The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism.

The hydride transfer from C6 of tetrahydrofolate to the reaction's exocyclic methylene-dUMP intermediate is the rate limiting step in thymidylate synthase (TSase) catalysis. This step has been studied by means of QM/MM molecular dynamics simulations to generate the corresponding free energy surfaces. The use of two different initial X-ray structures has allowed exploring different conformational spaces and the existence of chemical paths with not only different reactivities but also different reaction mechanisms. The results confirm that this chemical conversion takes place preferentially via a concerted mechanism where the hydride transfer is conjugated to thiol-elimination from the product. The findings also confirm the labile character of the substrate-enzyme covalent bond established between the C6 of the nucleotide substrate and a conserved cysteine residue. The calculations also reproduce and rationalize a normal H/T 2° kinetic isotope effect measured for that step. From a computational point of view, the results demonstrate that the use of an incomplete number of coordinates to describe the real reaction coordinate can render biased results.