MICROCALORIMETRIC AND KINETIC STUDIES OF THE α-CHYMOTRYPSIN – HYDROCINNAMIC ESTER – HYDROCINNAMIC ACID SYSTEM

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Abstract
Using a microcalorimeter of the Tian–Calvet type the heat of binding of hydrocinnamic acid to a-chymotrypsin has been measured in aqueous solution over a range of pH values. The heat evolved was found to vary from about 5 to about 28 kcal per mole as the pH was varied from 5.1 to 7.8. In order to see whether these heats are due to the binding of the inhibitor at the active center of the enzyme, heats were also measured at pH 7.1 by a kinetic method, the inhibitor constant being determined over a range of temperatures; the heat calculated in this way was consistent with the microcalorimetric value. The kinetic work also gave rise to entropies of binding of the inhibitor and the substrate (methyl hydrocinnamate); these entropies are negative, the significance of which is discussed in terms of the mode of binding. It is suggested that the results are best explained on the hypothesis that the active center of the enzyme contains, in addition to the ordinary acidic and basic sites, a negatively charged group that interacts electrostatically with the anion of hydrocinnamic acid.