Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild‐type angiotensin‐converting enzyme

Abstract

Angiotensin-converting enzyme (ACE) in vertebrates is a zinc metallopeptidase involved in the release of angiotensin II and the inactivation of bradykinin, two peptide hormones that play a key role in blood pressure regulation and renal and cardiovascular function [1][2][3][4]. ACE inhibitors have been on the market for more than 20 years, with successful applications for conditions ranging from mild hypertension to postmyocardial infarction [5,6]. Somatic ACE is a very unusual enzyme which contains two active sites on the same polypeptide chain [7]. Since this discovery, there has been much speculation about the functional significance of the presence of two active sites in the same enzyme [8][9][10][11][12][13]. At the biochemical level, the presence of two active sites in the same enzyme has hampered the full characterization of substrate and inhibitor selectivity of somatic ACE. To circumvent these limitations, mutants of human ACE containing a single functional active site [14,15] or isolated ACE domains have been utilized to perform these studies [10,11]. Alternative approaches to study directly the properties of both domains in somatic form of ACE are still lacking. The development of the first highly N-domain-specific inhibitor, RXP407 [16], and the recent identification of a C-domain-specific inhibitor, RXPA380 [13], of human ACE may provide such an alternative strategy for studying inhibitor and substrate selectivity of any form of somatic ACE (Scheme 1). To demonstrate its interest, this approach was used to study the selectivity of somatic ACE purified from Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wildtype enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.Abbreviations ACE, angiotensin-converting enzyme; DpaOH...

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…For example, cleavage of the heptopeptide Met 5 -enkephalin-Arg 6 -Phe 7 is about 5-fold faster by the ACE N-domain than by the C-domain at a physiologic pH (Deddish et al, 1997). In addition, domain-specific fluorogenic substrates have been developed, including the C-domain substrate Oaminobenzoic acid-Phe-Arg-Lys(2,4-dinitrophenyl)-Prohydroxide and the murine N-domain-specific substrate (7-methoxycoumarin-4-yl)acetyl-Ala-Ser-Asp-Lys-N 3 - (Araujo et al, 2000;Bersanetti et al, 2004;Jullien et al, 2006).…”

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide

“…Potency of inhibitors towards C-ACE and N-ACE, as well as potency towards NEP and ECE-1, were determined as described by Jullien et al [15,34]. K i values were determined at 25°C in 50 mM Hepes (pH 6.8), 200 mM NaCl, 10 μM ZnCl 2 and 0.02% Brij-35.…”

Different contributions of the angiotensin-converting enzyme C-domain and N-domain in subjects with the angiotensin-converting enzyme II and DD genotype

“…7) [24]. The curve consisted of two parts, reflecting the titration of the Ndomain and C-domain, respectively.…”

“…All studies with HHL, Ang I, and bradykinin were repeated in the presence of increasing concentrations of quinaprilat. Finally, the inhibitory effects of RXP407 and RXPA380 were evaluated in serum diluted 1 : 20 (RXP407) or 1 : 100 (RXPA380) in 50 mmol/l HEPES buffer as described by Jullien et al [24], using the N-domain and C-domain-selective substrates Mca-Ala-Ser-Asp-LysDpaOH (Mca-Ala) and Mca-Arg-Pro-Pro-Gly-Phe-SerPro-DpaOH (Mca-BK(1-8)) at a concentration of 15 and 10 mmol/l, respectively.…”