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ones most susceptible for proteolysis. No enzyme activity was observed for library with Proin discussed position.StpA and StpB displayed broad specificity in position P 1 . They were able to hydrolyzewith almost the same rate substrates with Lys, Leu, Asp (StpA) and Gly, Lys and Pro(StpB) in this position. This is not the case for StpC which very efficiently hydrolyzed onlyone peptide with Asp in position P 1 . Fluorescence of all other peptides is several timeslower.Based on the performed kinetic investigations, several FRET peptides that could serveas efficient substrates of Staphylococcal aureus cystein proteinases were selected. For StpAthe best substrate sequence is ABZ-Phe-Gly-Ala-Lys-ANB-NH 2 , StpB: ABZ-Ile-Ala-Ala-Gly-ANB-NH 2 and StpC ABZ-Ile-Ala-Lys-Asp-ANB-NH 2 . Their determined kineticparameters are listed in Table 1.Table 1. Kinetic characteristic of fluorescent substratesSubstrateEnzymek cat[s -1 ]×10 1 K M[M]×10 6k cat / K M[s -1 ×M -1 ] ×10 -3ABZ-Phe-Gly-Ala-Lys-ANB-NH 2ABZ-Ile-Ala-Ala-Gly-ANB-NH 2ABZ-Ile-Ala-Lys-Asp-ANB-NH 2StpA 7.16±8.26×10 -2 5.6±0.4 127.8±7.4StpB 1.12±0.47×10 -2 154.6±15.5 0.7±0.1StpC 0.23±0.03×10 -2 385.3±20.4 below 0.1StpA 0.56±0.03×10 -2 163.9±12.2 3.4±0.2StpB 8.86±6.02×10 -2 7.6±1.1 118.0±12.2StpC 0.02±0.01×10 -2 271.4±10.2 below 0.1StpA 0.32±0.11×10 -2 587.1±48.1 below 0.1StpB 0.12±0.07×10 -2 612.9±52. 7 below 0.1StpC 8.91±5.86×10 -3 14.3±1.35 62.4±3.1Primary specificity of enzymes studied are in good agreement with crystallographic dataprovided by Filipek and coworkers [5] for staphopain B – staphostatin B (inhibitor)complex. In position P 1 they observed specific conformation of the peptide chain that couldbe adopted only by Gly residue. It's worth emphasizing that staphostatin is a largepolypeptide consisted of more than 100 amino acid residues with defined structure. Sincethe library used in this study are tetrapeptides that display much broader flexibility withinits peptide chain, there is no surprise that we observed more than one rapidly hydrolyzedsequence.We would like to emphasize that to our knowledge this is the first comparative reportthat describes the substrate specificity of three cysteine proteases originated fromStaphylococcus aureus.AcknowledgmentsThis work was supported by Ministry of Science and Higher Education under grant1600/B/H03/2009/36.References1. Potempa, J., Dubin, A., Korzus, G., Travis, J. J. Biol. Chem. 263, 2664-2667 (1988).2. Arvidson, S., Fischetti, V.A., Novick, R.P., Ferretti, J.J., Potrnoy, D.A., Rood, J.I. (Eds.) Grampositivepathogens. American Society for Microbiology, Washington D.C, 2000, p.379.3. Maeda, H., Yamamoto, T. Biol. Chem. Hoppe-Seyler 377, 217-226 (1996).4. Dubin, G. Acta Biochim. Pol. 50, 715-724 (2003).5. Filipek, R., Rzychon, M., Oleksy, A., Gruca, M., Dubin, A., Potempa, J., Bochtler, M. J. Biol.Chem. 278, 40959-40966 (2008).203