Structure Prediction and Active Site Analysis of the Metal Binding Determinants in γ-Glutamylcysteine Synthetase*

γ-Glultamylcysteine synthetase (γ-GCS) catalyzes the first step in the de novo biosynthesis of glutathione. In trypanosomes, glutathione is conjugated to spermidine to form a unique cofactor termed trypanothione, an essential cofactor for the maintenance of redox balance in the cell. Using extensive similarity searches and sequence motif analysis we detected homology between γ-GCS and glutamine synthetase (GS), allowing these proteins to be unified into a superfamily of carboxylate-amine/ammonia ligases. The structure of γ-GCS, which was previously poorly understood, was modeled using the known structure of GS. Two metal-binding sites, each ligated by three conserved active site residues (n1: Glu-55, Glu-93, Glu-100; and n2: Glu-53, Gln-321, and Glu-489), are predicted to form the catalytic center of the active site, where the n1 site is expected to bind free metal and the n2 site to interact with MgATP. To elucidate the roles of the metals and their ligands in catalysis, these six residues were mutated to alanine in the Trypanosoma brucei enzyme. All mutations caused a substantial loss of activity. Most notably, E93A was able to catalyze thel-Glu-dependent ATP hydrolysis but not the peptide bond ligation, suggesting that the n1 metal plays an important role in positioning l-Glu for the reaction chemistry. The apparent K m values for ATP were increased for both the E489A and Q321A mutant enzymes, consistent with a role for the n2 metal in ATP binding and phosphoryl transfer. Furthermore, the apparentK d values for activation of E489A and Q321A by free Mg2+ increased. Finally, substitution of Mn2+for Mg2+ in the reaction rescued the catalytic deficits caused by both mutations, demonstrating that the nature of the metal ligands plays an important role in metal specificity.