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.