Substrate Loop in Glutathione Synthetase: Mutation Analysis

Abstract

Glutathione (GSH), a small tripeptide, is a required cellular antioxidant. Cellular GSH is synthesized by two ATP‐dependent enzymes. The first enzyme, gamma‐glutamylcysteine synthetase, ligates glutamate and cysteine to form gamma‐glutamylcysteine. The second enzyme, glutathione synthetase (GS), ligates glycine to gamma‐glutamylcysteine to form GSH. Human GS, a homodimer, contains a conserved eleven amino acid residue loop region (266‐FRDGYMPRQYS‐276) called the Substrate Loop (S‐loop). In order to study the role of the conserved amino acid residues, several GS mutant enzymes, such as Gly269Val and Tyr270Phe, within this region were prepared using PCR site‐directed mutagenesis. These were expressed, purified and assayed for activity. Our findings show that mutations at Gly269 cause a significant decrease in GS activity (< 5% wild type). (Supported by Welch Foundation Chemistry Department grant; TWU A&S Research Development Fund (MEA).)