QM/MM MD Study on the Reaction Mechanism of Thymidine Phosphorylation Catalyzed by the Enzyme <i>Thermotoga maritima</i> Thymidine Kinase 1
Samanta Makurat, Rui P. P. Neves, Maria J. Ramos, Janusz Rak
Abstract
High Resolution Image Download MS PowerPoint Slide Here, we report mechanistic studies on type II thymidine kinase, Thermotoga maritima Tm TK, 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 S N 2 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 Mg 2+ ion.