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

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione 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 trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p-(4,6-diamino-s-triazin-2-yl)aminophenylarsenoxide] 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(inact) of 14.3x10(-4) s(-1) and 1.06X10(-4) s(-1) and K-i of 17.2 mu M and 9.6 mu M for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of the characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH(4) form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme-inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur-arsenic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.