Biosynthesis of the trypanosomatid metabolite trypanothione: purification and characterization of trypanothione synthetase from Crithidia fasciculata

Abstract

Trypanothione synthetase from Crithidia fasciculata has been purified ca. 14 500-fold to homogeneity in an overall yield of 40%. The pure enzyme catalyzed the synthesis of N1- and N8-glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine(trypanothione) from ATP/magnesium, glutathione (GSH), and spermidine, N1- and N8-glutathionylspermidines being intermediates of trypanothione synthesis. The enzyme showed a sharp pH optimum of 7.5-7.75 for the synthesis of both mono-and diglutathionylspermidine conjugates. It was highly specific for its physiological substrates ATP/Mg2+, GSH, spermidine, and N1- and N8-glutathionylspermidine with Km values of 400 .mu.M, 914 .mu.M, 1.07 mM, 20 .mu.M, and 7.MU.M, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87 000 and absorption maxima .lambda.max = 225 and 280 nm (A280/A260 = 1.85). Trypanothione synthetase is a new member of the ATP-dependent class of ligases which form amide linkage with concomitant production of ADP and orthophosphate.