Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides.
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.
Barros
Nilana M T
NM
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020 São Paulo, Brazil.
Campos
Marcelo
M
Bersanetti
Patrícia A
PA
Oliveira
Vitor
V
Juliano
Maria A
MA
Boileau
Guy
G
Juliano
Luiz
L
Carmona
Adriana K
AK
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
D009842:Oligopeptides; D062367:ortho-Aminobenzoates; D012965:Sodium Chloride; D008780:Methyltransferases; C021439:indoleethylamine N-methyltransferase; D015260:Neprilysin; D019297:2,4-Dinitrophenol; D001920:Bradykinin