Thymidylate synthase (TS) is a promising
cancer target, due to its crucial function in thymine synthesis. It
performs the reductive methylation of 2′-deoxyuridine-5′-phosphate
(dUMP) to thymidine-5′-phosphate (dTMP), using <i>N</i>-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After
the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent
conformational activation, this complex has been proposed to react
via nucleophilic attack (Michael addition) by Cys146, followed by
methylene-bridge formation to generate the ternary covalent intermediate.
Herein, QM/MM (B3LYP-D/6-31+G(d)-CHARMM27) methods are used to model
the formation of the ternary covalent intermediate. A two-dimensional
potential energy surface reveals that the methylene-bridged intermediate
is formed via a concerted mechanism, as indicated by a single transition
state on the minimum energy pathway and the absence of a stable enolate
intermediate. A range of different QM methods (B3LYP, MP2 and SCS-MP2,
and different basis sets) are tested for the calculation of the activation
energy barrier for the formation of the methylene-bridged intermediate.
We test convergence of the QM/MM results with respect to size of the
QM region. Inclusion of Arg166, which interacts with the nucleophilic
thiolate, in the QM region is important for reliable results; the
MM model apparently does not reproduce energies for distortion of
the guanidinium side chain correctly. The spin component scaled-Møller–Plessett
perturbation theory (SCS-MP2) approach was shown to be in best agreement
(within 1.1 kcal/mol) while the results obtained with MP2 and B3LYP
also yielded acceptable values (deviating by less than 3 kcal/mol)
compared with the barrier derived from experiment. Our results indicate
that using a dispersion-corrected DFT method, or a QM method with
an accurate treatment of electron correlation, increases the agreement
between the calculated and experimental activation energy barriers,
compared with the semiempirical AM1 method. These calculations provide
important insight into the reaction mechanism of TS and may be useful
in the design of new TS inhibitors