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 N(epsilon)-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 P(1) position. To examine the selectivity of S(1)('), S(2), and S(3) 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 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.
Cotrin
Simone Silva
SS
Department of Biophysics, Escola Paulista de Medicina, UNIFESP, Rua Três de Maio, 100, São Paulo 04044-020, Brazil.
Puzer
Luciano
L
de Souza Judice
Wagner Alves
WA
Juliano
Luiz
L
Carmona
Adriana K
AK
Juliano
Maria Aparecida
MA
D002268:Carboxypeptidases / Q000378:metabolism*; D002401:Cathepsin B / Q000378:metabolism*; D004150:Dipeptidases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates
D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C007493:epsilon-dinitrophenyllysine; D002268:Carboxypeptidases; D004150:Dipeptidases; D002401:Cathepsin B; D008239:Lysine