Human glutathione synthetase reaction order and kinetics examined using spectroscopic and calorimeteric techniques

Glutathione (GSH) is a tripeptide important in preventing cellular oxidative damage. Human glutathione synthetase (hGS) catalyzes the second stage of GSH biosynthesis. Homodimeric human glutathione synthetase is negatively cooperative with respect to its L-γ-Glu-Cys substrate. Although the allosteric effects of substrates binding to hGS have been studied, the order of substrate binding has not. GS in plants and prokaryotes are reported to exhibit opposing random ter and ordered ter ter reaction orders respectively; currently little is known about the reaction order in humans. Knowledge of the mechanism and reaction order of hGS is vital to understand how it contributes to the regulation of the levels of the limiting amino acid cysteine and of glutathione. Using ITC binding studies the mechanism of action and reaction order of hGS has been evaluated and suggests a semi-ordered reaction in human GS.