Role of Vibrational Motions on Thymidylate Synthase Catalysis

Thymidylate Synthase (TSase) catalyzes the last committed step in thymine biosynthesis, i.e., the conversion of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), using 5,10-methylenetetrahydrafolate as both the methylene and hydride donor. dTMP is an essential DNA building block, thus TSase activity is critical is fast proliferating cells and is thus a target for chemotherapeutic drugs and antibiotics.

Recent studies investigated the role of vibrational motions of proteins in enzyme catalyzed reactions. Studies isolating effects of fast protein vibrations in the catalyzed reaction are conducted by comparing light (natural distribution of isotopes) and heavy (¹³C, ¹⁵N, ²H labeled) TSase. A broad spectrum of kinetic properties and activation parameters have been measured to compare these two isozymes.

Initial velocity data were analyzed to obtain the k_cat value for each enzyme at four temperatures (5°C, 25°C, 30°C, 40°C).  Rates for both light and Heavy TSases were measured at different dUMP concentrations as a function of 5,10-methylenetetrahydrafolate concentration and vise versa. Rates were fit to the Michaelis-Menten equation, with or without substrate inhibition, as  5,10-methylenetetrahydrafolate showed substrate inhibition. Parameters K_M and K_cat were compared for both heavy and light TSases at all temperatures and were identical (within the experimental error) at 20°C, but varied at all other temperatures. To further assess whether or not the mass difference within the enzyme changes the rate-limiting step for the specific reaction, the kinetic isotope effect was measured on k_cat. Intrinsic isotope effects were also measured and findings indicate that k_catin the heavy TSase was no longer rate limited by the hydride transfer. The temperature independence of the isotope effects for the light enzyme indicated ideal enzyme dynamics. A large temperature dependency for heavy TSase, on the other hand, indicated that those fast vibrations of the light TSase are altered by the slower dynamics in the heavy TSase enzyme. Future work will include analysis and further study on the role of vibrational motions versus the role of binding between the heavy and light TSase through the use of isothermal titration calorimetry experiments.