Substrate binding determinants of Trypanosoma brucei γ-glutamylcysteine synthetase

γ-Glutamylcysteine synthetase (γ-GCS) catalyzes the ATP-dependent ligation of L-Glu and L-Cys, which is the first step in de novo biosynthesis of the tripeptide glutathione. Recently it was demonstrated that γ-GCS is a structural homologue of glutamine synthetase (GS), providing the basis to build a model for the γ-GCS active site [Abbott et al. (201) J. Biol. Chem. 276, 42099-42107]. Substrate binding determinants in the active site of γ-GCS have been identified and characterized in the enzyme from the parasitic protozoa Trypanosoma brucei using this model as a guide for site-directed mutagenesis. R366 and R491 were identified as key determinants of L-Glu binding. Mutation of R366 to Ala increases the K_d for L-Glu by 160-fold and eliminates the positive cooperativity observed for the binding of L-Glu and ATP to the wild-type enzyme, based on a rapid equilibrium random mechanism of substrate binding. Unlike the wild-type enzyme, the R366A mutant enzyme was able to form product using the substrate analogue γ-aminobutyric acid, suggesting that R366 interacts with the α-carboxylate of L-Glu. Mutation of R491 to Ala decreased k_cat for ATP hydrolysis by 70-fold; however, dipeptide product was only formed in 5% of these turnovers. These data suggest that R491 stabilizes the phosophorylated γ-carboxylate of L-Glu during nucleophilic attack by the L-Cys to form the dipeptide product. T323, R474, and R487 were predicted to be ATP binding determinants. Mutation of each of these residues to Ala increased the apparent K_m for ATP by 20-100-fold while having only modest effects on k_cat or the apparent K_m's for the other substrates. Finally, mutation of R179, a conserved residue that is present in γ-GCS, but not in GS, increased the apparent K_m for both L-Cys and L-Glu.