ATP-dependent ligases in trypanothione biosynthesis - kinetics of catalysis and inhibition by phosphinic acid pseudopeptides

Sandra L. Oza, Shoujun Chen, Susan Wyllie, James K. Coward, Alan H. Fairlamb

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    20 Citations (Scopus)

    Abstract

    Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K-GSH = 609 mu M, K-Spd = 157 mu M and K-ATP = 215 mu M. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K-i similar to 156 mu M), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) m(-1).s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K-i = 18.6 nm. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K-i(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.

    Original languageEnglish
    Pages (from-to)5408-5421
    Number of pages14
    JournalFEBS Journal
    Volume275
    Issue number21
    DOIs
    Publication statusPublished - Nov 2008

    Keywords

    • drug discovery
    • enzyme mechanism
    • glutathionylspermidine synthetase
    • slow-binding inhibition
    • trypanothione synthetase
    • BIFUNCTIONAL GLUTATHIONYLSPERMIDINE SYNTHETASE/AMIDASE
    • MULTISUBSTRATE ANALOG INHIBITORS
    • GLUTATHIONE-LIKE TRIPEPTIDES
    • GAMMA-GLUTAMATE SYNTHETASE
    • ESCHERICHIA-COLI
    • TRYPANOSOMA-BRUCEI
    • CRITHIDIA-FASCICULATA
    • LEISHMANIA-MAJOR
    • SLOW-BINDING
    • FOLYLPOLYGLUTAMATE SYNTHETASE

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