Aspartate 458 and the glycine triad of human glutathione synthetase

Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH). Human GS displays negative cooperative to the γ-glutamyl substrate (GAB). The hGS active site has three highly conserved catalytic loops, notably the G- and A-loop. Herein, with experimental and computational investigations, we report the impact of mutation of A-loop residue Asp458 and the glycine triad (Gly369, Gly370 and Gly371) on hGS structure, activity, cooperativity and stability. The Michaelis-Menten constant (Km) was determined for all three substrates (Glycine, GAB, ATP). For Asp458 mutant hGS, ATP Km was unchanged, glycine Km increased, and GAB Km decreased along with a change in cooperativity (negative- to non-cooperative). The glycine triad valine mutations (G369V, G370V and G371V) displayed dramatic decreased activity compared to wild-type. These findings indicate that Asp458 and the glycine triad are essential for hGS catalysis and that mutation of Asp458 directly impacts the allostery of this enzyme.