Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: assays with human cathepsin B

2004 ◽  
Vol 335 (2) ◽  
pp. 244-252 ◽  
Author(s):  
Simone Silva Cotrin ◽  
Luciano Puzer ◽  
Wagner Alves de Souza Judice ◽  
Luiz Juliano ◽  
Adriana K. Carmona ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Substrate Specificity ◽  
Fluorescence Resonance Energy Transfer ◽  
Cathepsin B ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Combinatorial Libraries ◽  
Positional Scanning ◽  
Human Cathepsin ◽  
Fluorescence Resonance

Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides

2012 ◽  
Vol 393 (12) ◽  
pp. 1547-1554 ◽  
Author(s):  
Patrícia A. Bersanetti ◽  
Regiane A. Sabatini ◽  
Beatriz S. Matos ◽  
Ross G. Douglas ◽  
Aloysius Nchinda ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Combinatorial Libraries ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Angiotensin I Converting Enzyme ◽  
Positional Scanning ◽  
Fluorescence Resonance

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 P3 to P1′ positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz=ortho-aminobenzoic acid; Dnp=2,4-dinitrophenyl) showed the highest selectivity for ACE N-domain (kcat/Km=1.76 μm-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.

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