Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides

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dc.contributor.authorCezari, Maria Helena S [UNIFESP]
dc.contributor.authorPuzer, Luciano [UNIFESP]
dc.contributor.authorJuliano, Maria Aparecida [UNIFESP]
dc.contributor.authorCarmona, Adriana Karaoglanovic [UNIFESP]
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T12:33:35Z
dc.date.available2016-01-24T12:33:35Z
dc.date.issued2002-11-15
dc.description.abstractWe 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.en
dc.description.affiliationUniversidade Federal de São Paulo, Dept Biophys, Escola Paulista Med, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Biophys, Escola Paulista Med, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent365-369
dc.identifierhttp://dx.doi.org/10.1042/BJ20020840
dc.identifier.citationBiochemical Journal. London: Portland Press, v. 368, p. 365-369, 2002.
dc.identifier.doi10.1042/BJ20020840
dc.identifier.issn0264-6021
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/27038
dc.identifier.wosWOS:000179424700039
dc.language.isoeng
dc.publisherPortland Press
dc.relation.ispartofBiochemical Journal
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectfluorescent peptideen
dc.subjectfluorogenic substrateen
dc.subjectproteinaseen
dc.subjectthiol proteaseen
dc.titleCathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptidesen
dc.typeinfo:eu-repo/semantics/article
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