Glutathione (GSH; L-γ -glutamyl-L-cysteinylglycine), an essential antioxidant, is synthesized intracellularly in two ATP-dependent steps. The second enzyme in GSH biosynthesis, human glutathione synthetase (hGS), ligates γ glutamylcysteine (γGC) to glycine to form GSH. hGS is negatively cooperative with respect to only the γGC substrate. Molecular modeling using the crystal structure of hGS shows that the three substrates (γ GC, gly and ATP) bind and are covered by several flexible loops (G-. A-, S-, E-, and H-loops). Our results have show that the G-, A- and S-loop, as in other loop structures and many proteins and enzymes, have interactions that facilitate binding of substrates and protect reaction intermediates in the protein active sites. Our results have indicated that the G-loop and A-loop regulate access to the active sites and the S-loop assists in γGC substrate binding. Correspondingly, the G-, A, and S-loops all show a good amount of homology for the most conserved residues in both mammalian and eukaryotic species. Our current research investigates the four most highly conserved residues (Q211, E214, R215, N216) on the E-loop (210-AQEKERN-216). The E-loop is near the carboxyl of the γGC substrate; therefore, we hypothesize it is important for binding and catalysis. Thus, point mutations of the four highly conserved residues were prepared (Q211A, E214A/S, R215A, N216A/V). After expression, purification and assays, our results show E-loop mutations cause decreases in negative cooperativity, activity, and γ-GC substrate binding. Thus, E-loop is vital to hGS function.

E-loop of human glutathione synthetase

ACS Spring Meeting 2021

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