Human glutathione synthetase is responsible for catalyzing the final step in glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweaver-Burk double reciprocal plot for the binding of gamma -glutamyl substrate, indicating cooperative binding. The measured apparent K-m values for gamma -glutamyl-alpha -aminobutyrate (an analog of gamma -glutamyl-alpha -aminobutyrate) are 63 and 164 muM, respectively. Neither ATP (K-m of 248 muM) nor glycine (K-m of 452 muM) exhibits such cooperative binding behavior. Although 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 -glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for gamma -glutamyl substrate binding 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 substrate.