Thermodynamics of the binding of D-glucose to yeast hexokinase.

The binding of D-glucose to baker's yeast hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) was studied by isothermal and differential scanning calorimetry (DSC) and by fluorometric titration. The enthalpy and heat capacity changes associated with the binding of glucose were found to be nearly zero at both low and high ionic strengths over the temperature range from 7 to 29 degrees C. Thus, the free-energy change, amounting to -5.1 kcal mol(-1) at 25 degrees C and high ionic strength, is nearly independent of the temperature and is primarily of entropic origin. DSC study of the thermal unfolding of the free enzyme at low ionic strength gave an excess heat capacity curve with two maxima. This result appears to reflect a difference in thermal stability of the two domains in the hexokinase molecule which are indicated by X-ray crystallography [Bennett, W.S., & Steitz, T. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852]. In contrast, the unfolding of free enzyme at high ionic strength was fully cooperative. The excess heat capacity curve for the unfolding of the glucose-bound enzyme had only one peak at both low and high ionic strengths. This is consistent with the X-ray result that the binding of glucose induces a conformational change in the enzyme which brings the two lobes into close proximity. It is interesting that such a significant, molecule-wide conformational change is accompanied by only very small net changes in enthalpy and heat capacity.