Artigo
Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides
Fecha
2007-04-01Registro en:
Biological Chemistry. Berlin: Walter de Gruyter & Co, v. 388, n. 4, p. 447-455, 2007.
1431-6730
10.1515/BC.2007.048
WOS:000245407000011
Autor
Barros, Nilana M. T.
Campos, Marcelo
Bersanetti, Patricia A.
Oliveira, Vitor
Juliano, Maria A.
Boileau, Guy
Juliano, Luiz
Carmona, Adriana K.
Institución
Resumen
We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P, (cleavage at the X-F bond) and P-2 (cleavage at R-F bond) specificity, respectively. in these peptides a Phe residue was fixed in P-1' to fulfill the well-known NEP S-1' site requirement for a hydrophobic amino acid. in addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp 2,4-dinitrophenyl ethylenediamine). the enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. the results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S-2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates that permit continuous measurement of the enzyme activity, even in crude tissue extracts.