INTERACTION WITH D-GLUCOSE AND THERMAL-DENATURATION OF YEAST HEXOKINASE-B - A DSC STUDY

DSC measurements have been performed on the monomeric form of yeast he xokinase B in the absence and presence of increasing concentrations of D-glucose, The hexokinase, in the absence of D-glucose, at both pH 8. 0 and 8.5, shows reproducible calorimetric profiles characterized by t he presence of two partially overlapped peaks, These can be ascribed t o the presence of two structural domains in the native conformation of the enzyme, that possess different thermal stabilities and are denatu red more or less independently, In the presence of saturating and incr easing concentrations of D-glucose, the shape of the DSC profiles dram atically changes, since a single well-shaped peak is present, The bind ing of D-glucose enhances the interaction between the two lobes, as ev idenced by the shrinking of the protein in overall dimensions, and giv es rise to DSC profiles resembling those of a single domain protein. T o deconvolve the DSC curves we considered a denaturation model consist ing of two sequential steps with three macroscopic states of the prote in and the binding of D-glucose only to the native state. We carried o ut two-dimensional nonlinear regression of the excess heat capacity su rface constructed with the experimental DSC curves. This approach allo ws the calculation of a unique set of thermodynamic parameters charact erizing both the thermal denaturation of hexokinase, and the binding e quilibrium between D-glucose and the enzyme, It was found that the ass ociation constant is 9,800 +/- 1,500 M(-1) at pH 8.0, The binding of D -glucose is entropy-driven, since the binding enthalpy is zero, This f inding is rationalized by a thermodynamic cycle for the association of two molecules in aqueous solution.