title: 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.
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(epsilon)-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 microM(-1) s(-1)) versus the N-domain (k(cat)/K(m) = 0.51 microM(-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.
authors.affiliation: Department of Biophysics, Division of Nephrology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 3 de Maio 100, São Paulo 04044-020, Brazil.
authors.forename: Patrícia A
authors.initials: PA
authors.lastname: Bersanetti
authors.forename: Maria Claudina C
authors.initials: MC
authors.lastname: Andrade
authors.forename: Dulce E
authors.initials: DE
authors.lastname: Casarini
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Aloysius T
authors.initials: AT
authors.lastname: Nchinda
authors.forename: Edward D
authors.initials: ED
authors.lastname: Sturrock
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
mesh_terms: D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D019151:Peptide Library; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D009842:Oligopeptides; D019151:Peptide Library; D007703:Peptidyl-Dipeptidase A