Kinetics and redox-sensitive oligomerisation reveal negative subunit cooperativity in tryparedoxin peroxidase of Trypanosoma brucei brucei.
Tryparedoxin peroxidases (TXNPx) are peroxiredoxin-type enzymes that detoxify hydroperoxides in trypanosomatids. Reduction equivalents are provided by trypanothione [T(SH)2] via tryparedoxin (TXN). The T(SH)2-dependent peroxidase system was reconstituted from TXNPx and TXN of T. brucei brucei (TbTXN-Px and TbTXN). TbTXNPx efficiently reduces organic hydroperoxides and is specifically reduced by TbTXN, less efficiently by thioredoxin, but not by glutathione (GSH) or T(SH)2. The kinetic pattern does not comply with a simple rate equation but suggests negative co-operativity of reaction centers. Gel permeation of oxidized TbTXNPx yields peaks corresponding to a decamer and higher aggregates. Electron microscopy shows regular ring structures in the decamer peak. Upon reduction, the rings tend to depolymerise forming open-chain oligomers. Co-oxidation of TbTXNPx with TbTXNC43S yields a dead-end intermediate mimicking the catalytic intermediate. Its size complies with a stoichiometry of one TXN per subunit of TXNPx. Electron microscopy of the intermediate displays pentangular structures that are compatible with a model of a decameric TbTXNPx ring with ten bound TbTXN molecules. The redox-dependent changes in shape and aggregation state, the kinetic pattern and molecular models support the view that, upon oxidation of a reaction center, other subunits adopt a conformation that has lower reactivity with the hydroperoxide.
Department of Biochemistry, Technical University of Braunschweig, Mascheroder Weg 1, D-38124 Braunschweig.
Heike
H
Budde
Leopold
L
Flohé
Hans-Jürgen
HJ
Hecht
Birgit
B
Hofmann
Matthias
M
Stehr
Josef
J
Wissing
Heinrich
H
Lünsdorf
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000096:biosynthesis / Q000378:metabolism*; D015800:Protozoan Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D015800:Protozoan Proteins; D004247:DNA; D006861:Hydrogen Peroxide; D010544:Peroxidases; C110959:tryparedoxin peroxidase; D013917:Thrombin