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 hexokinase 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 the presence of two partially overlapped peaks. These can be ascribed to the presence of two structural domains in the native conformation of the enzyme, that possess different thermal stabilities and are denatured more or less independently. In the presence of saturating and increasing concentrations of D-glucose, the shape of the DSC profiles dramatically changes, since a single well-shaped peak is present. The binding of D-glucose enhances the interaction between the two lobes, as evidenced by the shrinking of the protein in overall dimensions, and gives rise to DSC profiles resembling those of a single domain protein. To deconvolve the DSC curves we considered a denaturation model consisting of two sequential steps with three macroscopic states of the protein and the binding of D-glucose only to the native state. We carried out two-dimensional nonlinear regression of the excess heat capacity surface constructed with the experimental DSC curves. This approach allows the calculation of a unique set of thermodynamic parameters characterizing both the thermal denaturation of hexokinase, and the binding equilibrium between D-glucose and the enzyme. It was found that the association 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 finding is rationalized by a thermodynamic cycle for the association of two molecules in aqueous solution.
Catanzano
F
F
Dipartimento di Chimica, Università di Napoli Federico II Via Mezzocannone, Italy.
Gambuti
A
A
Graziano
G
G
Barone
G
G
D002152:Calorimetry, Differential Scanning; D005947:Glucose / Q000737:chemistry*; D006593:Hexokinase / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013816:Thermodynamics
D006593:Hexokinase; D005947:Glucose