Inter-Active Site Communication Mediated by the DimerInterface β‑Sheet in the Half-the-Sites Enzyme, ThymidylateSynthase

Thymidylate synthase (TS) is a dimeric enzyme conserved in all life forms that exhibits the allosteric feature of half-the-sites activity. Neither the reason for, nor the mechanism of this phenomenon is understood. We used a combined NMR and molecular dynamics approach to study a stable intermediate preceding hydride transfer, which is the rate limiting, and half-the-sites step. In NMR titrations with ligands leading to this intermediate, we measured chemical shifts of the apo enzyme (lig0), the saturated holo-enzyme (lig2), and the typically elusive singly bound (lig1) states. ~40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes greater than 30 A away in the distal subunit. Quartet peak patterns have symmetrical character, indicating reciprocity in communicating the first and second binding events to the distal protomer. Quartets include key catalytic residues and map to the dimer interface β-sheet, which also represents the shortest path between...