Reaction Kinetics of Protease with Substrate Phage

摘要

Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin. Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin. bovine serum albumin colony-forming unit counts per second horseradish peroxidase mouse monoclonal antibody 179 enzyme-linked immunosorbent assay Since the first reports of the display of randomized peptide libraries on the surface of filamentous bacteriophages (1Cwirla S.E. Peters E.A. Barrett R.W. Dower W.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6378-6382Crossref PubMed Scopus (854) Google Scholar, 2Devlin J.J. Panganiban L.C. Delvin P.E. Science. 1990; 249: 404-406Crossref PubMed Scopus (824) Google Scholar, 3Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1900) Google Scholar), phage display has become a widely adopted approach for the mapping of antibody epitopes and macromolecule-binding peptides (4Cochran A.G. Chem. Biol. 2000; 7: R85-R94Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 5Zwick M.B. Shen J. Scott J.K. Curr. Opin. Biotechnol. 1998; 9: 427-436Crossref PubMed Scopus (117) Google Scholar, 6Souroujon M.C. Mochlyrosen D. Nat. Biotechnol. 1998; 16: 919-924Crossref PubMed Scopus (201) Google Scholar), the selection of peptide agonists or antagonists against potential drug targets (7Lowman H.B. Annu. Rev. Biophys