PURIFICATION OF GLUTATHIONYLSPERMIDINE AND TRYPANOTHIONE SYNTHETASES FROM CRITHIDIA-FASCICULATA

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione - glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase - have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1, N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a k(cat) of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a k(cat) of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions 0.0-0.15 M NaCl), consistent with a functional complex in vivo.