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.
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.
Bersanetti
Patrícia A
PA
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.
Andrade
Maria Claudina C
MC
Casarini
Dulce E
DE
Juliano
Maria A
MA
Nchinda
Aloysius T
AT
Sturrock
Edward D
ED
Juliano
Luiz
L
Carmona
Adriana K
AK
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
D000806:Angiotensin-Converting Enzyme Inhibitors; D009842:Oligopeptides; D019151:Peptide Library; D007703:Peptidyl-Dipeptidase A