Characterization of the peptide substrate specificity of glutathionylspermidine synthetase from Crithidia fasciculata

Sofie De Craecker, Christophe Verbruggen, Padinchare K. Rajan, Keith Smith, Achiel Haemers (Lead / Corresponding author), Alan H. Fairlamb

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    9 Citations (Scopus)


    Trypanothione, a metabolite specific to trypanosomatid parasites, is enzymatically synthesized from spermidine and glutathione by the consecutive action of the ATP-dependent carbon-nitrogen ligases, glutathionylspermidine synthetase and trypanothione synthetase. As part of our programme aimed at developing inhibitors of these enzymes, we have synthesized a series of analogues of glutathione (γ-L-Glu-L-Cys-Gly) and tested them as substrates or inhibitors of glutathionylspermidine synthetase. Recognition at the γ-glutamyl moiety appears to be essential, as any modification of this part of glutathione results in a total loss of activity as a substrate. Alkylation of the thiol side chain of cysteine wilh methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (kcat/Km) as substrates equivalent to or better than glutathione. In contrast, the bulkier S-butyl analogue was a much poorer substrate. Substitution of L-Cys by amino acids with an alkyl side-chain is also well tolerated giving relative catalytic efficiencies of 1.1 and 1.5 for peptide analogues containing L-Val and L-Ile respectively. Other analogues, where the bulk of the alkyl chain is increased further (as in L-Leu or L-Phe) or where the glycine moiety is replaced with L-Ala, are inhibitors rather than substrates.

    Original languageEnglish
    Pages (from-to)25-32
    Number of pages8
    JournalMolecular and Biochemical Parasitology
    Issue number1
    Publication statusPublished - Jan 1997


    • Crithidia fasciculata
    • glutathione-binding site
    • glutathionylspermidine synthetase
    • kinetic analysis
    • substrate specificity
    • trypanothione

    ASJC Scopus subject areas

    • Parasitology
    • Molecular Biology


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