Thermodynamic studies of the interaction of alpha-chymotrypsin with water. I. Determination of the isosteric enthalpies and entropies of water binding to the native enzyme.

The enthalpies (deltaH) and entropies (deltaS) of the interaction of water with alpha-chymotrypsin were evaluated from multitemperature sorption isotherms in the temperature range 283--313 K, determined in a fully automatized, computer controlled sorption apparatus. The temperature dependence of deltaH and deltaS shows a marked anomaly in the temperature range 295--298 K. The experimental results are interpreted by a phase transition of the enzyme protein, and by the existence of a low- and high-temperature conformer of alpha-chymotrypsin below and above the transition region. The two conformers differ significantly in their water binding energetics, as proved by two F-tests based on analyses of variance. The deltaH and deltaS versus water content functions of the high and low temperature conformer show markedly different anomalies at and below 70 mol H2O per mol protein. This water content corresponds closely to the monolayer volume vm, (as defined by Brunauer, Emmett and Teller). The protein surface covered by one water-monolayer, agrees well with the polar and charged surface area of chymotrypsin, computed from X-ray data. The experimental results suggest that the interaction of the first water-monolayer with the protein surface induces major conformational changes. The energetic contributions of these structural changes dominate the deltaH and deltaS terms below vm, giving rise to the anomalies observed. Above this water content, their influence is drastically reduced, and the isosteric quantities are predominantly determined by the water binding process per se. This process is possibly related to the pronounced enthalphy-entropy compensation pattern exhibited by the deltaH and deltaS terms. A more detailed analysis and discussion of this compensation effect will be given.