An Analysis of All the Relevant Facts and Arguments Indicates that Enzyme Catalysis Does Not Involve Large Contributions from Nuclear Tunneling.

Enzymatic reactions play a major role in controlling and performing most life processes. Thus, understanding how enzymes really work has both fundamental and practical importance. In this respect, it is crucial to understand the origin of the enormous catalytic power of enzymes, and this is currently one of the major challenges of modern biophysics[1, 2]. Although many elements of this puzzle have been elucidated by biochemical and structural studies, the source of the catalytic power of enzymes has not been widely understood, and clearly remains controversial within the scientific community (see e.g. Ref. [3]). At the present stage, all consistent simulation studies appear to indicate that electrostatic effects play a major role in stabilizing the transition states of enzymatic reactions (for reviews of this matter, see for instance Refs. [4, 5]). However, this idea has not been generally accepted, perhaps due to the difficulties of understanding the effect and also due to general difficulties in the clarification of the nature of polar pre-organization effects[6]. Thus, there is a great interest in exotic alternative proposals. For instance, the idea that enzyme catalysis involves special factors such as nuclear quantum mechanical (NQM) effects, and, in particular, nuclear tunneling, has gained popularity in recent years[7–14]. Additionally, it has been suggested that transition state theory (TST) cannot be used in studies of enzyme catalysis, it has actually been demonstrated to provide an excellent tool for such studies[15]. While there is no argument about the fact that NQM effects play a significant role in enzymatic reactions in which the rate limiting steps involves the transfer of either a proton, a hydride or hydrogen, rigorous and systematic theoretical studies have demonstrated that the NQM contributions are similar in solutions and in proteins and thus do not make a significant contribution to catalysis (where catalysis is defined as the difference between the enzymatic and the solution reactions). Additionally, is has been established that TST provides an excellent tool for quantifying enzymatic catalysis[16, 17]. Nevertheless, there are still major misunderstandings remaining in the field with regards to the catalytic tunneling proposal[18]. We would like to point out at the beginning of this paper that assessing the catalytic contributions of NQM effects must, naturally, reproduce all key experimental observations. However, it cannot be based on accepting the interpretation of experimental data as fact. Here, one needs reliable models that can actually simulate any type of experimental observations, and that allow one to dissect the overall catalytic effect in order to identify its constituent contributions. In other words, once a model is able to reproduce what is actually observed experimentally, it can then be used to separate the overall effect into its individual contributions. Since enzymes are very complex systems, the most effective models should probably be based on computational simulation approaches. In this work, we will consider arguments that support the catalytic NQM idea, and point out the ways in which these arguments are extremely problematic (and sometimes even self-contradictory in some of the most basic cases). Our analysis aims to clarify the misunderstandings of this issue by summarizing the results of our recent studies, as well as by discussing some other relevant works. We clarify that calculations of quantized activation free energies converge much more rapidly than calculations of the temperature dependence of these quantities, and that the calculated activation free energies support our conclusions about the contribution of NQM effects to catalysis. We then proceed to clarify the difference between calculations that evaluate the temperature dependence of isotope effects by consistent microscopic simulations, and studies that reproduce such dependence by using adjustable phenomenological parameters. We point out that, at present, our studies of the temperature dependence of isotope effects are as good as (if not better than) any of the reported consistent calculations, including in the challenging case of lipoxygenase. Furthermore, we will summarize our systematic study of kinetic isotope effects (KIEs) and their temperature dependence in mutants of dihydrofolate reductase (DHFR). This study provides a unique example of the reproduction of the observed change in the trend of the temperature dependence of KIEs that is wholly based on microscopic calculations. Ultimately, our aim is to clarify the main misunderstandings in this field with simple non-polemic logical arguments. We believe that addressing the validity of the main arguments and facts in the field is the best way in order to reach a constructive scientific consensus.