Concerted versus Stepwise Mechanism in Thymidylate Synthase

Abstract

Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.

The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism

“…Keeping in mind that the limit that we could obtain if the C-S bond was completely broken, computed as equilibrium isotope effects (EIE), is a small normal effect (1.095 ± 0.011 at AM1/MM level), our results would be in agreement with a very asynchronous but concerted mechanism. Our results appear to be slightly underestimated by comparison with the published experimental measurements (Exp 2° KIE = 1.104 ±0.004), 12 although showing the same trend. The inverse effect obtained from structures of the step-wise mechanism can be explained keeping in mind the character of the C5-C6 bond, between double and single that is developed in the TS.…”

“…The results show no measurable difference between the two mechanism when substituting 16 O4 oxygen by its 18 O4 isotope, but different trends are obtained on KIEs for 12 C6 to 13 C6 substitution when computed between reactants and TS in concerted and step-wise mechanism: a normal effect in the concerted mechanism and no effect in the step-wise one. As observed in Figure 6, a larger change in hybridization on C6 from reactants to TS is observed for the concerted mechanism (Figure 6B) than in the step-wise mechanism (Figure 6A) since the C6-S bond is elongated in the former and no change is detected in the step-wise TS.…”

“…It is important to mention that without tunneling correction the predicted KIEs are probably deflated (as in ref 48 ), but their trend is likely to be correct. Finally, heavy atoms KIEs, computed for 12 C to 13 C substitution at C6 position predict a normal effect (KIE=1.011 at AM1/MM and 1.0108 at M06-2X/MM) in the concerted mechanism and no effect (KIE=1) in the step-wise one. This result is not surprising as a change in hybridization has been observed from reactants to the concerted TS (the C6-S is slightly elongated in the TS), while no change has suffered C6 atom in the step-wise TS.…”

Examinations of the Chemical Step in Enzyme Catalysis

“…Additionally, an observed normal secondary (2°) KIE on C6 of dUMP supported the concerted mechanism for the hydride transfer (Islam et al, 2014). As a control, mutation to several other residues closer to the reaction center were found to produce no or very minor changes in the temperature dependence of KIE int (Abeysinghe & Kohen, 2015; Agrawal, Hong, et al, 2004; Z.…”

“…A mutation at R166 should have altered the temperature dependence of KIE int only if it was involved in TSs of H-transfers. KIE obs s for H/T and D/T were measured over a range of temperatures for the both the hydride transfer and the proton abstraction in WT and in a variant of R166 (R166K, the only active mutant of R166) (Agrawal, Hong, et al, 2004; Islam, Strutzenberg, Ghosh, & Kohen, 2015; Islam, Strutzenberg, Gurevic, & Kohen, 2014; Z. Wang & Kohen, 2010).…”