Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis
Open Access
- 1 July 2000
- journal article
- Published by FapUNIFESP (SciELO) in Brazilian Journal of Medical and Biological Research
- Vol. 33 (7), 765-770
- https://doi.org/10.1590/s0100-879x2000000700006
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.Universidade de São PauloUniversidade Federal de São Paulo (UNIFESP)Universidade Estadual de São PauloInstituto ButantanUNIFESPSciELKeywords
This publication has 10 references indexed in Scilit:
- Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substratesBiochemical Journal, 2000
- Characterisation of cholera toxin by liquid chromatography—Electrospray mass spectrometryToxicon, 1999
- Potential virulence factors of Proteus bacilli.Microbiology and Molecular Biology Reviews, 1997
- Molecular analysis of a metalloprotease from Proteus mirabilisJournal of Bacteriology, 1995
- Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairsLetters in Peptide Science, 1995
- [24] Serralysin and related bacterial proteinasesMethods in Enzymology, 1995
- Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreinsAnalytical Biochemistry, 1991
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- On the size of the active site in proteases. I. PapainBiochemical and Biophysical Research Communications, 1967
- Statistical estimations in enzyme kineticsBiochemical Journal, 1961