Concentration‐dependent effects of zinc on angiotensin‐converting enzyme‐2 activity (1067.4)

Angiotensin‐converting enzyme‐2 (ACE‐2) degrades angiotensin II (Ang II) and forms Ang (1‐7), which antagonizes much of the pathophysiology of Ang II. ACE‐2, a member of the M2 family of metallopeptidases, contains a HEXXH motif that functions as the zinc binding domain at its active site. To ascertain the importance of zinc on ACE‐2 activity, we measured metabolism of an artificial substrate of ACE‐2 (MCA‐APK‐Dnp) by rat kidney and lung, as well as recombinant human ACE‐2 (rhACE‐2) at various concentrations of zinc. Frozen rat tissues were homogenized in 19 volumes of 20 mM NaPO 4 at pH 7.15 with 0.05% Triton X‐100 detergent. The 48,000 x g supernatant was diluted 40‐fold in assay buffer, final concentrations: 50 mM sodium phosphate (pH 7.0); 100 mM NaCl; 0, 10, 100, or 1000 µM zinc acetate; with or without the ACE‐2 inhibitor MLN‐4760. rhACE‐2 was present at a concentration of 4 pg/µL. Metabolism of the fluorogenic substrate (50 µM) was measured at 393 nm with excitation at 328 nm at 37 o C. In both rat tissues and rhACE‐2 the rate of MLN‐4760‐blockable metabolism of substrate was highest with no zinc or 10 µM zinc. However, in the presence of 100 µM zinc, activity was significantly (p<0.05) decreased in rat lung and rhACE‐2 compared to 0 or 10 µM zinc. In the presence of 1000 µM zinc, activity was further reduced (p<0.05) in all three preparations compared to 0, 10 and 100 µM zinc. These results suggest that ACE‐2 may have additional lower affinity binding site(s) for zinc that interfere with its ability to metabolize its substrates. Assays of ACE‐2 activity should not be run in the presence of zinc concentrations greater than 10 µM.