can complement the defect, the mutant strain shows a reduced GSH level Drosophila melanogaster

Inhibitors

EC Number Inhibitors Comment Organism Structure
6.3.2.2 GSH feedback inhibition, the elimination of disulfide bridges between the subunits renders the enzyme more sensitive to inhibition Drosophila melanogaster

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.2.2 0.91
-
 L-glutamate 25°C, recombinant His-tagged wild-type enzyme Drosophila melanogaster
6.3.2.2 0.92
-
 L-glutamate 25°C, recombinant His-tagged wild-type catalytic subunit Drosophila melanogaster
6.3.2.2 0.97
-
 L-glutamate 25°C, recombinant His-tagged wild-type catalytic subunit with recombinant His-tagged mutant modifier subunit C213S/C214S/C267S Drosophila melanogaster

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
6.3.2.2 28000
-
 1 * 80000, about, catalytic subunit, + 1 * 28000, about, modifier subunit, SDS-PAGE Drosophila melanogaster
6.3.2.2 35000
-
 x * 35000, mutant enzyme modifier subunit, SDS-PAGE Drosophila melanogaster
6.3.2.2 80000
-
 1 * 80000, about, catalytic subunit, + 1 * 28000, about, modifier subunit, SDS-PAGE Drosophila melanogaster
6.3.2.2 140000
-
 holoenzyme, gel filtration Drosophila melanogaster

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 Drosophila melanogaster the enzyme has key influence on glutathione homeostasis ADP + phosphate + gamma-L-Glu-L-Cys
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.3.2.2 Drosophila melanogaster
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.3.2.2 recombinant HIs-tagged wild-type and mutant enzymes Drosophila melanogaster

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
6.3.2.2 additional information
-
-
 Drosophila melanogaster
6.3.2.2 8.39
-
 Drosophila catalytic subunit as a hybrid with the wild-type human modifier subunit, substrate L-alpha-aminobutyrate Drosophila melanogaster
6.3.2.2 8.45
-
 wild-type holoenzyme Drosophila melanogaster

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.2.2 ATP + L-Glu + L-Cys
-
 Drosophila melanogaster ADP + phosphate + gamma-L-Glu-L-Cys
-
 ?
6.3.2.2 ATP + L-Glu + L-Cys the enzyme has key influence on glutathione homeostasis Drosophila melanogaster ADP + phosphate + gamma-L-Glu-L-Cys
-
 ?

Subunits

EC Number Subunits Comment Organism
6.3.2.2 dimer 1 * 80000, about, catalytic subunit, + 1 * 28000, about, modifier subunit, SDS-PAGE Drosophila melanogaster
6.3.2.2 monomer x * 35000, mutant enzyme modifier subunit, SDS-PAGE Drosophila melanogaster
6.3.2.2 More enzyme is build of 2 subunits, a modifier and a catalytic subunit, the modifier subunit DmGCLM possesses cysteine residues, Cyys213, Cys214, and Cys267, which can form covalent interactions with the catalytic subunit DmGCLC and modify its activity, the activity of the holoenzyme is enhanced compared to the catalytic subunit alone Drosophila melanogaster

Synonyms

EC Number Synonyms Comment Organism
6.3.2.2 GCL
-
 Drosophila melanogaster

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.2.2 25
-
 assay at Drosophila melanogaster

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.2.2 ATP
-
 Drosophila melanogaster