Biosynthesis of the Trypanosomatid Metabolite Trypanothione: Purification and Characterization of Trypanothione Synthetase from Crithidia fasciculated

Graeme B. Henderson, Miyuki Yamaguchi, Louis Novoa, Alan H. Fairlamb, Anthony Cerami

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    38 Citations (Scopus)

    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-gluta-thionylspermidine 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 μM, 914 μM, 1.07 mM, 20 μM, and 7 μM, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87 000 and absorption maxima λmax = 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.

    Original languageEnglish
    Pages (from-to)3924-3929
    Number of pages6
    JournalBiochemistry
    Volume29
    Issue number16
    DOIs
    Publication statusPublished - 24 Apr 1990

    ASJC Scopus subject areas

    • Biochemistry

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