Cooperative Binding of γ-Glutamyl Substrate to Human Glutathione Synthetase

Abstract

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 γ-glutamyl substrate, indicating cooperative binding. The measured apparent Km values for γ-glutamyl-α-aminobutyrate (an analog of γ-glutamyl-α-aminobutyrate) are 63 and 164 μM, respectively. Neither ATP (Km of 248 μM) nor glycine (Km of 452 μM) exhibits such cooperative binding behavior. Although ATP is proposed to play a key role in the sequential binding of γ-glutamyl substrate to the enzyme, the cooperative binding of the γ-glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for γ-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 γ-glutamyl substrate.