Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions

Abstract

The proposal that enzymatic catalysis is due to conformational fluctuations has been previously promoted by means of indirect considerations. However, recent works have focused on cases where the relevant motions have components toward distinct conformational regions, whose population could be manipulated by mutations. In particular, a recent work has claimed to provide direct experimental evidence for a dynamical contribution to catalysis in dihydrofolate reductase, where blocking a relevant conformational coordinate was related to the suppression of the motion toward the occluded conformation. The present work utilizes computer simulations to elucidate the true molecular basis for the experimentally observed effect. We start by reproducing the trend in the measured change in catalysis upon mutations (which was assumed to arise as a result of a "dynamical knockout" caused by the mutations). This analysis is performed by calculating the change in the corresponding activation barriers without the need to invoke dynamical effects. We then generate the catalytic landscape of the enzyme and demonstrate that motions in the conformational space do not help drive catalysis. We also discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. Finally, we point out that the changes in the reaction potential surface modify the reorganization free energy (which includes entropic effects), and such changes in the surface also alter the corresponding motion. However, this motion is never the reason for catalysis, but rather simply a reflection of the shape of the reaction potential surface.

Fig. 1.

A superimposition of WT DHFR in the closed [blue, from Protein Data Bank (PDB) ID code 1RX2] and occluded (gray, from PDB ID code 1RX4) conformations. The mobile Met20 loop (red, residues 9–24) and the sites of mutation (N23 and S148) are indicated in the closed conformation. The DHF-H⁺ and NADPH ligands are shown in the closed RS configuration.

Fig. 2.

Average EVB free-energy profiles for the reference reaction in solution (long gray dashes), WT EcDHFR (long black dashes), the N23PP-S148A mutant (short black dashes), and the S148A mutant (solid black line). The numbers in the brackets denote the corresponding activation barriers, in kilocalories per mole.

Fig. 3.

Free-energy landscapes (in kilocalories per mole) for both (A) wild-type EcDHFR, as well as the (B) N23PP/S148A mutant. The energetics along the conformational coordinate was examined using a specialized version of the LRA approach, which allowed us to estimate the conformational energy for the transition between the closed and occluded conformations (see also, e.g., refs.  and 17). Note that the results for the full (CL → OC) transition are also provided in Table S2.

Fig. 4.

An illustration of the relevant motions in the landscape of the conformational and chemical space. Shown here are two different possible paths, comprising (1) trajectories that move directly from the closed state to the closed TS, (2) trajectories that first move from the closed state in the direction toward the occluded state, and then move back to the closed state and the closed TS.