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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 nuclear magnetic resonance (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 apoenzyme (lig 0 ), the saturated holoenzyme (lig 2 ), and the typically elusive singly bound (lig 1 ) states. Approximately 40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes &gt;30 Å 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 the two active sites. Simulations corroborate the coupling observed in solution in that there is excellent overlap between quartet residues and main-chain atoms having intersubunit cross-correlated motions. Simulations identify five hot spot residues, three of which lie at the kink in the unique β-bulge abutting the active sites on either end of the sheet. Interstrand cross-correlated motions become more organized and pronounced as the enzyme progresses from lig 0 to lig 1 and ultimately lig 2 . Coupling in the apparently symmetrical complex has implications for half-the-sites reactivity and potentially resolves the paradox of inequivalent TS active sites despite the vast majority of X-ray structures appearing to be symmetrical.

Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase