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 positional-scanning 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 mu 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.