Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme

Quenched fluorescence peptides were used to investigate the substrate speci ficity requirements for recombinant wild-type angiotensin I-converting enzy me (ACE) and two full-length mutants bearing a single functional active sit e (N- or C-domain). We assayed two series of bradykinin-related peptides fl anked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amin o acids), in which the fluorescence appeared when Abz/EDDnp are separated b y substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also syn thesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitroph enyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K-m = 1.1 mu M-1 s(- 1)) being practically resistant to hydrolysis by the C-domain. The importan ce of hydroxyl-containing amino acids at the Pt position for N-domain speci ficity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P- OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K-m values of 5.1 and 4.0 mu M and k(cat) values of 246 and 210 s(-1), respectively, have been shown to b e excellent substrates for ACE. The differentiation of the catalytic specif icity of the C- and N-domains of ACE seems to depend on very subtle variati ons on substrate-specific amino acids. The presence of a free C-terminal ca rboxyl group or an aromatic moiety at the same substrate position determine s specific interactions with the ACE active site which is regulated by chlo ride and seems to distinguish the activities of both domains.