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 similar to10-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 (1-8) (k(cat)/K-m = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K-m = 2.1 x 10(6) M-1 s(-1)), and dynorphin A 1-13 (k(cat)/K-m = 3.1 x 10(6) M-1 s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin 11 (1-8) as a substrate than with angiotensin I (1-10). ACE2 also efficiently hydrolyzes des-Are-bradykinin (k(cat)/K-m = 1.3 x 10(5) m(-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.