Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Abstract

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 chromatographymass 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) (kcat/Km = 1.9 x 106 M-1 S-1), apelin-13 (kcat/Km = 2.1 x 106 M-1 S-1), and dynorphin A 1-13 (kcat/Km = 3.1 x 106 M-1 S-1). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II (1-8) as a substrate than with angiotensin I (1-10). ACE2 also efficiently hydrolyzes des-Arg9-bradykinin (kcat/Km = 1.3 x 105 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.