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Role of human glutathione synthetase H‐loop residues in substrate binding and activity (773.4)
Author(s) -
Feyen Jessica,
Ingle Brandall,
Berthold Meghan,
Davis Myra,
Sandoval Esmeralda,
Hinojos Caleb,
Crutchfield Amanda,
Cundari Thomas,
Anderson Mary
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.773.4
Subject(s) - glutathione , glutathione synthetase , cooperativity , mutagenesis , mutant , biochemistry , cooperative binding , substrate (aquarium) , glycine , chemistry , binding site , enzyme , intracellular , biophysics , biology , amino acid , gene , ecology
Glutathione (GSH) is an antioxidant essential for mammalian cell survival and is found in every cell of almost all organisms. GSH cannot be taken up by the cell, so understanding the intercellular synthesis of this important antioxidant is crucial. GSH is synthesized in a two‐step process. In the second step, human glutathione synthetase (hGS) binds γ‐glutamylcysteine and glycine and then uses the energy of ATP to make GSH. hGS is a homodimer that displays negative cooperativity, toward the γ‐glutamylcysteine substrate, a phenomenon which contributes to metabolic regulation but is not yet fully understood. The H‐loop (147‐TISASF‐152) of hGS is highly conserved and located near the binding site of γ‐glutamylcysteine. The role of H‐loop residues was examined using site‐directed mutagenesis of H‐loop residues, followed by purification and kinetic characterization of H‐loop mutant hGS enzymes. Our results show that the H‐loop mutant hGS enzymes have altered binding affinity (Km) for the negatively cooperative substrate, γ‐glutamylcysteine. Grant Funding Source : Supported by NIH R15GM086833 (MEA), REP Grant (TWU, MEA), Welch Chem Dept Grant (TWU)