CONVENIENT ISOLATION AND KINETIC MECHANISM OF GLUTATHIONYLSPERMIDINE SYNTHETASE FROM CRITHIDIA-FASCICULATA

Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathio nylspermidine synthetase and trypanothione synthetase. Glutathionylspe rmidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography . The enzyme showed a specific activity of 38 mu mol of glutathionylsp ermidine formed per min per mg of protein. Its molecular mass was 78 k Da in SDS polyacrylamide gel electrophoresis, and it appeared predomin antly monomeric in native polyacrylamide gel electrophoresis and gel f iltration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6, Partial amino acid sequencing revealed homology with, but l ow similarity to, the glutathionylspermidine synthetase/amidase of Esc herichia coli, and amidase activity was not detected in glutathionylsp ermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting K-m values for Mg2+-ATP, GSH, and spermidine o f 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0.09 mM, respectively, an d a k(cat) of 415 +/- 78 min(-1). Partial reactions at restricted cosu bstrate supply were not detected by P-31 NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited compe titively with respect to ATP (K-i = 0.08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (K-i = 0.48 mM).