THERMODYNAMIC ANALYSIS OF THE BINDING OF 5-FLUORO-2'-DEOXYURIDINE 5'-MONOPHOSPHATE TO THYMIDYLATE SYNTHASE OVER A RANGE OF TEMPERATURES

The binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) to La ctobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16-35 degrees C. Calorimetric measurements in various buffer sys tems with different heats of ionization suggest that a proton uptake i s involved in the binding process of the nucleotide. In the temperatur e range investigated, the mol protons bound/mol nucleotide increases a s the temperature decreases. A model of two equal and independent site s fits well with the binding isotherms for thymidylate synthase. The b inding constants, the changes in Gibbs energy, enthalpy, and entropy/s ite for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contribu tions in the range 16-35 degrees C. The enthalpy changes become more n egative as the temperature increases, with Delta C-p = -170+/-20 J . K -1 (mol FdUMP bound)(-1). The behavior of the system supports the obse rvation that FdUMP binds to thymidylate synthase without producing pro found conformational changes in the protein dimer.