Mechanism of surface peptide proton exchange in bovine pancreatic trypsin inhibitor. Salt effects and O-protonation.

The acid-catalyzed hydrogen exchange rate constants kH, and the base-catalyzed rate constants kOH, have been determined (in the preceding paper) for the 25 most rapidly exchanging NH groups of bovine pancreatic trypsin inhibitor. Most of these NH groups are at the protein-solvent interface. The correlation of kH, but not kOH, with the static accessibility and hydrogen bonding of the peptide carbonyl O atom indicates that the mechanism of acid catalysis in proteins involves O-protonation. Agreement between the ionic strength dependence observed for kH and kOH and the ionic strength dependence calculated for an O-protonation mechanism supports this conclusion. N-protonation for acid catalysis, as well as N-deprotonation for base catalysis, have traditionally been assumed in the mechanism of the chemical step in peptide amide proton exchange. A preference for the alternative O-protonation mechanism has far-reaching implications in the interpretation of protein hydrogen exchange kinetics. With an O-protonation mechanism, acid-catalyzed rates of surface NH groups are primarily a function of the average solvent accessibility of the carbonyl O atoms in the dynamic solution structure, while base-catalyzed rates of surface NH groups measure solvent accessibility of the peptide N. The relative dynamic accessibilities of peptide O atoms, as measured by relative values of kH (corrected for electrostatic effects), correlate with O static accessibilities in the crystal structure. A lower correlation of static accessibility of N atoms with kOH is observed for surface NH groups in peptide groups in which the carbonyl O is not hydrogen bonded. For some surface NH groups, the observed pH of minimum rate, pHmin, deviates widely from the pHmin of model compounds. This is explained as the combined result of electrostatic effects and of the differences in accessibility of the carbonyl O and N atoms that result in a change in the relative values of kH and kOH as compared to those of model peptides. A mechanism whereby exchange of interior sites is catalyzed by interactions of catalysis ions with protein surface atoms via charge transfer is suggested.