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ATP‐dependent ligases in trypanothione biosynthesis – kinetics of catalysis and inhibition by phosphinic acid pseudopeptides
Author(s) -
Oza Sandra L.,
Chen Shoujun,
Wyllie Susan,
Coward James K.,
Fairlamb Alan H.
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06670.x
Subject(s) - phosphinate , crithidia fasciculata , chemistry , stereochemistry , spermidine , glutathione , biochemistry , suramin , enzyme , organic chemistry , receptor , fire retardant
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 ( Cf GspS) obeys a rapid equilibrium random ter‐ter model with kinetic constants K GSH = 609 μ m , K Spd = 157 μ m and K ATP = 215 μ m . Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli , are equally potent against Cf GspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) ( K i ∼ 156 μ 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 × 10 4 m −1 ·s −1 and k 4 (off rate) = 1.3 × 10 −3 s −1 , providing a dissociation constant K i = 18.6 n m . 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 Cf GspS. 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.