CALORIMETRIC DETERMINATION OF LINKAGE EFFECTS INVOLVING AN ACYL-ENZYME INTERMEDIATE

Enthalpy changes of α-chymotrypsin acylation by 3-(2-furyl)acryloyliniidazole (FAI) wore calorimetrically determined as a function of pH. By observing the functional dependence of acylation enthalpies on buffer ionization heats, a complex pH profile was obtained describing proton release accompanying formation of acyl-enzyme. A pKa of 4.0 for FAI ionization and apparent pKa values of 6.8, 7.55 and 8.8 on the enzyme were used to account for the proton release data. A model which accounts for the proton release behavior was used to fit the acylation enthalpy data and values for the apparent dissociation enthalpies of the groups involved were obtained along wih a pH-independent intrinsic enthalpy of acylation. This model suggests a group with an apparent pK = 6.8 and ΔHion = 8.7 kcal/mol which is perturbed to a pK of 7.55 and ΔHion = 7.6 kcal/mol on attachment of the acyl moiety to the enzyme. The apparent ionization enthalpy change for the active-inactive transition (pK3 = 8.8; ΔH= 3.0 kcal/mol) corresponds with that calculated from the data of Fersht (J. Mol. Biol. 64 (1972) 497). The pH-independent intrinsic enthalpy of acylation (ΔH = -7.9 kcal/mol) is corrected for group ionizations linked to the acylation process. Consequently, it more closely reflects molecular processes of interest such as substrate binding, covalent bond rearrangement, and product release. © 1990.