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.T he enormous catalytic power of enzymes has been attempted to be rationalized by several proposals. Here, we would like to focus on a specific proposal that appears to be gaining significant support. Namely, there exists a long-standing assumption that enzyme dynamics and flexibility are important to the chemical step of catalysis (see, e.g., refs. 1-4 and references cited therein). This hypothesis has emerged in several forms, ranging from the assumption that enzymatic catalysis can be linked to lid closures upon binding (e.g., ref. 5) to more recent studies (6, 7) that considered the effect of modifying the accessibility of conformational states separated by relatively small structural differences. It was then argued that the observed changes in the rate of the chemical step upon mutations that appear to prevent the flexibility of the active-site residues could be interpreted as evidence for a dynamical coupling to catalysis. This proposal is particularly well defined in a recent study (6) that focused on dihydrofolate reductase (DHFR). That is, ref. 6 demonstrated that the N23PP, S148A, and N23PP/S148A mutants of DHFR have more limited conformational flexibility than the WT and cannot access the occluded (OC) conformation from the closed (CL) conformation, which is available to th...

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

“…Small, non‐conjugated substrates are “good” substrates, resulting in higher k cat constants than those for the bulky ones. In line with our proposal,3b, 13 dynamic coupling was found to be less significant for small, non‐conjugated substrates at 20 °C ( k cat LE / k cat HE ≤1.2; Figure 3 C). Sampling an ideal configuration for hydride transfer is likely less energetically demanding for “good” substrates where less reorganization of the active site residues is required, and thus they lead to higher values of k cat and lower enzyme KIEs (Figure 3 C and Tables S8 and S9).…”

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

“…For example, analysis of the catalytic landscape of DHFR (and its mutants) has been reported in Ref. 73. This work illustrated that, in contrast to the implications of Ref.…”

“…More specifically, some of the changes in the catalytic effect of DHFR upon mutation are due to the entropic effect of having more crossing ridges at the TS than at the RS, or vice versa (see the analysis in Ref. 73). Obviously, this is a thermodynamic effect, as the configurational space is not dynamical.…”

“…40 In particular, this work argued that the mutational induced reduction in catalysis is due to dynamical effects, and also that the reorganization change upon mutation is very small and thus presumably not responsible for this change in catalysis (based on inspecting the observed structural changes upon mutation). However, our subsequent simulation study 73 has shown that the observed catalysis simply reflects the alternation of the free energy surface, and the corresponding changes in preorganization. It was also pointed out 73 that the preorganization cannot be estimated by just examining structural changes by experimental methods that cannot accurately determine the reorganization of hydrogen bonds, but rather, it is necessary to perform free energy calculations.…”

“…However, our subsequent simulation study 73 has shown that the observed catalysis simply reflects the alternation of the free energy surface, and the corresponding changes in preorganization. It was also pointed out 73 that the preorganization cannot be estimated by just examining structural changes by experimental methods that cannot accurately determine the reorganization of hydrogen bonds, but rather, it is necessary to perform free energy calculations. Our clear analysis of these problems was subsequently confirmed by other groups, 55,74,75 although some obfuscated the fact that they have merely reproduced our previous results.…”