Vibrationally Enhanced Hydrogen Tunneling in the Escherichia coli Thymidylate Synthase Catalyzed Reaction

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Abstract
The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5−45 °C. The observed H/T (TV/KH) and D/T (TV/KD) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain−Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20−30 °C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant kcat (Dk = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry36, 4212−4222]. Additionally, the Swain−Schaad relationships between that Dk and the V/K KIEs reported here suggested that at 20 °C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (ΔEa = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were AH/AT = 6.8 ± 2.8 and AD/AT = 1.9 ± 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on kcat (4.0 ± 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.