Positional-Scanning Combinatorial Libraries of Fluorescence Resonance Energy Transfer Peptides for Defining Substrate Specificity of the Angiotensin I-Converting Enzyme and Development of Selective C-Domain Substrates ² Patrı ´cia A. Bersanetti, ‡ Maria Claudina C. Andrade, § Dulce E. Casarini, § Maria A. Juliano, ‡ Aloysius T. Nchinda, | Edward D. Sturrock, | Luiz Juliano, ‡ and Adriana K. Carmona* ,‡ Department of Biophysics and Department of Medicine, DiVision of Nephrology, Escola Paulista de Medicina, UniVersidade Federal de Sa ˜ o Paulo, Rua 3 de Maio 100, Sa ˜ o Paulo 04044-020, Brazil, and DiVision of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, UniVersity of Cape Town, ObserVatory 7925, Cape Town, South Africa ReceiVed July 23, 2004; ReVised Manuscript ReceiVed September 20, 2004 ABSTRACT: Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides were used for the analyses of the S 3 to S 1 ′ subsites of the somatic angiotensin I-converting enzyme (ACE). Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to design selective substrates for the C-domain. Initially, we defined the S 1 specificity by preparing a library with the general structure Abz-GXXZXK(Dnp)-OH [Abz ) o-aminobenzoic acid, K(Dnp) ) N ǫ -2,4- dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19 natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P 1 position had higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P 1 so we could define the C-domain selectivity of the S 1 ′,S 2 , and S 3 subsites. On the basis of the results from these libraries, we synthesized peptides Abz- GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domain selectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, which demonstrated the highest selectivity for the recombinant ACE C-domain (k cat /K m ) 36.7 µM -1 s -1 ) versus the N-domain (k cat /K m ) 0.51 µM -1 s -1 ). The substrate binding of Abz-LFK(Dnp)-OH with testis ACE using a combination of conformational analysis and molecular docking was examined, and the results shed new light on the binding characteristics of the enzyme. The angiotensin I-converting enzyme (ACE, 1 EC 3.4.15.1) is a zinc-dipeptidyl carboxypeptidase that plays an important role in blood pressure regulation by converting the inactive decapeptide angiotensin I to the potent vasopressor angio- tensin II (1). The enzyme is also able to hydrolyze bradykinin (2, 3) and other naturally occurring peptides such as substance P and luteinizing hormone-releasing hormone (4). Several ACE forms are present in mammalian tissues. A somatic isoform (150-180 kDa), which is expressed in endothelial, epithelial, and neuroepithelial cells, consists of two homologous domains (N- and C-domains) within a single polypeptide sequence, each domain possessing its own catalytic site (5-8). A smaller isoenzyme (90-110 kDa), found exclusively in male germinal cells, contains a single active site and corresponds to the C-domain of the somatic enzyme with the exception of a short unique N-terminal sequence (9-11). The recent description of the crystal structure of human testis ACE provided new details at the molecular level (12). Another form of human ACE that consists of only the N-domain was found in ileal fluid of patients undergoing surgery and is believed to be a result of limited proteolysis of the parent somatic form (13). ACE is primarily a membrane-bound protein, but a soluble form is found in many body fluids and is released from the cell surface by the action of a protein secretase or sheddase that cleaves the Arg1203-Ser1204 bond, 27 residues from the cell surface (14). The cytoplasmic domain of ACE is not necessary for sheddase recognition but is involved in modulation of ACE shedding (15). The C- and N-domains of ACE are functional and share a high degree of homology, particularly at the active centers, but they differ in substrate specificities, inhibitor and chloride profiles (16-19), and their stability under denaturing condi- tions (20). The active sites of both domains cleave angio- tensin I, substance P, and bradykinin with similar efficiency ² This work was supported by the Brazilian Agencies Fundac ¸ a ˜o de Amparo a Pesquisa do Estado de Sa ˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico (CNPq) and by the Wellcome Trust, U.K., and National Research Foundation. * To whom correspondence should be addressed: Universidade Federal de Sa ˜o Paulo, Departamento de Biofı ´sica, Rua Tre ˆs de Maio 100, Sa ˜o Paulo 04044-020, SP Brazil. Telephone: 55-11-55764450. Fax: 55-11-55759617. E-mail: adriana@biofis.epm.br. ‡ Department of Biophysics, Division of Nephrology, Escola Paulista de Medicina, Universidade Federal de Sa ˜o Paulo. § Department of Medicine, Division of Nephrology, Escola Paulista de Medicina, Universidade Federal de Sa ˜o Paulo. | University of Cape Town. 1 Abbreviations: ACE, angiotensin I-converting enzyme; Abz, o-aminobenzoic acid; CVFF, consistent valence force field; Dnp, 2,4- dinitrophenyl; ESFF, extensible systematic force field; FRET, fluores- cence resonance energy transfer; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PS-SC, positional-scanning syn- thetic combinatorial. 15729 Biochemistry 2004, 43, 15729-15736 10.1021/bi048423r CCC: $27.50 © 2004 American Chemical Society Published on Web 11/20/2004