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Site‐Directed Mutagenesis Studies of Human Glutathione Synthetase
Author(s) -
Brown Teresa,
Bhansali Vikas S.,
Anderson Mary E.
Publication year - 2006
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.20.4.a472-d
Subject(s) - glutathione synthetase , biochemistry , mutagenesis , dna ligase , glutathione , amino acid , enzyme , site directed mutagenesis , alanine , protein subunit , mutant , chemistry , directed mutagenesis , biology , gene
Glutathione Synthetase (GS) catalyses the final step in the synthesis of glutathione (GSH; γ‐L‐Glutamyl‐L‐cysteinylglycine), an important cellular antioxidant. Human GS (hGS) is the only eukaryotic member of the ATP‐grasp superfamily, an important class of amine‐carboxylase ligase enzymes, whose function is known. GS is a homodimer, with each subunit having an active site and being composed of 474 amino acids. There are three loop structures; one is alanine‐rich (A‐loop). The A‐loop consists of 13 amino acid residues, many of which are conserved in eukaryotes. There is a known inborn error of metabolism in this region, Gly464Val. Using site‐directed mutagenesis and affinity chromatography, we have purified and characterized the patient enzyme (Gly464Val), as well as hGS enzymes with mutations in this conserved region. Our studies show a decrease in activity for these mutant enzymes, as compared to the wild type. Our studies provide a better understanding of GSH deficiency and provide information on the role of the A‐loop. (Supported by a Faculty Enhancement Program Grant from Texas Woman’s University (MEA) and a Welch Foundation Departmental grant to the Chemistry Dept at TWU.)