EFFECT OF INHIBITOR COMPLEX-FORMATION ON THE HYDRATION PROPERTIES OF ALPHA-CHYMOTRYPSIN - CHANGES INDUCED IN PROTEIN HYDRATION BY TOSYLATION OF THE NATIVE ENZYME

The effect of enzyme‐inhibitor complex formation on the hydration properties of the macromolecular moiety was investigated on the model system of α‐chymotrypsin and its Ser‐195 tosyl derivative. The primary (A‐shell) hydration of the native and modified enzyme was compared by sorption measurements. The secondary (B‐shell) hydration water was investigated by differential scanning calorimetry. Tosylation is known to induce pronounced conformational changes in the chymotrypsin molecule. These structural modifications have the following effects on the hydration of the native enzyme. The water binding capacity of the protein surface is significantly increased, as shown by both the calorimetric and the sorption results. The amount of unfreezable water of primary hydration is increased by 50 mol H2O/mol chymotrypsin. The heats (ΔH) and entropies (ΔS) of the interaction of water with chymotrypsin are strongly reduced in the modified enzyme. This effect is interpretable by a reduction of the H bonding potential of the protein surface. Parallel to this decrease in δH, the heats of fusion of the secondary hydration water (Qfus) are significantly increased by tosylation (Qfus = 256.2 ± 7.8 and 294.2 ± 4.8 J g−1 H2O for the native and the tosylated enzyme, respectively). This increase in Qfus reflects an increase in the extent of H bonding in the B‐shell hydration sphere. These changes in the hydration of the native enzyme, associated with the reaction: native chymotrypsin → tosylchymotrypsin, are interpreted by cooperative phase transitions of water molecules in the primary and secondary hydration water. One of these transitions was found to exhibit a significant, linear enthalpy–entropy compensation effect. The compensation temperature documentclass{article}pagestyle{empty}begin{document}$ hat{beta} $end{document} is 290.7 ± 2.8°K. This documentclass{article}pagestyle{empty}begin{document}$ hat{beta} $end{document} value agrees well with compensation temperatures reported in the literature for a series of biochemical reactions in aqueous solution (250–320° K). This agreement in documentclass{article}pagestyle{empty}begin{document}$ hat{beta} $end{document} may point to a common source of both compensation phenomena. Copyright © 1979 John Wiley & Sons, Inc.