Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Author Fernandes, B.l. Google Scholar
Anéas, M.a.f. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Palma, M.s. Google Scholar
Lebrun, Ivo Google Scholar
Portaro, Fernanda Calheta Vieira Autor UNIFESP Google Scholar
Institution Universidade de São Paulo (USP)
Universidade Federal de São Paulo (UNIFESP)
Universidade Estadual de São Paulo
Instituto Butantan
Abstract 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.
Keywords metalloprotease
substrate specificity
quenched fluorescence peptides
Proteus mirabilis
Language English
Date 2000-07-01
Published in Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 33, n. 7, p. 765-770, 2000.
ISSN 0100-879X (Sherpa/Romeo, impact factor)
Publisher Associação Brasileira de Divulgação Científica
Extent 765-770
Origin http://dx.doi.org/10.1590/S0100-879X2000000700006
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000088236100006
SciELO ID S0100-879X2000000700006 (statistics in SciELO)
URI http://repositorio.unifesp.br/handle/11600/992

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