New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops

Página do item simplificado
dc.contributor.authorRehault, S.
dc.contributor.authorBrillard-Bourdet, M.
dc.contributor.authorJuliano, Maria Aparecida [UNIFESP]
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.authorGauthier, F.
dc.contributor.authorMoreau, T.
dc.contributor.institutionUniv Tours
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T12:30:49Z
dc.date.available2016-01-24T12:30:49Z
dc.date.issued1999-05-14
dc.description.abstractCathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha(1)-antichymotrypsin loop were the most sensitive for cathepsin G; with k(cat)/K-m values of 5-20 mM(-1) s(-1). Substitutions were introduced at positions P-1 and P-2 in alpha(1)-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant k(cat)/K-m of 150 mM(-1) s(-1). Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S-1 subsite, subsites S-1' and S-2' significantly contribute to the definition of the substrate specificity of cathepsin G.en
dc.description.affiliationUniv Tours, Enzymol & Prot Chem Lab, F-37032 Tours, France
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent13810-13817
dc.identifierhttp://dx.doi.org/10.1074/jbc.274.20.13810
dc.identifier.citationJournal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 274, n. 20, p. 13810-13817, 1999.
dc.identifier.doi10.1074/jbc.274.20.13810
dc.identifier.issn0021-9258
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/26080
dc.identifier.wosWOS:000080322200014
dc.language.isoeng
dc.publisherAmer Soc Biochemistry Molecular Biology Inc
dc.relation.ispartofJournal of Biological Chemistry
dc.rightsinfo:eu-repo/semantics/openAccess
dc.titleNew, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loopsen
dc.typeinfo:eu-repo/semantics/article
Arquivos
Coleções
EPM - Artigos