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J. Biol. Chem., Vol. 275, Issue 21, 16139-16145, May 26, 2000

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Compulsory Order of Substrate Binding to Herpes Simplex Virus Type 1 Thymidine Kinase
A CALORIMETRIC STUDY^*

Remo Perozzo, Ilian Jelesarov^§, Hans Rudolf Bosshard^§, Gerd Folkers, and Leonardo Scapozza^¶

From the  Department of Applied Biosciences, Swiss Federal Institute of Technology, Winterthurerstr. 190, CH-8057 Zurich, Switzerland and the ^§ Department of Biochemistry, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK·dT complex. At pH 7.5 and 25 °C, the binding constants are 1.9 × 10⁵ M¹ for dT and 3.9 × 10⁶ M¹ for ATP binding to the binary HSV1 TK·dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (Cp) obtained from H in the range of 10-25 °C is 360 cal K¹ mol¹ for dT binding and 140 cal K¹ mol¹ for ATP binding. These large Cp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of Cp and TS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK·dT·ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

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