Hydrolysis of Biological Peptides by Human Angiotensin-converting Enzyme-related Carboxypeptidase*

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased ∼10-fold by Cl⁻ and F⁻ but is unaffected by Br⁻. ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II (1., 2., 3., 4., 5., 6., 7., 8.) (k _cat/K _m = 1.9 × 10⁶m⁻¹ s⁻¹), apelin-13 (k _cat/K _m = 2.1 × 10⁶m⁻¹s⁻¹), and dynorphin A 1–13 (k _cat/K _m = 3.1 × 10⁶m⁻¹ s⁻¹). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II (1., 2., 3., 4., 5., 6., 7., 8.) as a substrate than with angiotensin I (1., 2., 3., 4., 5., 6., 7., 8., 9., 10.). ACE2 also efficiently hydrolyzes des-Arg⁹-bradykinin (k _cat/K _m = 1.3 × 10⁵m⁻¹ s⁻¹), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X _{(1–3 residues)}-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.