Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism

Abstract

In the RAS (renin-angiotensin system), Ang I (angiotensin I) is cleaved by ACE (angiotensin-converting enzyme) to form Ang II (angiotensin II), which has effects on blood pressure, fluid and electrolyte homoeostasis. We have examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, the homologue of ACE, ACE2, and NEP (neprilysin). The activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates. Ang I was effectively cleaved by NEP to Ang (1-7) (kcat/K(m) of 6.2x10(5) M(-1) x s(-1)), but was a poor substrate for ACE2 (kcat/K(m) of 3.3x10(4) M(-1) x s(-1)). Ang (1-9) was a better substrate for NEP than ACE (kcat/K(m) of 3.7x10(5) M(-1) x s(-1) compared with kcat/K(m) of 6.8x10(4) M(-1) x s(-1)). Ang II was cleaved efficiently by ACE2 to Ang (1-7) (kcat/K(m) of 2.2x10(6) M(-1) x s(-1)) and was cleaved by NEP (kcat/K(m) of 2.2x10(5) M(-1) x s(-1)) to several degradation products. In contrast with a previous report, Ang (1-7), like Ang I and Ang (1-9), was cleaved with a similar efficiency by both the N- and C-domains of ACE (kcat/K(m) of 3.6x10(5) M(-1) x s(-1) compared with kcat/K(m) of 3.3x10(5) M(-1) x s(-1)). The two active sites of ACE exhibited negative co-operativity when either Ang I or Ang (1-7) was the substrate. In addition, a range of ACE inhibitors failed to inhibit ACE2. These kinetic data highlight that the flux of peptides through the RAS is complex, with the levels of ACE, ACE2 and NEP dictating whether vasoconstriction or vasodilation will predominate.

Figure 1. Schematic diagram of the ACE constructs and ACE2

Human FL-ACE, C-ACE and N-ACE constructs are shown with their amino acid numbering. ACE2 is shown for comparison. Signal peptides are indicated by diagonal lines, transmembrane domains by a black box, and the bridge region of somatic ACE by an open box. The unique C-terminal domain of ACE2 is represented by a dotted box.

Figure 2. SDS/PAGE of the ACE preparations

The affinity-purified preparations of recombinant human ACE (5 μg of protein each) were analysed by SDS/PAGE as described in the Materials and methods section. Lane 1, FL-ACE; lane 2, C-ACE; lane 3, N-ACE. Molecular-mass markers (kDa) are indicated on the right.

Figure 3. Schematic diagram of the RAS

The role of ACE, ACE2 and NEP in the metabolism of the various angiotensin peptides is shown with the k_cat/K_m for each reaction. The thickness of the arrows is representative of the catalytic efficiency of each reaction.