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

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) (k_cat/K_m of 6.2×10⁵ M⁻¹·s⁻¹), but was a poor substrate for ACE2 (k_cat/K_m of 3.3×10⁴ M⁻¹·s⁻¹). Ang (1–9) was a better substrate for NEP than ACE (k_cat/K_m of 3.7×10⁵ M⁻¹·s⁻¹ compared with k_cat/K_m of 6.8×10⁴ M⁻¹·s⁻¹). Ang II was cleaved efficiently by ACE2 to Ang (1–7) (k_cat/K_m of 2.2×10⁶ M⁻¹·s⁻¹) and was cleaved by NEP (k_cat/K_m of 2.2×10⁵ M⁻¹·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 (k_cat/K_m of 3.6×10⁵ M⁻¹·s⁻¹ compared with k_cat/K_m of 3.3×10⁵ M⁻¹·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.