Biochemical and biophysical characterization of gamma-glutamylcysteine synthetase.

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 Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of and 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 Gcs binds substrate in order ATP >L-glutamate>L-cysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of Gcs.