Reaction kinetics of protease with substrate phage - Kinetic model developed using stromelysin

Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reac tion kinetics between the enzyme and substrate phage are not well understoo d. Using a quantitative ELISA assay to monitor the disappearance of substra te, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage, We found that under the proteo lytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage w as a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be describe d by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that t he phage display selection process enriches clones that have high binding a ffinity to the protease, and the selection may not discriminate those of di fferent chemical reactivity toward the enzyme. Considering that multiple su bstrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the mo del was ascertained when we successfully applied it to determine the bindin g affinity of a competitive inhibitor of stromelysin.