QM/MM calculations suggest a novel intermediate following the proton abstraction catalyzed by thymidylate synthase.

The cleavage of covalent C-H bonds is one of the most energetically demanding, yet biologically essential, chemical transformations. Two C-H bond cleavages are involved in the reaction catalyzed by thymidylate synthase (TSase), which provides the sole de novo source of thymidylate (i.e., the DNA base T) for most organisms. Our QM/MM free energy calculations show that the C-H → O proton transfer has three transition states that are energetically similar but structurally diverse. These characteristics are different from our previous calculation results on the C-H → C hydride transfer, providing an explanation for differences in temperature dependences of KIEs on these two C-H bond activation steps. The calculations also suggest that the traditionally proposed covalent bond between the protein and substrate (the C6-S bond) is very labile during the multistep catalytic reaction. Collective protein motions not only assist cleavage of the C6-S bond to stabilize the transition state of the proton transfer step but also rearrange the H-bond network at the end of this step to prepare the active site for subsequent chemical steps. These computational results illustrate functionalities of specific protein residues that reconcile many previous experimental observations and provide guidance for future experiments to examine the proposed mechanisms. The synchronized conformational changes in the protein and ligands observed in our simulations demonstrate participation of protein motions in the reaction coordinate of enzymatic reactions. Our computational findings suggest the existence of new reaction intermediates not covalently bound to TSase, which may lead to a new class of drugs targeting DNA biosynthesis.