QM/MM MD Study on the Reaction Mechanism of Thymidine Phosphorylation Catalyzed by the Enzyme Thermotoga maritima Thymidine Kinase 1

Here, we report mechanistic studies on type II thymidine kinase, Thermotoga maritima TmTK, aiming to predict barriers for the enzyme-catalyzed reaction. Extensive umbrella sampling QM/MM MD simulations (PBE/GPW/DZVP-GTH-PBE:AMBER) resulted in a free energy barrier for the phosphorylation reaction's rate-limiting step of 16.6 kcal·mol–1, which is in an excellent agreement with the experimentally reported value. An atomistic picture provided by our simulations reveals that the reaction follows a concerted, dissociative SN2 reaction mechanism in which the 5′-oxygen of the ribose moiety in thymidine is phosphorylated by the γ-phosphate of ATP, while assisted by an asynchronous deprotonation of the 5′-hydroxyl by a GLU84 base. The reaction was calculated to be endergonic, with a reaction free energy of 10.8 kcal·mol–1, and it can be followed by low-barrier processes that promote the unbinding of the phosphorylated thymidine product, namely, the deprotonation of the GLU84 by the thymidine-phosphate that is accompanied by a weaker binding of the product to the Mg2+ ion.