title: Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides.
abstract: Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ′ positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 μ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.
authors.affiliation: Health Informatics Department, Universidade Federal de S ã o Paulo, Rua Botucatu 862, São Paulo, 04023-062, SP, Brazil.
authors.forename: Patrícia A
authors.initials: PA
authors.lastname: Bersanetti
authors.forename: Regiane A
authors.initials: RA
authors.lastname: Sabatini
authors.forename: Beatriz S
authors.initials: BS
authors.lastname: Matos
authors.forename: Ross G
authors.initials: RG
authors.lastname: Douglas
authors.forename: Aloysius
authors.initials: A
authors.lastname: Nchinda
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: João Bosco
authors.initials: JB
authors.lastname: Pesquero
authors.forename: Edward D
authors.initials: ED
authors.lastname: Sturrock
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D019151:Peptide Library; D010455:Peptides; D007703:Peptidyl-Dipeptidase A