Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease closely related to angiotensin I-converting enzyme (ACE). This study characterized ACE2's catalytic activity and identified its substrate preferences. ACE2 has a pH optimum of 6.5 and is enhanced by monovalent anions, particularly Cl⁻ and F⁻, but not Br⁻. ACE2 hydrolyzes 11 biological peptides, with cleavage occurring only at the C-terminal residue. Three peptides—angiotensin II (1–8), apelin-13, and dynorphin A 1–13—were hydrolyzed with high catalytic efficiency (kcat/Km values of 1.9 × 10⁶, 2.1 × 10⁶, and 3.1 × 10⁶ M⁻¹s⁻¹, respectively). ACE2 also efficiently hydrolyzes des-Arg⁹-bradykinin (kcat/Km = 1.3 × 10⁵ M⁻¹s⁻¹), but not bradykinin. A consensus sequence for ACE2 substrates was identified as Pro-X (1–3 residues)-Pro-Hydrophobic, with hydrolysis between proline and the hydrophobic amino acid. ACE2 was purified to >98% purity and characterized using a fluorogenic substrate, Mca-APK(Dnp), which showed a kcat/Km of 7.7 × 10⁵ M⁻¹s⁻¹. ACE2's activity was influenced by pH and anion concentration, with optimal activity at pH 6.5 and enhanced by Cl⁻ and F⁻. ACE2 hydrolyzed 126 biological peptides, with 11 showing cleavage at the C-terminal residue. These substrates included angiotensin II, apelin-13, dynorphin A 1–13, and des-Arg⁹-bradykinin. ACE2's catalytic efficiency for angiotensin II was 400-fold higher than for angiotensin I. ACE2's substrate specificity suggests a role in regulating peptides with biological activity, such as angiotensin II, which is converted to angiotensin 1–7 by ACE2. This conversion may have opposing physiological effects, with angiotensin II being a vasoconstrictor and angiotensin 1–7 a vasodilator. ACE2 also hydrolyzes apelin-13 and neurotensin 1–8, with apelin-13 being a potent vasoconstrictor. The study highlights ACE2's potential role in modulating peptide signaling and its implications for physiological processes such as blood pressure regulation. Further research is neededHuman angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease closely related to angiotensin I-converting enzyme (ACE). This study characterized ACE2's catalytic activity and identified its substrate preferences. ACE2 has a pH optimum of 6.5 and is enhanced by monovalent anions, particularly Cl⁻ and F⁻, but not Br⁻. ACE2 hydrolyzes 11 biological peptides, with cleavage occurring only at the C-terminal residue. Three peptides—angiotensin II (1–8), apelin-13, and dynorphin A 1–13—were hydrolyzed with high catalytic efficiency (kcat/Km values of 1.9 × 10⁶, 2.1 × 10⁶, and 3.1 × 10⁶ M⁻¹s⁻¹, respectively). ACE2 also efficiently hydrolyzes des-Arg⁹-bradykinin (kcat/Km = 1.3 × 10⁵ M⁻¹s⁻¹), but not bradykinin. A consensus sequence for ACE2 substrates was identified as Pro-X (1–3 residues)-Pro-Hydrophobic, with hydrolysis between proline and the hydrophobic amino acid. ACE2 was purified to >98% purity and characterized using a fluorogenic substrate, Mca-APK(Dnp), which showed a kcat/Km of 7.7 × 10⁵ M⁻¹s⁻¹. ACE2's activity was influenced by pH and anion concentration, with optimal activity at pH 6.5 and enhanced by Cl⁻ and F⁻. ACE2 hydrolyzed 126 biological peptides, with 11 showing cleavage at the C-terminal residue. These substrates included angiotensin II, apelin-13, dynorphin A 1–13, and des-Arg⁹-bradykinin. ACE2's catalytic efficiency for angiotensin II was 400-fold higher than for angiotensin I. ACE2's substrate specificity suggests a role in regulating peptides with biological activity, such as angiotensin II, which is converted to angiotensin 1–7 by ACE2. This conversion may have opposing physiological effects, with angiotensin II being a vasoconstrictor and angiotensin 1–7 a vasodilator. ACE2 also hydrolyzes apelin-13 and neurotensin 1–8, with apelin-13 being a potent vasoconstrictor. The study highlights ACE2's potential role in modulating peptide signaling and its implications for physiological processes such as blood pressure regulation. Further research is needed