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

• Published in: Biochemistry (Easton) Vol. 29; no. 16; pp. 3924 - 3929
• Main Authors: Henderson, Graeme B, Yamaguchi, Miyuki, Novoa, Louis, Fairlamb, Alan H, Cerami, Anthony
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 24-04-1990
• Subjects: Amide Synthases; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography; Crithidia - enzymology; Crithidia fasciculata; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glutathione - analogs & derivatives; Glutathione - blood; Kinetics; Ligases; Ligases - isolation & purification; Molecular Weight; Spermidine - analogs & derivatives; Spermidine - blood; trypanothione synthase; Characterization; Rhizoflagellata; Protozoa; Crithidia fasciculata; Purification; Kinetic parameter
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00468a019

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 microM, 914 microM, 1.07 mM, 20 microM, and 7 microM, 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.