Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis
Autor(a) principal: | |
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Data de Publicação: | 2000 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1590/S0100-879X2000000700006 http://hdl.handle.net/11449/19666 |
Resumo: | The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2. |
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spelling |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilismetalloproteasesubstrate specificityquenched fluorescence peptidesProteus mirabilisThe protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.Universidade de São Paulo (USP)Universidade Federal de São Paulo (UNIFESP)Universidade Estadual Paulista (UNESP)Instituto ButantanAssociação Brasileira de Divulgação Científica (ABRADIC)Universidade de São Paulo (USP)Universidade Federal de São Paulo (UNIFESP)Universidade Estadual Paulista (Unesp)Instituto ButantanFernandes, B. L.Anéas, M. A. F.Juliano, L.Palma, Mario Sergio [UNESP]Lebrun, I.Portaro, F. C. V.2014-05-20T13:54:58Z2014-05-20T13:54:58Z2000-07-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article765-770application/pdfhttp://dx.doi.org/10.1590/S0100-879X2000000700006Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 33, n. 7, p. 765-770, 2000.0100-879Xhttp://hdl.handle.net/11449/1966610.1590/S0100-879X2000000700006S0100-879X2000000700006S0100-879X2000000700006.pdf2901888624506535SciELOreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengBrazilian Journal of Medical and Biological Research1.492info:eu-repo/semantics/openAccess2023-12-13T06:22:55Zoai:repositorio.unesp.br:11449/19666Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-12-13T06:22:55Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
title |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
spellingShingle |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis Fernandes, B. L. metalloprotease substrate specificity quenched fluorescence peptides Proteus mirabilis |
title_short |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
title_full |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
title_fullStr |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
title_full_unstemmed |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
title_sort |
Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis |
author |
Fernandes, B. L. |
author_facet |
Fernandes, B. L. Anéas, M. A. F. Juliano, L. Palma, Mario Sergio [UNESP] Lebrun, I. Portaro, F. C. V. |
author_role |
author |
author2 |
Anéas, M. A. F. Juliano, L. Palma, Mario Sergio [UNESP] Lebrun, I. Portaro, F. C. V. |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Universidade de São Paulo (USP) Universidade Federal de São Paulo (UNIFESP) Universidade Estadual Paulista (Unesp) Instituto Butantan |
dc.contributor.author.fl_str_mv |
Fernandes, B. L. Anéas, M. A. F. Juliano, L. Palma, Mario Sergio [UNESP] Lebrun, I. Portaro, F. C. V. |
dc.subject.por.fl_str_mv |
metalloprotease substrate specificity quenched fluorescence peptides Proteus mirabilis |
topic |
metalloprotease substrate specificity quenched fluorescence peptides Proteus mirabilis |
description |
The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2. |
publishDate |
2000 |
dc.date.none.fl_str_mv |
2000-07-01 2014-05-20T13:54:58Z 2014-05-20T13:54:58Z |
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.uri.fl_str_mv |
http://dx.doi.org/10.1590/S0100-879X2000000700006 Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 33, n. 7, p. 765-770, 2000. 0100-879X http://hdl.handle.net/11449/19666 10.1590/S0100-879X2000000700006 S0100-879X2000000700006 S0100-879X2000000700006.pdf 2901888624506535 |
url |
http://dx.doi.org/10.1590/S0100-879X2000000700006 http://hdl.handle.net/11449/19666 |
identifier_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 33, n. 7, p. 765-770, 2000. 0100-879X 10.1590/S0100-879X2000000700006 S0100-879X2000000700006 S0100-879X2000000700006.pdf 2901888624506535 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Brazilian Journal of Medical and Biological Research 1.492 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
765-770 application/pdf |
dc.publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica (ABRADIC) |
publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica (ABRADIC) |
dc.source.none.fl_str_mv |
SciELO reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
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1799965274411106304 |