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

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Cotrin, S. S.
Puzer, L.
Judice, WAD
Juliano, L.
Carmona, A. K.
Juliano, M. A.
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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.
Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 335, n. 2, p. 244-252, 2004.