Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides

Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides

Author Cezari, Maria Helena S Autor UNIFESP Google Scholar
Puzer, Luciano Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Carmona, Adriana Karaoglanovic Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
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.
Keywords fluorescent peptide
fluorogenic substrate
proteinase
thiol protease
Language English
Date 2002-11-15
Published in Biochemical Journal. London: Portland Press, v. 368, p. 365-369, 2002.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press
Extent 365-369
Origin http://dx.doi.org/10.1042/BJ20020840
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000179424700039
URI http://repositorio.unifesp.br/handle/11600/27038

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