Trypanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate.1. Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the k,,,/K, ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 x lo6 M-' s-' 1 and trypanothione disulfide (4.63 x lo6 M-' s f l 1.2. Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH-reduced forms, were irreversibly modified by the cytostatic agent, 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki = 40 pM) than of trypanothione reductase (ICso = 200 pM).3. Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374-53781 a nitrofuran derivative, 2-(5 -nitro -2-furanylmethy1idene)-N,W-[1,4-piperazinediylbis (1,3 -propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki = 0.5 pM) than for glutathione reductase (ICso = 10 pM). A naphthoquinone derivative, 2,3-bis[3-(2-amidinohydrazono)-butyl]-l,4-naphthoquinone dihydrochloride, turned out to be both an inhibitor = 1 pM) and an NADPHoxidation-inducing substrate (K, = 14 pM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three-dimensional structures of glutathione and trypanothione reductases. Enzymes. Trypanothione reductase (EC 1.6.4.-); glutathione reductase (EC 1.6.4.2). donovani) and oriental sore (Leishmania tropica) are caused by these protozoa.In contrast to all other known eukaryotes [l, 21 Trypanosomatidae do not possess the flavoenzyme glutathione reductase that is responsible for a high intracellular glutathione/ glutathione disulfide ratio. The thiol metabolism of trypanosomes and leishmanias is based on conjugates between glutathione and spermidine, namely trypanothione [3] and glutathionylspermidine [4]. These thiols are kept in the reduced state by trypanothione reductase which catalyzes the following reactions: trypanothione disulfideTrypanothione reductase is an FAD-cystine oxidoreductase which has been purified from the insect parasite Crithidia fasciculata [5] and, in crystalline form, from T . cruzi [6]. The DNA sequence is known for the enzyme from T. congolense [7].In vitro studies on trypanothione reductase are hampered by the fact that the natural dis...

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