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

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 Nε-2,4-dinitrophenyl-lysine with free carboxyl group, the Z position was successively oc...

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Bibliographic Details
Published in:Analytical biochemistry Vol. 335; no. 2; pp. 244 - 252
Main Authors: Cotrin, Simone Silva, Puzer, Luciano, de Souza Judice, Wagner Alves, Juliano, Luiz, Carmona, Adriana K., Juliano, Maria Aparecida
Format: Journal Article
Language:English
Published: United States Elsevier Inc 15-12-2004
Subjects:
Amino acids
Carboxypeptidases - metabolism
Cathepsin B - metabolism
Cysteine peptidase
Dipeptidases - metabolism
Exopeptidase
Fluorescence Resonance Energy Transfer
Humans
Hydrolysis
Lysine - analogs & derivatives
Lysosome
ortho-Aminobenzoates
Peptide Library
Peptides - chemical synthesis
Peptides - metabolism
Recombinant Proteins - metabolism
Substrate Specificity
Cysteine peptidase
Amino acids
Exopeptidase
Lysosome
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Summary: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 Nε-2,4-dinitrophenyl-lysine with free carboxyl group, the Z position was successively occupied with 1 of 19 amino acids (cysteine 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 P1 position. To examine the selectivity of S1′, S2, and S3 subsites, the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH were then synthesized. The peptide Abz-GIVRAK(Dnp)-OH, which contains the most favorable residues in the P3–P1′ positions identified by screening of the libraries with cathepsin B, was hydrolyzed by this enzyme with kcat/Km=7288mM−1s−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.
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ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2004.09.012