Enzymes play a key role in almost all biological processes, accelerating a variety of metabolic reactions as well as controlling energy transduction, transcription and translation of genetic information, and signaling. They possess the remarkable capacity to accelerate reactions by many orders of magnitude compared to their uncatalyzed counterparts, making feasible crucial processes that would otherwise not occur on biologically relevant timescales. Thus, there is broad interest in understanding the catalytic power of enzymes on a molecular level. Several proposals have been put forward to try to explain this phenomenon, and one that has rapidly gained momentum in recent years is the idea that enzyme dynamics somehow contributes to catalysis. This review examines the dynamical proposal in a critical way considering basically all reasonable definitions, including (but not limited to) such proposed effects as "coupling between conformational and chemical motions", "landscape searches" and "entropy funnels". It is shown that none of these proposed effects have been experimentally demonstrated to contribute to catalysis, nor are they supported by consistent theoretical studies. On the other hand, it is clarified that careful simulation studies have excluded most (if not all) dynamical proposals. This review places significant emphasis on clarifying the role of logical definitions of different catalytic proposals, and on the need for a clear formulation in terms of the assumed potential surface and reaction coordinate. Finally, it is pointed out that electrostatic preorganization actually accounts for the observed catalytic effects of enzymes, through the corresponding changes in the activation free energies.

Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

Hybrid quantum mechanical / molecular mechanical (QM/MM) approaches have been used to provide a general scheme for chemical reactions in proteins. However, such approaches still present a major challenge to computational chemists, not only because of the need for very large computer time in order to evaluate the QM energy but also because of the need for proper computational sampling. This review focuses on the sampling issue in QM/MM evaluations of electrostatic energies in proteins. We chose this example since electrostatic energies play a major role in controlling the function of proteins and are key to the structure-function correlation of biological molecules. Thus, the correct treatment of electrostatics is essential for the accurate simulation of biological systems. Although we will be presenting here different types of QM/MM calculations of electrostatic energies (and related properties), our focus will be on pK a calculations. This reflects the fact that pK a of ionizable groups in proteins provide one of the most direct benchmarks for the accuracy of electrostatic models of macromolecules. While pK a calculations by semimacroscopic models have given reasonable results in many cases, existing attempts to perform pK a calculations using QM/ MM-FEP have led to large discrepancies between calculated and experimental values. In this work, we accelerate our QM/MM calculations using an updated mean charge distribution and a classical reference potential. We examine both a surface residue (Asp3) of the bovine pancreatic trypsin inhibitor, as well as a residue buried in a hydrophobic pocket (Lys102) of the T4-lysozyme mutant. We demonstrate that by using this approach, we are able to reproduce the relevant sidechain pK a s with an accuracy of 3 kcal/mol. This is well within the 7 kcal/mol energy difference observed in studies of enzymatic catalysis, and is thus sufficient accuracy to determine the main contributions to the catalytic energies of enzymes. We also provide an overall perspective of the potential of QM/ MM calculations in general evaluations of electrostatic free energies, pointing out that our approach should provide a very powerful and accurate tool to predict the electrostatics of not only solution but also enzymatic reactions, as well as the solvation free energies of even larger systems, such as nucleic acid bases incorporated into DNA.

The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+–O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase I as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.