TRYPANOTHIONE REDUCTASE FROM LEISHMANIA-DONOVANI - PURIFICATION, CHARACTERIZATION AND INHIBITION BY TRIVALENT ANTIMONIALS

Trypanothione reductase was purified to homogeneity from Leishmania do novani promastigotes transfected with the expression plasmid pTEX-LdTR . The physical, spectral and kinetic properties were found to be simil ar to those obtained from other pathogenic trypanosomatids. The substr ates trypanothione disulfide and NADPH exhibit Michaelis-Menten satura tion kinetics with K-m values of 36 mu M and 9 mu M, respectively, the former yielding a k(cat)/K-m of 5.0X10(6) M(-1) s(-1). Like other try panothione reductases, the leishmania enzyme is unable to use glutathi one disulfide as substrate. Both trypanothione reductase and the analo gous mammalian enzyme, glutathione reductase, are inhibited by trivale nt but not pentavalent anti-leishmanial antimonials. Inhibition by tri valent sodium antimonyl gluconate (Triostam) occurs in a time-dependen t manner, with the pseudo-first-order rate constants of inhibition bei ng linearly related to drug concentration. Inhibition proceeds until a n apparent equilibrium between active enzyme/free drug and inactive en zyme-drug complex is reached. MelT, an adduct of melarsen oxide and di hydrotrypanothione which is a competitive inhibitor of the disulfide b inding site of trypanothione reductase, confers protection against Tri ostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the b road absorbance band centred on 530 nm, characteristic of the charge-t ransfer complex in the two-electron-reduced EH, enzyme, is lost upon a ddition of Triostam. Further spectral changes resemble those associate d with reduction of the FAD prosthetic group to FADH(2). 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.