Compulsory order of substrate binding to herpes simplex virus type 1 thymidine kinase - A calorimetric study

Isothermal titration calorimetry has been used to investigate the thermodyn amic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK), Binding follows a sequential pathw ay in which dT binds first and ATP second. The free enzyme does not bind AT P, whose binding site becomes only accessible in the HSV1 TK dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) M-1 for dT and 3.9 x 10(6) M-1 for ATP binding to the binary HSV1 TK dT complex. Bindi ng of both substrates is enthalpy-driven and opposed by a large negative en tropy change. The heat capacity change (Delta Cp) obtained from Delta H in the range of 10-25 degrees C is -360 cal K-1 mol(-1) for dT binding and -14 0 cal K-1 mol(-1) for ATP binding. These large Delta Cp values are incompat ible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of Delta Cp and T Delta S strongly ind icate large scale conformational adaptation of the active site in sequentia l substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal s tructure of the ternary HSV1 TK dLT ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain ar e proposed to take place when the enzyme changes from the substrate-free, p resumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.