Cooperative binding of gamma-glutamyl substrate to human glutathione synthetase

Human glutathione synthetase is responsible for catalyzing the final step i n glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweav er-Burk double reciprocal plot for the binding of gamma -glutamyl substrate , indicating cooperative binding. The measured apparent K-m values for gamm a -glutamyl-alpha -aminobutyrate (an analog of gamma -glutamyl-alpha -amino butyrate) are 63 and 164 muM, respectively. Neither ATP (K-m of 248 muM) no r glycine (K-m of 452 muM) exhibits such cooperative binding behavior. Alth ough ATP is proposed to play a key role in the sequential binding of gamma -glutamyl substrate to the enzyme, the cooperative binding of the gamma -gl utamyl substrate is not affected by alterations of ATP concentration. Quant itative analysis of the kinetic results for gamma -glutamyl substrate bindi ng gives a Hill coefficient (h) of 0.75, indicating negative cooperativity. Our studies, for the first time, show that human glutathione synthetase is an allosteric enzyme with cooperative binding for gamma -glutamyl substrat e. (C) 2001 Academic Press.