MECHANISM OF ISOTOPE EXCHANGE KINETICS OF SINGLE PROTONS IN BOVINE PANCREATIC TRYPSIN-INHIBITOR

The hydrogen isotope exchange kinetics of the slowest exchanging proton resonances in the bovine pancreatic trypsin inhibitor nuclear magnetic resonance spectrum were measured from pH 1 to 12, 33-68 °C. The pH dependence and the apparent activation energy for each proton vary with temperature. The kinetics for each proton are explained by a model in which exchange is governed by two discrete conformational processes that differ in temperature dependence. One process is related to thermal unfolding, and kinetics for exchange by this pathway are of high activation energy,~60 kcal/mol, and about half-order in OH- ion. The second is a dynamical process of the folded conformation, and kinetics for exchange by this process give an activation energy of 20-35 kcal/mol with variable pH dependence approaching first order in catalyst ion. Since the chemical exchange step has an@r@nactivation energy of ≃ 20 kcal/mol, the enthalpies of the two conformational processes are ≃ 40 and 0-15 kcal/mol, respectively. The model is simple, has a precedent in the hydrogen-exchange literature, and predicts the complex features of the pH and temperature dependence of the single proton exchange rates, including the data of Richarz et al. [Richarz, P., Sehr, P., Wagner, G., & Wuthrich, K. (1979) J. Mol. Biol. 130, 19] and Wagner & Wuthrich [Wagner, G„ & Wuthrich, K. (1979) J. Mol. Biol. 130, 31], For the two slowest exchanging protons, the rates at 51 °C show a pH-independent plateau between pH 8.4 and 9.6. In the context of our model, comparison with data for the same resonances at 45 °C suggests that the high activation energy conformational process is rate limiting at pH >8.4, 51 °C, and the rate of exposure to solvent is equal to the observed exchange rate, 5ËŸ10-2 h-1. © 1979, American Chemical Society. All rights reserved.