Abstract
The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of -249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys(40), the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to < or =4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.
Highlights
Tryparedoxins have been found only in parasitic protozoa of the family Trypanosomatidae, to which belong the causative agents of tropical diseases such as African sleeping sickness (Trypanosoma brucei gambiense and T. brucei rhodesiense), Nagana cattle disease (T. brucei and Trypanosoma congolense), Chagas’ disease (Trypanosoma cruzi), and the three manifestations of leishmaniasis (Leishmania donovani, Leishmania major, and Leishmania mexicana)
Its first elucidated role was as a component of a cascade composed of trypanothione, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase that catalyzes the detoxication of organic hydroperoxides in the parasites (1, 13)
We report on the catalytic activities, the determination of the pK value of Cys40, and the redox potential of T. brucei tryparedoxin
Summary
Reduction of Dithiol Proteins by NaBH4—In a total volume of 400 l, 200 M T. brucei tryparedoxin or E. coli thioredoxin was incubated with 20 mM NaBH4 in 100 mM potassium phosphate, pH 7.0, for 5 min at room temperature. Glutathione-dependent Dehydroascorbate Reductase Assays—The reaction mixtures contained in a total volume of 80 l of 100 mM potassium phosphate, 1 mM EDTA, pH 6.5, 100 M dehydroascorbate, 1 mM GSH, and 0.1–5.6 M T. brucei tryparedoxin, E. coli glutaredoxin, and thioredoxin, respectively. In a total volume of 800 l of 100 mM Tris/HCl, pH 7.9, 0.1–20 M E. coli thioredoxin and glutaredoxin as well as T. brucei tryparedoxin and thioredoxin, respectively, were incubated with 10 – 80 l of malate dehydrogenase solution and 5 mM DTE for 30 min at 25 °C. For each mutant forward and reverse primers were derived covering the active site
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