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

doi:10.1074/jbc.M200581200

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

From the Departments of ^‡Metabolic Disease,^¶Lead Discovery, ^‖Protein Sciences, ^**Technology Platform, ^‡Cardiovascular Biology, and ^§§Medicinal Chemistry, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139

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 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 × 10⁶ m ⁻¹ s⁻¹), apelin-13 (k _cat/K _m = 2.1 × 10⁶ m ⁻¹s⁻¹), and dynorphin A 1–13 (k _cat/K _m = 3.1 × 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 (k _cat/K _m = 1.3 × 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Both authors contributed equally to this work.
↵¶¶ To whom correspondence should be addressed: Dept. of Metabolic Disease, Millennium Pharmaceuticals, Inc., 270 Albany St., Cambridge, MA 02139. Tel.: 617-551-8757; E-mail: tummino@mpi.com.
Published, JBC Papers in Press, January 28, 2002, DOI 10.1074/jbc.M200581200
↵2 E. Calderwood, N. Dales, A. Gould, B. Guan, T. Ocain, and M. Patane, unpublished results.
↵3 Biologically active peptides not hydrolyzed by ACE2 (115 in total) are as follows: adrenocorticotrophic hormone 1–39 (human), adrenocorticotrophic hormone fragment 1–14, allatostatin I, allatostatin II, alytesin, amylin, β-amyloid peptide (1–28), angiotensin 1–9, angiotensin 1–7, angiotensin 1–5, antiflammin-1, antiflammin-2, atrial natriuretic peptide, bombesin, bradykinin, bradykinin fragment 1–7, brain injury-derived neurotrophic peptide brain natriuretic peptide-32 (porcine), caerulein, calcitonin (human), α-calcitonin gene-related peptide (human), cholecystokinin-8 sulfated, corticotropin-releasing factor (human), CSH 103, dermorphin, dynorphin A 1–17, dynorphin B (porcine), eledoisin, endomorphin-1, endomorphin-2, α-endorphin, β-endorphin (human), β-neoendorphin, endothelin, Met-enkephalin, enterostatin (human), fibronectin adhesion-promoting peptide, fibronectin fragment 1371–1382,N-formyl-Met-Leu-Phe, galanin (human), galantide, gastric inhibitory polypeptide, gastrin I (human), Arg-Arg-gastrin fragment 22–30 (human), gastrin-releasing peptide (human), glucagon, glucagon-like peptide 1 (7–37), glucagon-like peptide 1 fragment (7–36), glucagon-like peptide 2 guanylin (rat), histidyl-proline (cyclized form), inhibin β-subunit fragment 67–94 (human), isotocin, kemptide, (Trp⁴)-kemptide, (Val⁶, Ala⁷)-kemptide, kinetensin (human), leptin (full-length), leptin fragment 22–56 (human), Leu-enkephalin, litorin, luteinizing hormone-releasing hormone, malantide, mast cell-degranulating peptide HR1, mastoparan (wasp), melanin-concentrating hormone (human), α-melanocyte-stimulating hormone, β-melanocyte-stimulating hormone (human), γ-melanocyte-stimulating hormone, morphiceptin, motilin (porcine), myelin basic protein fragment 4–14 (bovine), neurogranin fragment 28–43, α-neurokinin, neurokinin A, neurokinin B, neuromedin B, neuromedin C, neuromedin K, neuromedin N, neuropeptide FF (porcine), neuropeptide K, neuropeptide Y (human), neurotensin (1–13), nociceptin, nocistatin (bovine), orcokinin, orexin A, orexin B, oxytocin, pancreastatin fragment 37–52, pancreatic polypeptide, parathyroid hormone 1–34 (human), peptide histidine methionine-27 (human), peptide T, peptide YY (human), pituitary adenylate cyclase-activating peptide 1–27, protein kinase C substrate, ranatensin, RGDS, sauvagine (frog), secretin (human), small, cardioactive peptide A, somatostatin-14, somatostatin-28, substance P, thyrotropin-releasing hormone, tyrosine protein kinase substrate, urocortin, urodilatin, valosin (porcine), vasoactive, intestinal peptide (human), (Arg⁸)-vasopressin, and (Arg⁸)-vasotocin.
Abbreviations:

ACE2

angiotensin-converting enzyme-related carboxypeptidase

ACE

angiotensin I-converting enzyme

Ang I

angiotensin I (1-10)

Ang II

angiotensin II (1-8)

Mca-APK(Dnp)

((7-methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH)

Mca-YVADAPK(Dnp)

(7-methoxycoumarin-4-yl)acetyl-Tyr-Val-Ala-Asp-Ala-Pro-Lys(2,4-dinitrophenyl)-OH

MALDI-TOF

matrix-assisted laser desorption ionization time-of-flight

HPLC

high pressure liquid chromatography

MES

4-morpholineethanesulfonic acid

CHES

2- (cyclohexylamino)ethanesulfonic acid

CAPS

3-(cyclohexylamino) propanesulfonic acid

bis-Tris propane

1,3-bis[tris(hydroxymethyl)methylamino]propane
- Received January 18, 2002.
The American Society for Biochemistry and Molecular Biology, Inc.