Glutathione synthetase: Conserved residues of the substrate loop
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The important antioxidant tripeptide glutathione (GSH) is synthesized in two ATP-dependent steps; the second enzyme in the biosynthetic pathway, glutathione synthetase (GS), ligates glycine to γ-glutamylcysteine (γ-GC). Human glutathione synthetase (hGS) deficiency causes hemolytic anemia, metabolic acidosis, 5-oxprolinuria and a total deficiency may be lethal. Three flexible loops (A, G and S) surround the substrates (ATP, glycine and γ-GC). Human glutathione synthetase is negatively cooperative to one substrate, γ-GC. The Substrate- or S-loop is proximal to γ-GC and thought to participate in γ-GC binding. The S-loop (266-FRDGYMPRQYS-276) contains 11 residues, some of which are highly conserved (F266, R267, G269, Y270, P272 and Y275). Site directed mutagenesis was used to change these highly conserved S-loop residues, and then their roles in substrate binding, enzyme activity and stability were assessed.