Please use this identifier to cite or link to this item: https://repositorio.unifesp.br/handle/11600/27038
Title: Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides
Authors: Cezari, Maria Helena S [UNIFESP]
Puzer, Luciano [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Carmona, Adriana Karaoglanovic [UNIFESP]
Juliano, Luiz [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
Keywords: fluorescent peptide
fluorogenic substrate
proteinase
thiol protease
Issue Date: 15-Nov-2002
Publisher: Portland Press
Citation: Biochemical Journal. London: Portland Press, v. 368, p. 365-369, 2002.
Abstract: We have examined in detail the specificity of the subsites S-1, S-2, S-1' and S-2' for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Drip (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o-aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or epsilon-amino-Dnp-Lys, as the fluorescence donor-receptor pair. the higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. the subsite S-1 accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P-1 had lower K-m values. Despite the presence of Glu(245) at S-2, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P-2 was hydrolysed better than that containing an arginine residue. S-1' is essentially a hydrophobic subsite, and S-2' has particular preference for phenylalanine or tryptophan residues.
URI: http://repositorio.unifesp.br/handle/11600/27038
ISSN: 0264-6021
Other Identifiers: http://dx.doi.org/10.1042/BJ20020840
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