A‐Loop Residues in Glutathione Synthetase

Abstract

Glutathione (GSH), an important tripeptide found in all living cells, functions as an antioxidant and intracellular thiol source. GSH is synthesized in two ATP-dependent steps. The first step is the formation of the dipeptide γ-glutamylcysteine from glutamate and cysteine, catalyzed by γ-glutamylcysteine synthetase. Glutathione synthetase (GS) ligates the dipeptide with glycine to form GSH. Human GS (hGS) is a homodimer. Each subunit has an active site and is composed of 474 residues. The subunit has three important loop structures. One is the alanine-rich, A-loop. It is located near the C-terminal and the glycine binding site. The A-loop is composed of thirteen residues. For higher eukaryotes, the A-loop sequence has ~90% identity with hGS. However, lower eukaryotes and prokaryotes only have ~45%. In addition, there is a patient with a mutation in the A-loop, G464V; symptoms are recurrent bacterial infections, mental retardation, and severe metabolic acidosis (5-oxoprolinuria). These suggest that the A-loop is important for GS function. We prepared several A-loop mutants (G464V and G459S) and have found significantly lowered activity (<10%). Our A-loop studies further the understanding of its role in the production of GSH. (Supported by a Faculty Enhancement Program Grant TWU (MEA), a Welch Foundation Grant (Chem. Dept. TWU), U.S. Dept. Ed (CASCaM UNT, T.R.C.), Faculty Research Grant UNT (T.R.C.).