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Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei.
Biochem J. 2007 Aug 01; 405(3):445-54.BJ

Abstract

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 catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is 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 Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.

Authors+Show Affiliations

Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 504, 69120 Heidelberg, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17456049

Citation

Schlecker, Tanja, et al. "Catalytic Mechanism of the Glutathione Peroxidase-type Tryparedoxin Peroxidase of Trypanosoma Brucei." The Biochemical Journal, vol. 405, no. 3, 2007, pp. 445-54.
Schlecker T, Comini MA, Melchers J, et al. Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei. Biochem J. 2007;405(3):445-54.
Schlecker, T., Comini, M. A., Melchers, J., Ruppert, T., & Krauth-Siegel, R. L. (2007). Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei. The Biochemical Journal, 405(3), 445-54.
Schlecker T, et al. Catalytic Mechanism of the Glutathione Peroxidase-type Tryparedoxin Peroxidase of Trypanosoma Brucei. Biochem J. 2007 Aug 1;405(3):445-54. PubMed PMID: 17456049.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei. AU - Schlecker,Tanja, AU - Comini,Marcelo A, AU - Melchers,Johannes, AU - Ruppert,Thomas, AU - Krauth-Siegel,R Luise, PY - 2007/4/26/pubmed PY - 2007/8/19/medline PY - 2007/4/26/entrez SP - 445 EP - 54 JF - The Biochemical journal JO - Biochem J VL - 405 IS - 3 N2 - 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 catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is 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 Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/17456049/Catalytic_mechanism_of_the_glutathione_peroxidase_type_tryparedoxin_peroxidase_of_Trypanosoma_brucei_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20070259 DB - PRIME DP - Unbound Medicine ER -