MECHANISM OF INHIBITION OF TRYPANOTHIONE REDUCTASE AND GLUTATHIONE-REDUCTASE BY TRIVALENT ORGANIC ARSENICALS

The dithiol trypanothione, novel to trypanosomatids and analogous to g lutathione in mammalian systems, has been shown to interact with anti- trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothio ne reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous g lutathione reductase both contain catalytically active sulphydryl grou ps we have examined the ability of several arsenicals to differentiall y inhibit these enzymes. Melarsen oxide (4,6-diamino-s-triazin-2-yl)am inophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo-first-order kinetics with k(ina ct) of 14.3x10(-4) s(-1) and 1.06X10(-4) s(-1) and K-i of 17.2 mu M an d 9.6 mu M for trypanothione reductase and glutathione reductase, resp ectively. Inhibition requires prior reduction of the enzyme by NADPH a nd can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of th e characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess N ADPH leads to a spectrum resembling the EH(4) form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibi tion is proposed where, first, free arsenical and previously reduced e nzyme immediately establish an equilibrium with an inactive monothioar sane enzyme-inhibitor complex involving the interchange cysteine dista l to the FAD; second, a subsequent rearrangement about the sulphur-ars enic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane com plex. Molecular modelling suggests that the differences in kinetic beh aviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypa nothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotryp anothione acts as a protectant in vivo, preventing the direct modifica tion of trypanothione reductase by sequestering the arsenical as MelT.