Studies on the E-loop of human glutathione synthetase

Glutathione (GSH; L-γ-glutamyl-L-cysteinylglycine), an abundant antioxidant, is synthesized intracellularly in two sequential ATP-dependent steps. Human glutathione synthetase (hGS), the second step, ligates γ-glutamylcysteine (γ-GC) to glycine forming GSH. hGS, an obligate homodimer, displays negative cooperativity to the γ-glutamyl-substrate; thus, binding of γ-GC to one subunit decreases its affinity in the second subunit. The recently found E-loop (A210-Q211-E212-K213-E214-R215-N216) is highly conserved (>66%) in mammals; however, only four residues are conserved in other eukaryotes (>49%). E-loop is near the carboxyl of the γ-GC substrate; therefore, we hypothesize it is important for binding and catalysis. Point mutations of these conserved E-loop residues (Q211A, E214S/A, R215A, N216A/V) were prepared (site-directed mutagenesis). After expression, purification and assays, our results show mutations on the E-loop cause changes in activity, γ-GC substrate binding, and negative cooperativity relative to wild-type hGS. Thus, E-loop is crucial to hGS function.