Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides
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Patrícia A. Bersanetti
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.
©2012 by Walter de Gruyter Berlin Boston
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Articles in the same Issue
- Masthead
- Masthead
- Guest Editorial
- Highlight: 29th Winter School on Proteinases and Their Inhibitors
- Highlight: 29th Winter School on Proteinases and their Inhibitors
- Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases
- Effects of cathepsin K deficiency on intercellular junction proteins, luminal mucus layers, and extracellular matrix constituents in the mouse colon
- Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins
- Cysteine cathepsins are not critical for TRAIL- and CD95-induced apoptosis in several human cancer cell lines
- Dissecting the impact of Frizzled receptors in Wnt/β-catenin signaling of human mesenchymal stem cells
- Overexpression of the urokinase receptor splice variant uPAR-del4/5 in breast cancer cells affects cell adhesion and invasion in a dose-dependent manner and modulates transcription of tumor-associated genes
- IGF-I receptor phosphorylation is impaired in cathepsin X-deficient prostate cancer cells
- Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF-β1 activation and reduces regulatory ID gene expression
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