title: Novel internally quenched fluorogenic substrates for angiotensin I-converting enzyme and carboxypeptidase Y.
abstract: Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is one of the most important enzymes in the renin-angiotensin system, a major blood pressure control system in mammals. We synthesized novel internally quenched fluorogenic (IQF) substrates for ACE based on the cleavage site of an angiotensin I, introducing N-methyl anthranic acid (Nma) and N(ε)-2,4-dinitrophenyl-lysine (Lys(Dnp))at the N- and C-terminal regions. Kinetic parameters of the synthesized IQF substrates Nma-Phe-His-Lys(Dnp) and Nma-His-Pro-Phe-Lys(Dnp)-Pro were compared with those of a common peptide substrate for ACE, hippuryl (Hip)-His-Leu. The k(cat)/K(m) values of Nma-Phe-His-Lys(Dnp), Nma-His-Pro-Phe-Lys(Dnp)-Pro, and Hip-His-Leu were 5.12, 1.90, and 0.80 µM(-1) s(-1) for rabbit lung ACE, and 16.0, 7.36, and 0.30 µM(-1) s(-1) for recombinant human (rh)-ACE, respectively.  These results indicate that Nma-Phe-His-Lys(Dnp) is an excellent substrate for rh-ACE. Carboxypeptidase Y also hydrolyzed Nma-Phe-His-Lys(Dnp) efficiently with K(m), k(cat), and k(cat)/K(m) values of 60.2 µM, 105 s(-1), and 1.74 µM(-1) s(-1), respectively. On the other hand, carboxypeptidase B did not hydrolyze IQF substrates. The newly developed IQF substrate, Nma-Phe-His-Lys(Dnp), is a valuable tool for ACE and carboxypeptidase studies.
authors.affiliation: Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan. saori@arif.pref.akita.jp
authors.forename: Saori
authors.initials: S
authors.lastname: Takahashi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ono
authors.forename: Takeshi
authors.initials: T
authors.lastname: Gotoh
authors.forename: Kumiko
authors.initials: K
authors.lastname: Yoshizawa-Kumagaye
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Sugiyama
mesh_terms: D000818:Animals; D043402:Cathepsin A / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011817:Rabbits
chemical_list: D005456:Fluorescent Dyes; D007703:Peptidyl-Dipeptidase A; D043402:Cathepsin A