Human Glutathione Synthetase Substrate Loop Analysis

Abstract

Glutathione (GSH), a small tripeptide, is a required cellular antioxidant. Normally, the biosynthesis of GSH keeps pace with its cellular use. However, conditions such as inborn genetic deficiencies, disease, and presence of toxic compounds, cause low GSH levels. Cellular GSH levels are maintained by two ATP-dependant enzymes. The first enzyme, gamma-glutamyl cysteine synthetase, ligates glutamate and cysteine to form gamma-glutamylcysteine. The second enzyme, glutathione synthetase (GS), adds glycine to the gamma-glutamylcysteine dipeptide to form GSH. GS subunits, of the homodimer, contain three conserved loop regions: the glycine-rich or G-loop, the alanine-rich or A-loop, and the substrate or S-loop. GS deficiencies, such as 5-oxoprolinuria, are found in patients with mutations within the S-loop at residues R267W and Y270C. To study role of these mutations we have prepared GS mutations at the residue R267 and neighboring residues, using PCR site directed mutagenesis. These enzymes were then purified and assayed for activity. Our findings show that mutations at the residue R267 (such as R267A) cause significant decrease in GS activity (< 5% of wild type). (Supported in part by a Dept. Welch Foundation Grant (TWU), a Willis Clark Scholarship & Fellowship in Chemistry (KS), the Research Enhancement Program TWU (MEA), U.S. Dept. EDU. (CASCaM,UNT), and Faculty Research Grant UNT (TRC)).