Aspartate 458 and the glycine triad of human glutathione synthetase




Brown, Teresa Rae

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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.



Pure sciences, Biosynthesis of glutathione, Glycine triad valine mutations, Pure sciences