The dimer loop and electrostatic interactions at the dimer interface of human glutathione synthetase
Human glutathione synthetase (hGS) is homodimeric and negatively cooperative toward its γ-glutamyl substrate making it a good model to study protein-protein interactions. The allosteric pathway between hGS active sites is hypothesized to travel through the dimer interface. To better understand the allostery of hGS and interactions at the dimer interface, two regions, the dimer loop [35-TSQEPTSSE-43] and key amino acid residues (Serine42, Arginine221 and Aspartate24) were studied using site-directed mutagenesis and analyzed for effects on cooperativity, activity and stability. Alanine mutant enzymes of dimer loop residues did not greatly affect activity or stability of hGS, nor change the cooperativity of hGS, but did affect γ-glutamyl substrate affinity, while alanine mutant enzymes of residues Ser42, Arg221 and Asp24 did not change cooperativity, but did decrease in activity and stability, and increase in γ-glutamyl affinity, with Asp24 having the greatest loss in activity and stability, followed by Arg221, and then Ser42.