Verification of the interaction of a tryparedoxin peroxidase with tryparedoxin by ESI-MS/MS.
Biol Chem
; 384(9): 1305-9, 2003 Sep.
Article
em En
| MEDLINE
| ID: mdl-14515993
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peroxidases
/
Tiorredoxinas
/
Trypanosoma brucei brucei
/
Proteínas de Protozoários
/
Espectrometria de Massas por Ionização por Electrospray
Limite:
Animals
Idioma:
En
Revista:
Biol Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2003
Tipo de documento:
Article