Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity

► Mutations of Val45 have a greater impact on human glutathione synthetase activity and stability than mutations at Val44. ► Km for γ-glutamyl-α-aminobutyrate substrate are nearly equal in V44A and V45A and decrease in V44W and V45W mutant hGS. ► Residues V44 and V45 are located along the allosteric pathway of hGS. ► Mutations at residues 44 and 45 impact the allosteric pathway more than the hGS active site. ► The dimer interface of hGS is intimately responsible for the stability of hGS.