C‐Terminal Residues of Glutathione Synthetase

Abstract

Glutathione (GSH:γ‐L‐Glutamyl‐L‐cysteinylglycine) is an important antioxidant found in living cells. Low cellular levels of GSH are associated with diseases such as stroke, AIDS and diabetes. Glutathione Synthetase (GS) catalyses the final ATP‐dependent step to form GSH. Human GS is the only mammalian member of the ATP‐grasp super‐family (an important class of amine carboxylate ligases) whose function is known; other family members are involved in nucleotide and cell wall synthesis. Each monomer of the dimeric GS has an active site, and GS displays negative cooperativity. There are three highly conserved loop regions (G‐,A‐,S‐loops). Higher eukaryotes have high A‐loop (alanine‐rich) identity (∼90%), while there is less identity in lower eukaryotes and prokaryotes (∼45%). The thirteen residue (Ile454‐Ala466) A‐loop is near the C‐terminal portion of GS and the glycine binding site. Several residues of the A‐loop were computationally modeled, mutated by site‐directed mutagenesis, and the mutant GS enzymes purified and assayed to characterize their role in GS function. We studied several GS mutants and found Glu 445 Arg and His 456 Ala enzymes have lowered activity (∼30% and ∼50%, respectively) compared to wild type GS. This A‐loop is important for GS activity. (Supported by a Welch Foundation Grant (Chem. Dept. TWU), U.S. Dept. Ed (CASCaM UNT, T.R.C.), Faculty Research Grant UNT (TRC).