Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity.

Thymidylate synthase (TSase) is a clinically important enzyme because it catalyzes synthesis of the sole de novo source of deoxy-thymidylate. Without this enzyme, cells die a "thymineless death" since they are starved of a crucial DNA synthesis precursor. As a drug target, TSase is well studied in terms of its structure and reaction mechanism. An interesting mechanistic feature of dimeric TSase is that it is "half-the-sites reactive", which is a form of negative cooperativity. Yet, the basis for this is not well-understood. Some experiments point to cooperativity at the binding steps of the reaction cycle as being responsible for the phenomenon, but the literature contains conflicting reports. Here we use ITC and NMR to resolve these inconsistencies. This first detailed thermodynamic dissection of multisite binding of dUMP to E. coli TSase shows the nucleotide binds to the free and singly bound forms of the enzyme with nearly equal affinity over a broad range of temperatures and in multiple buffers. While small but significant differences in ΔC°P for the two binding events show that the active sites are not formally equivalent, there is little-to-no allostery at the level of ΔG°bind. In addition NMR titration data reveal that there is minor intersubunit cooperativity in formation of a ternary complex with the mechanism based inhibitor, 5F-dUMP, and cofactor. Taken together, the data show that functional communication between subunits is minimal for both binding steps of the reaction coordinate.