Pepsinogen activation: genesis of the binding site

Abstract

Previous experiments have suggested that a substrate binding site may appear relatively early in the sequence of transformations tha pepsinogen undergoes during its unimolecular activation. To test, this possibility, tert-Boc-L-valyl-[3 (S)-hydroxy-4 (S)-amino-5-(2-naphthyl)pentanoyl]-L-alanylisoamylamide, a fluorescent analogue of pepstatin, has been used to measure the rate of appearance of the binding site. This probe is a potent inhibitor of pepsin, and its naphthyl group is an environmentally sensitive fluorophore that permits binding to be detected by fluorescence spectroscopy. In stopped-flow experiments, the fluorescence change following acidification of pepsinogen due to binding of the probe was found to obey a two-term expotential decay law. Analysis of the data obtained at various pH values permits us to rule out a sequential model for the kinetics. Rather, we were able to demonstrate that a concurrent model, with two species undergoing simultaneous transformation at different rates, is consistent with the results. The two species are related to each other by the protonation of a single site with a pKa of approximately 2.2. We obtained essentially identical results in an analysis of the early events observed in the acidification of pepsinogen labeled with 6-(p-toluidinyl)naphthalene-2-sulfonyl chloride (Auer and Glick, 1984). The protonic equilibrium probably occurs early after exposure to acid and is proposed to lead to a conformational change that freezes the distribution between the two species. It may be that these two conformers persist as identifiable entities at later stages of activation as well. In accord with this hypothesis, we find that Arrhenius energy of activation for the conversion to alkaline lability to vary over a range of pH values.