title: Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides.
abstract: 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 P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' 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 S2' 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.
authors: Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020 São Paulo, Brazil.
authors: Nilana M T
authors: NM
authors: Barros
authors: Marcelo
authors: M
authors: Campos
authors: Patrícia A
authors: PA
authors: Bersanetti
authors: Vitor
authors: V
authors: Oliveira
authors: Maria A
authors: MA
authors: Juliano
authors: Guy
authors: G
authors: Boileau
authors: Luiz
authors: L
authors: Juliano
authors: Adriana K
authors: AK
authors: Carmona
mesh_terms: D019297:2,4-Dinitrophenol / Q000378:metabolism; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008297:Male; D008780:Methyltransferases; D015260:Neprilysin / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D062367:ortho-Aminobenzoates; D012965:Sodium Chloride; D008780:Methyltransferases; C021439:indoleethylamine N-methyltransferase; D015260:Neprilysin; D019297:2,4-Dinitrophenol; D001920:Bradykinin