Proton NMR studies of Cucurbita maxima trypsin inhibitors: evidence for pH-dependent conformational change and His25-Tyr27 interaction

A pH-dependent His25-Tyr27 interaction was demonstrated in the Cucurbita maxima trypsin inhibitors (CMTI-I and CMTI-III) by nuclear magnetic resonance spectroscopy. On the basis of peak shapes, line widths, deuterium exchange kinetics and nuclear Overhauser effect data, it was shown that an interaction between His25 and Tyr27 is triggered by the ionization of the histidine residue and that a hydrogen bond is formed between OH of Tyr27 and N(epsilon) of His25. Moreover, the relative orientations of His25 and Tyr27 arerelevant, because trypsin is active only above pH 6.