Thermodynamic stabilization of nucleotide binding to thymidylate synthase by a potent benzoquinazoline folate analogue inhibitor.

The stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymidylate synthase (TS) in the presence and absence of a folate analogue inhibitor of TS, 1843U, was determined by differential scanning calorimetry. When the enzyme is thermally unfolded in the presence of dUMP, two separate temperature transitions are evident, although only one binding site/dimer was detected in equilibrium dialysis experiments. In the absence of dUMP, TS shows a major peak of unfolding at 45 degrees C with a shoulder at 47 degrees C. In the presence of increasing amounts of dUMP progressive changes in the size of each peak occur, each associated with a higher temperature of unfolding. At a ratio of dUMP/TS of 100, a major peak predominates with an unfolding temperature (Td) of 60 degrees C. FdUMP shows a similar profile, while dGMP does not alter the Td of the enzyme since dGMP alone does not bind to TS. Despite the fact that 1843U binds tightly to TS in the absence of nucleotide ligands [Dev, I. K., Dallas, W.S., Ferone, R., Hanlon, H., McKee, D.D., & Yates, B. B. (1994) J.Biol. Chem. 269, 1873-1882], it exhibits only a small effect on the Td profile of TS. However, when 1843U is present, in addition to the nucleotides (dUMP, FdUMP, or dGMP), a Td of 72 degrees C is achieved and the enthalpy of unfolding is increased by one-third. The stabilizing effect of substrate binding to TS by 1843U examined by thermodynamic parameters can be attributed to the considerable extra amount of free energy released on formation of the ternary complex of TS-1843U-nucleotide. The tightness of this complex is due to the stacking energy that results from Van der Waals contacts between the nucleotide purine or pyrimidine ring and the benzoquinazoline ring of 1843U [Weichsel, A., Montfort, W. R., Cieśla, J., & Maley, F. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3493-3497], which induces a local conformational change in the protein. This conformational change is associated with a significant positive entropy change, which suggests that water is expelled from the active site region.