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...

The crucial process of aminoacyl-tRNA delivery to the ribosome is energized by the GTPase reaction of the elongation factor Tu (EF-Tu). Advances in the elucidation of the structure of the EF-Tu/ribosome complex provide the rare opportunity of gaining a detailed understanding of the activation process of this system. Here, we use quantitative simulation approaches and reproduce the energetics of the GTPase reaction of EF-Tu with and without the ribosome and with several key mutants. Our study provides a novel insight into the activation process. It is found that the critical H84 residue is not likely to behave as a general base but rather contributes to an allosteric effect, which includes a major transition state stabilization by the electrostatic effect of the P loop and other regions of the protein. Our findings have general relevance to GTPase activation, including the processes that control signal transduction.enzymatic catalysis | preorganization | allostery T he elongation cycle of protein synthesis uses the elongation factor Tu (EF-Tu) with GTP to deliver aminoacyl-tRNA (aa-tRNA) to the mRNA-programmed ribosome. More specifically, the GTP-bound state of EF-Tu forms a high-affinity ternary complex with the aa-tRNA. Upon binding of the ternary complex to the ribosome, the aa-tRNA occupies the A site and, when the codon-anticodon interaction is cognate, the GTPase activity of the EF-Tu is increased significantly. The conformational change following GTP hydrolysis to GDP and a leaving phosphate group (P i ) leads to dissociation of EF-Tu from the ribosome and accommodation of the aa-tRNA on the A site for peptidyl transfer (see Fig. 1 for a schematic description; for additional details, see refs. 1-3).Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1-3, 14-25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level. However, despite major biochemical and structural breakthroughs, a detailed explanation of how the codon recognition in the 30S subunit leads to GTP hydrolysis remains elusive. That is, although it is known that the precise positioning of H84 is critical for efficient catalysis (1,9,(11)(12)(13)(23)(24)(25)(26), the energetics of this positioning and its ultimate role in stabilizing the transition state (TS) are unclear. More precisely, it is frequently assumed that the conserved H84 is moved to a catalytic configuration and then serves as a general base, but this assumption is problematic (see ref. 1 and Reproducing the Overall Catalytic and Mutational Effects below) and has similar pitfalls as in the highly related case of the Ras-RasGAP (RasGAP) system. That is, where it was originally assumed that Q61 in the RasGAP system serves as a general base, but this assumption has been shown to ...

Monoclonal antibodies (mAbs) are one of the most lucrative pharmacologics currently on the market due to their diverse array of applications. However, the diversity of these therapeutics is often limited...

Accurate rate coefficients for 35 1,2-hydrogen shift reactions for hydrides containing up to 10 silicon atoms have been calculated using G3//B3LYP. The overall reactions exhibit two distinct barriers. Overcoming the first barrier results in the formation of a hydrogen-bridged intermediate species from a substituted silylene and is characterized by a low activation energy. Passing over the second barrier converts this stable intermediate into the double-bonded silene. Values for the single event Arrhenius pre-exponential factor, A, and the activation energy, E(a), were calculated from the G3//B3LYP rate coefficients, and a group additivity scheme was developed to predict A and E(a). The values predicted by group additivity are more accurate than structure/reactivity relationships currently used in the literature, which rely on a representative A value and the Evans-Polanyi correlation to predict E(a). The structural factors that have the most pronounced effect on A and E(a) were considered, and the presence of rings was shown to influence these values strongly.

The thermochemical properties for selected hydrogenated silicon clusters (Si(x)H(y), x = 3-13, y = 0-18) were calculated using quantum chemical calculations and statistical thermodynamics. Standard enthalpy of formation at 298 K and standard entropy and constant pressure heat capacity at various temperatures, i.e., 298-6000 K, were calculated for 162 hydrogenated silicon clusters using G3//B3LYP. The hydrogenated silicon clusters contained ten to twenty fused Si-Si bonds, i.e., bonds participating in more than one three- to six-membered ring. The hydrogenated silicon clusters in this study involved different degrees of hydrogenation, i.e., the ratio of hydrogen to silicon atoms varied widely depending on the size of the cluster and/or degree of multifunctionality. A group additivity database composed of atom-centered groups and ring corrections, as well as bond-centered groups, was created to predict thermochemical properties most accurately. For the training set molecules, the average absolute deviation (AAD) comparing the G3//B3LYP values to the values obtained from the revised group additivity database for standard enthalpy of formation and entropy at 298 K and constant pressure heat capacity at 500, 1000, and 1500 K were 3.2%, 1.9%, 0.40%, 0.43%, and 0.53%, respectively. Sensitivity analysis of the revised group additivity parameter database revealed that the group parameters were able to predict the thermochemical properties of molecules that were not used in the training set within an AAD of 3.8% for standard enthalpy of formation at 298 K.

The mechanism of H(2) addition and elimination reactions in selected silicon hydrides (Si(x)H(y), x = 1-10, y = 4-20) was modeled using quantum chemical calculations, statistical thermodynamics, transition state theory and transition state group additivity. Rate coefficients for 25 H(2) addition reactions were calculated using G3//B3LYP. For nearly every reaction, the overall conversion exhibits two steps. In the addition direction, the reactants first meet to form an adduct which then converts into a saturated silicon hydride via homolytic H-H bond cleavage. Values for the single-event Arrhenius pre-exponential factor, Ã, and the activation energy, E(a), were calculated from the G3//B3LYP rate coefficients, and a group additivity scheme was developed to predict à and E(a). The values predicted by group additivity are more accurate than kinetic correlations currently used in the literature, which rely on representative à values and the Evans-Polanyi correlation. The factors that have the most pronounced effect on à and E(a) were investigated, and stabilization of the divalent silicon atom of the unsaturated silicon hydride with electron-donating substituents was found to influence kinetic parameters considerably. The rate coefficients for H(2) addition reactions were found to correlate reasonably well with the difference in energy between the highest occupied molecular orbital of H(2) (E(HOMO)) and the lowest unoccupied molecular orbital of the reactant silylene (E(LUMO)).

Kinetic parameters for the dominant pathways during the addition of the four Si(2)H(2) isomers, i.e., trans-HSiSiH, SiSiH(2), Si(H)SiH, and Si(H(2))Si, to monosilane, SiH(4), and disilane, Si(2)H(6), have been calculated using G3//B3LYP, statistical thermodynamics, conventional and variational transition state theory, and internal rotation corrections. The direct addition products of the multifunctional Si(2)H(2) isomers were monofunctional substituted silylenes, hydrogen-bridged species, and silenes. During addition to monosilane and disilane, the SiSiH(2) isomer was found to be most reactive over the temperature range of 800 to 1200 K. Revised parameters for the Evans-Polanyi correlation and a representative pre-exponential factor for multifunctional silicon hydride addition and elimination reaction families under pyrolysis conditions were regressed from the reactions in this study. This revised kinetic correlation was found to capture the activation energies and rate coefficients better than the current literature methods.

Accurate rate coefficients for 40 bimolecular substituted silylene addition reactions for silicon hydrides containing up to nine silicon atoms are calculated using the G3//B3LYP method. The overall reactions exhibit two steps: the reactants first meet to form an adduct, which then converts into a saturated silicon hydride. Values for the single-event Arrhenius pre-exponential factor, A, and the activation energy, E(a), are calculated from the G3//B3LYP rate coefficients corrected for internal rotations, and a group additivity scheme is developed to predict A and E(a). The values predicted by group additivity are more accurate than structure-reactivity relationships currently used in the literature, which rely on representative A values and the Evans-Polanyi correlation. The structural factors that have the most pronounced effect on A and E(a) are considered, and the presence of rings is shown to influence these values strongly.