Static and Kinetic Studies on the Binding between Pepsin and Streptomyces Pepsin Inhibitor with a Fluorescent Probe

The fluorescence of pepsin-bound 2-p-toluidinylnaphthalene-6-sulfonate (TNS) decreases upon the binding of Streptomyces pepsin inhibitor (SPI) with the enzyme. Equilibrium dialysis experiments showed this decrease to arise from the release of TNS from pepsin due to the binding of SPI in a molar ratio of 1: 1. The results of stopped-flow kinetic study on the concentration dependence of the apparent rate constant for the binding reaction between pepsin and SPI using the fluorescence decrease of TNS as a probe were consistent with the interpretation that the binding reaction consists of at least two steps, including a fast bimolecular step followed by a slow unimolecular step. It was found that a fluorescence decrease occurred in both steps. The rate-pH profile sug-gested the participation of some ionizable group(s) (possibly a carboxyl group) in the binding. Studies of interactions between an enzyme and substrate analogs offer useful information on the. structure of the active site and the reaction mech-anism of the enzyme. Pepstatin (/), a pentapeptide from Streptomyces, is regarded as a substrate analog for pepsin and several other acid proteinases. Marciniszyn et al. suggested, based on their studies with pepstatin and its derivatives, that pepstatin can be regarded as a transition state analog (2). Streptomyces pepsin inhibitor (SPI) (3, 4), which was isolated by Murao and Satoi (5), has a molec-ular structure very similar to that of pepstatin (6)