New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops
Autor(a) principal: | |
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Data de Publicação: | 1999 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/26080 http://dx.doi.org/10.1074/jbc.274.20.13810 |
Resumo: | Cathepsin 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. |
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Rehault, S.Brillard-Bourdet, M.Juliano, Maria Aparecida [UNIFESP]Juliano, Luiz [UNIFESP]Gauthier, F.Moreau, T.Univ ToursUniversidade Federal de São Paulo (UNIFESP)2016-01-24T12:30:49Z2016-01-24T12:30:49Z1999-05-14Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 274, n. 20, p. 13810-13817, 1999.0021-9258http://repositorio.unifesp.br/handle/11600/26080http://dx.doi.org/10.1074/jbc.274.20.1381010.1074/jbc.274.20.13810WOS:000080322200014Cathepsin 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.Univ Tours, Enzymol & Prot Chem Lab, F-37032 Tours, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Science13810-13817engAmer Soc Biochemistry Molecular Biology IncJournal of Biological ChemistryNew, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loopsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP11600/260802023-01-12 21:52:29.262metadata only accessoai:repositorio.unifesp.br:11600/26080Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-01-13T00:52:29Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
title |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
spellingShingle |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops Rehault, S. |
title_short |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
title_full |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
title_fullStr |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
title_full_unstemmed |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
title_sort |
New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops |
author |
Rehault, S. |
author_facet |
Rehault, S. Brillard-Bourdet, M. Juliano, Maria Aparecida [UNIFESP] Juliano, Luiz [UNIFESP] Gauthier, F. Moreau, T. |
author_role |
author |
author2 |
Brillard-Bourdet, M. Juliano, Maria Aparecida [UNIFESP] Juliano, Luiz [UNIFESP] Gauthier, F. Moreau, T. |
author2_role |
author author author author author |
dc.contributor.institution.none.fl_str_mv |
Univ Tours Universidade Federal de São Paulo (UNIFESP) |
dc.contributor.author.fl_str_mv |
Rehault, S. Brillard-Bourdet, M. Juliano, Maria Aparecida [UNIFESP] Juliano, Luiz [UNIFESP] Gauthier, F. Moreau, T. |
description |
Cathepsin 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. |
publishDate |
1999 |
dc.date.issued.fl_str_mv |
1999-05-14 |
dc.date.accessioned.fl_str_mv |
2016-01-24T12:30:49Z |
dc.date.available.fl_str_mv |
2016-01-24T12:30:49Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 274, n. 20, p. 13810-13817, 1999. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/26080 http://dx.doi.org/10.1074/jbc.274.20.13810 |
dc.identifier.issn.none.fl_str_mv |
0021-9258 |
dc.identifier.doi.none.fl_str_mv |
10.1074/jbc.274.20.13810 |
dc.identifier.wos.none.fl_str_mv |
WOS:000080322200014 |
identifier_str_mv |
Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 274, n. 20, p. 13810-13817, 1999. 0021-9258 10.1074/jbc.274.20.13810 WOS:000080322200014 |
url |
http://repositorio.unifesp.br/handle/11600/26080 http://dx.doi.org/10.1074/jbc.274.20.13810 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.none.fl_str_mv |
Journal of Biological Chemistry |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
13810-13817 |
dc.publisher.none.fl_str_mv |
Amer Soc Biochemistry Molecular Biology Inc |
publisher.none.fl_str_mv |
Amer Soc Biochemistry Molecular Biology Inc |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
|
_version_ |
1802764118257565696 |