Human glutathione synthetase residues Valine 44 and Valine 45 are required for subunit stability and negative cooperativity

Valine 44 and Valine 45 serve as important residues for human glutathione synthetase (hGS) function and stability given their location at the dimer interface of this enzyme. Computational studies suggest Val45 has more impact on the structure and stability of hGS than Val44. Experimentally, enzymes with mutations at the 44 and/or 45 positions were prepared, purified and assayed for initial activity. The Val45 position mutations have a greater impact on enzyme activity than mutations at Val44. Differential scanning calorimetry experiments reveal a stability loss in all mutant enzymes. The γ-GluABA substrate affinity remains unaltered in V44A and V45A mutant enzymes, but increases with tryptophan introduction. Hill coefficients trend towards less negative cooperativity except with V45W. These results imply that residues V44 and V45 are located along the allosteric pathway of this negatively cooperative dimeric enzyme and that these residues are integral to the stability of human glutathione synthetase.