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γ-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP >L …