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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 126.96.36.199) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants KGSH = 609 μM, KSpd = 157 μM and KATP = 215 μM. 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) (Ki ∼ 156 μM), 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 k3 (on rate) = 6.98 × 104 M−1·s−1 and k4 (off rate) = 1.3 × 10−3 s−1, providing a dissociation constant Ki = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 188.8.131.52) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with Kiapp 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.