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

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
335
Issue
2
Identifiers
DOI: 10.1016/j.ab.2004.09.012
Keywords
  • Cysteine Peptidase
  • Lysosome
  • Amino Acids
  • Exopeptidase
Disciplines
  • Biology
  • Mathematics

Abstract

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 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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