The role of protein dynamics on structure and function of Thymidylate synthase

The relationship between the protein motions, structure and catalytic activity is of contemporary interest in enzymology. Here, a broad array of kinetic techniques, site-directed mutagenesis, and methods involving isotopic labeling of substrates and proteins were used in studying thymidylate synthase (TSase). TSase catalyzes the de novo biosynthesis of the DNA building block thymidine, 2'-deoxythymidine 5'-monophosphate (dTMP) in most organisms and is medicinally important as an antibiotic and a chemotherapeutic drug target. The mechanism of TSase involves several steps including two major C-H bond activations: a rate limiting hydride transfer and a non-rate limiting proton transfer. Therefore, it is a good model system to study the dynamic and structural effects on different hydrogen transfers in the same catalytic cycle. Our experiments of a structurally identical but dynamically altered remote mutant of Escherichia Coli (ec)TSase, Y209W, showed that none of these two H-transfer steps under was altered significantly. Yet other kinetic steps were dramatically affected and reveal the importance of long-range dynamics of the enzymatic complex and its catalytic function in ecTSase. We also found that Mg2+ affects the hydride transfer although it binds to the surface of ecTSase, further indicating the important of long range dynamics across the protein. Furthermore, a comparative studies of light vs. heavy WT ecTSase suggest a direct coupling of the altered fast protein vibrations on the hydride transfer. Studies of an active site mutant of a conserved histidine suggest that it plays a major role in the hydride transfer step in ecTSase, supporting mechanisms that involve charge accumulation at carbonyl 4. The studies of an evolutionary divergent TSase from Bacillus subtilis that has a valine at the equivalent position suggest that evolutionary pressure led to extensive polymorphism across the protein, which compensate for the His→Val substitution at the active site. Altogether, these findings could assist in rational inhibitor design as leads to more specific and potent chemotherapeutic or antibiotic drugs in the future, and the design of biomimetic catalysts that include the cross-protein dynamics into consideration.