Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei
Résumé
Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes - which are essential for the parasites - lack glutathione peroxidase activity but catalyze the trypanothione/tryparedoxin-dependent reduction of hydroperoxides. Cys-47, Gln-82, and Trp-137 correspond to the SeCys, Gln, and Trp catalytic triad of the mammalian selenoenzymes. Site directed mutagenesis revealed that Cys-47 and Gln-82 are essential. A glycine mutant of Trp-137 had 13% of wild-type activity which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys-95, conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys-76 by a serine resulted in a fully active enzyme species and its role remains unknown. Thr-50, proposed to stabilize the thiolate anion at Cys-47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H 2}O 2} induced formation of a sulfinic acid and covalent homodimers in accordance with Cys-47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys-47 and Cys-95. As shown by mass spectrometry, regeneration of the reduced enzyme by tryparedoxin involves a transient mixed disulfide between Cys-95 of the peroxidase and Cys-40 of tryparedoxin. The catalytic mechanism of the tryparedoxin peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.
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