Verification of the interaction of a tryparedoxin peroxidase with tryparedoxin by ESI-MS/MS.
Tryparedoxin peroxidases (TXNPx) catalyze hydroperoxide reduction by tryparedoxin (TXN) by an enzyme substitution mechanism presumed to involve three catalytic intermediates: (i) a transient oxidation state having C52 oxidized to a sulfenic acid, (ii) the stable oxidized form with C52 disulfide-bound to C173', and (iii) a semi-reduced intermediate with C40 of TXN disulfide-linked to C173' from which the ground state enzyme is regenerated by thiol/disulfide reshuffling. This kinetically unstable form was mimmicked by a dead-end intermediate generated by cooxidation of TXNPx of Trypanosoma brucei brucei with an inhibitory mutein of TXN in which C43 was replaced by serine (TbTXNC43S). Cleavage of the isolated dead-end intermediate by trypsin plus chymotrypsin yielded a fragment that complied in size with the TbTXNC43S sequence 36 to 44 disulfide-linked to the TbTXNPx sequence 169 to 177. The presumed nature of the proteolytic fragment was confirmed by MS/MS sequencing. The results provide direct chemical evidence for the assumption that the reductive part of the catalysis is initiated by an attack of the substrate's solvent-exposed C40 on C173' of the oxidized peroxidase and, thus, confirm the hypothesis on the interaction of 2-Cys-peroxiredoxins with their proteinaceous substrates.
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004791:Enzyme Inhibitors; D008958:Models, Molecular; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins; D021241:Spectrometry, Mass, Electrospray Ionization; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; C114110:tryparedoxin; D013879:Thioredoxins; D010544:Peroxidases; C110959:tryparedoxin peroxidase