Novel kinetics of mammalian glutathione synthetase: Characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase ( ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mam malian glutathione synthetase is a homodimer with each subunit containing a n active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase, It has the highest specific activity (11 mu mo l/min/mg) reported for any mammalian glutathione synthetase, The apparent K -m values for ATP and glycine are 37 and 913 mu M, respectively. The Linewe aver-Burk double reciprocal plot for gamma-glutamyl substrate binding revea led a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0.576, which shows the negative cooperativity. N either ATP, the other substrate involved in forming the enzyme-bound gamma- glutamyl phosphate intermediate, nor glycine, which at tacks this intermedi ate to form GSH, exhibit any cooperativity, The cooperative binding of gamm a-glutamyl substrate is not affected by ATP concentration, Thus, mammalian glutathione synthetase is an allosteric enzyme. (C) 2000 Academic Press.