Dependence on pH of the Free Energy and Enthalpy of Interaction of Monosaccharides and Disaccharides with Lysozyme

Abstract Heats of association of lysozyme with N-acetylglucosamine and β-methyl-N-acetylglucosaminide were measured calorimetrically at 0.1 ionic strength and 30° over the pH range 2 to 7. The values found for ΔH0 at pH 5 are -5900 cal per mole for N-acetylglucosamine and -5500 cal per mole for the glycoside. The value of ΔH0 for binding of the glycoside is 1600 cal per mole more positive at acid pH than at neutral pH. The pH dependence of ΔH0 defines an apparent pK of 3.8 for a group in the lysozyme active site. Kinetic studies of the lysozyme-catalyzed hydrolysis of the hexasaccharide of N-acetylglucosamine (Banerjee, S. K., Kregar, I., Turk, V., and Rupley, J. A. (1973) J. Biol. Chem. 248, 4786) previously have demonstrated that an ionization that is important for catalysis also exhibits an apparent pK of 3.8 and is assignable to Asp-52. Equilibrium constants for the association of lysozyme with N-acetylglucosamine and the β-methyl glycoside were determined using absorbance measurements over the pH range 0 to 9 at 0.1 ionic strength and 25°. The dependence on pH of the free energy of association for the methyl glycoside indicates that complex formation perturbs two ionizable groups of lysozyme, reducing the apparent pK of one (Asp-66) from 1.5 to 1.2 and increasing the apparent pK of the other (Glu-35) from 6.1 to 6.6. Because of enthalpyentropy compensation, the group of pK 3.8 that is observed in calorimetric experiments with β-methyl-N-acetylglucosaminide is not distinguished by the free energy measurements. The pH dependence of the interaction of N-acetylglucosamine with lysozyme is consistent with perturbation of three ionizable groups through complex formation. Heats of reaction and equilibrium constants for the association of lysozyme with the disaccharides N-acetylglucosaminyl-β(1→4)-glucose and -xylose were determined at 0.1 ionic strength, 30° and pH 2.5 or 5. The enthalpies of association at pH 5 for the disaccharides are -2700 and -500 cal per mole, respectively. The free energies of association are similar to that for N-acetylglucosamine.