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

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

Author Cotrin, S. S. Google Scholar
Puzer, L. Google Scholar
Judice, WAD Google Scholar
Juliano, L. Google Scholar
Carmona, A. K. Google Scholar
Juliano, M. A. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract We have developed positional scanning synthetic combinatorial libraries to define the substrate specificity of carboxydipeptidases. the library Abz-GXXZXK(Dnp)-OH, where Abz is ortho-aminobenzoic acid, K(Dnp) is N-c-2,4-dinitrophenyl-lysine with free carboxyl group, the Z position was successively occupied with 1 of 19 amino acids (eysteine was omitted), and X represents randomly incorporated residues, was assayed initially with human cathepsin B, and arginine was defined as one of the best residues at the P, position. To examine the selectivity of S-1(1) S-2, and S-3 subsites, the sublibraries Abz-GXXRZK(Dnp)-OH, AbzGXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH were then synthesized. the peptide Abz-GIVRAK(Dnp)-OH, which contains the most favorable residues in the P-3-P-1, positions identified by screening of the libraries with cathepsin B, was hydrolyzed by this enzyme with k(cat)/K-m = 7288 mM(-1) s(-1). This peptide is the most efficient substrate described for cathepsin B to this point, and it is highly selective for the enzyme among the lysosomal cysteine proteases. (C) 2004 Elsevier Inc. All rights reserved.
Keywords cysteine peptidase
lysosome
amino acids
exopeptidase
Language English
Date 2004-12-15
Published in Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 335, n. 2, p. 244-252, 2004.
ISSN 0003-2697 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 244-252
Origin http://dx.doi.org/10.1016/j.ab.2004.09.012
Access rights Closed access
Type Article
Web of Science ID WOS:000225502800009
URI http://repositorio.unifesp.br/handle/11600/28050

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