Trypanothione Reductase from Leishmania donovani Purification, Characterisation and Inhibition by Trivalent Antimonials

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX‐LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis‐Menten saturation kinetics with Km values of 36 μM and 9 μM, respectively, the former yielding a kcat/Km of 5.0×106 M−1 s−1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti‐leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time‐dependent manner, with the pseudo‐first‐order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme‐drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge‐transfer complex in the two‐electron‐reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3‐dimercaptopropanol, 2,3‐dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid‐unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.