A Critical Test of the “Tunneling and Coupled Motion” Concept in Enzymatic Alcohol Oxidation

The physical mechanism of C–H bond activation by enzymes is the subject of intense study, and we have tested the predictions of two competing models for C–H activation in the context of alcohol dehydrogenase. The kinetic isotope effects (KIEs) in this enzyme have previously suggested a model of quantum mechanical tunneling and coupled motion of primary (1°) and secondary (2°) hydrogens. Here we measure the 2° H/T KIEs with both H and D at the 1° position and find that the 2° KIE is significantly deflated with D-transfer, consistent with the predictions of recent Marcus-like models of H-transfer. The results suggest that the fast dynamics of H-tunneling result in a 1° isotope effect on the structure of the tunneling ready state: the trajectory of D-transfer goes through a shorter donor–acceptor distance than that of H-transfer

Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

Thymidylate synthase is an attractive target for antibiotic and anticancer drugs due to its essential role in the <i>de novo</i> biosynthesis of the DNA nucleotide thymine. The enzymatic reaction is initiated by a nucleophilic activation of the substrate via formation of a covalent bond to an active site cysteine. The traditionally accepted mechanism is then followed by a series of covalently bound intermediates, where that bond is only cleaved upon product release. Recent computational and experimental studies suggest that the covalent bond between the protein and substrate is actually quite labile. Importantly, these findings predict the existence of a noncovalently bound bisubstrate intermediate, not previously anticipated, which could be the target of a novel class of drugs inhibiting DNA biosynthesis. Here we report the synthesis of the proposed intermediate and findings supporting its chemical and kinetic competence. These findings substantiate the predicted nontraditional mechanism and the potential of this intermediate as a new drug lead

Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis

Molecular dynamics calculations and bionformatic studies of dihydrofolate reductase (DHFR) have suggested a network of coupled motions across the whole protein that is correlated to the reaction coordinate. Experimental studies demonstrated that distal residues G121, M42, and F125 in E. coli DHFR participate in that network. The missing link in our understanding of DHFR catalysis is the lack of a mechanism by which such remote residues can affect the catalyzed chemistry at the active site. Here, we present a study of the temperature dependence of intrinsic kinetic isotope effects (KIEs) that indicates synergism between a remote residue in that dynamic network, G121, and the active site’s residue I14. The intrinsic KIEs for the I14A–G121V double mutant showed steeper temperature dependence (Δ<i>E</i>_a(T‑H)) than expected from comparison of the wild type and two single mutants. That effect was nonadditive (i.e., Δ<i>E</i>_{a(T‑H) G121V} + Δ<i>E</i>_{a(T‑H) I14A} < Δ<i>E</i>_{a(T‑H) double mutant}), which indicates a synergism between the two residues. This finding links the remote residues in the network under investigation to the enzyme’s active site, providing a mechanism by which these residues can be coupled to the catalyzed chemistry. This experimental evidence validates calculations proposing that both remote and active site residues constitute a network of coupled promoting motions correlated to the bond activation step (C–H → C hydride transfer in this case). Additionally, the effect of I14A and G121V mutations on single turnover rates was additive rather than synergistic. Although single turnover rate measurements are more readily available and thus more popular than assessing intrinsic KIEs, the current finding demonstrates that these rates, which in DHFR reflect several microscopic rate constants, can fall short of revealing the nature of the C–H bond activation per se

Hydrogen Donor–Acceptor Fluctuations from Kinetic Isotope Effects: A Phenomenological Model

Kinetic isotope effects (KIEs) and their temperature dependence can probe the structural and dynamic nature of enzyme-catalyzed proton or hydride transfers. The molecular interpretation of their temperature dependence requires expensive and specialized quantum mechanics/molecular mechanics (QM/MM) calculations to provide a quantitative molecular understanding. Currently available phenomenological models use a nonadiabatic assumption that is not appropriate for most hydride and proton-transfer reactions, while others require more parameters than the experimental data justify. Here we propose a phenomenological interpretation of KIEs based on a simple method to quantitatively link the size and temperature dependence of KIEs to a conformational distribution of the catalyzed reaction. This model assumes adiabatic hydrogen tunneling, and by fitting experimental KIE data, the model yields a population distribution for fluctuations of the distance between donor and acceptor atoms. Fits to data from a variety of proton and hydride transfers catalyzed by enzymes and their mutants, as well as nonenzymatic reactions, reveal that steeply temperature-dependent KIEs indicate the presence of at least two distinct conformational populations, each with different kinetic behaviors. We present the results of these calculations for several published cases and discuss how the predictions of the calculations might be experimentally tested. This analysis does not replace molecular QM/MM investigations, but it provides a fast and accessible way to quantitatively interpret KIEs in the context of a Marcus-like model

Collective Reaction Coordinate for Hybrid Quantum and Molecular Mechanics Simulations: A Case Study of the Hydride Transfer in Dihydrofolate Reductase

The optimal description of the reaction coordinate in chemical systems is of great importance in simulating condensed phase reactions. In the current work, we present a collective reaction coordinate which is composed of several geometric coordinates which represent structural progress during the course of a hydride transfer reaction: the antisymmetric reactive stretch coordinate, the donor–acceptor distance (DAD) coordinate, and an orbital rehybridization coordinate. In this approach, the former coordinate serves as a distinguished reaction coordinate, while the latter two serve as environmental, Marcus-type inner-sphere reorganization coordinates. The classical free energy surface is obtained from multidimensional quantum mechanics–molecular mechanics (QM/MM) potential of mean force (PMF) simulations in conjunction with a general and efficient multidimensional weighted histogram method implementation. The minimum free energy path, or the collective reaction coordinate, connecting the dividing hypersurface to reactants and products, is obtained using an iterative scheme. In this approach, the string method is used to find the minimum free energy path. This path guides the multidimensional sampling, while the path is adaptively refined until convergence is achieved. As a model system, we choose the hydride transfer reaction in Escherichia coli dihydrofolate reductase (<i>ec</i>DHFR) using a recently developed accurate semiempirical potential energy surface. To estimate the advantages of the collective reaction coordinate, we perform activated dynamics simulations to obtain the reaction transmission coefficient. The results show that the combination of a distinguished reaction coordinate and an inner-sphere reorganization coordinate considerably reduces the dividing surface recrossing

Collective Reaction Coordinate for Hybrid Quantum and Molecular Mechanics Simulations: A Case Study of the Hydride Transfer in Dihydrofolate Reductase

The optimal description of the reaction coordinate in chemical systems is of great importance in simulating condensed phase reactions. In the current work, we present a collective reaction coordinate which is composed of several geometric coordinates which represent structural progress during the course of a hydride transfer reaction: the antisymmetric reactive stretch coordinate, the donor–acceptor distance (DAD) coordinate, and an orbital rehybridization coordinate. In this approach, the former coordinate serves as a distinguished reaction coordinate, while the latter two serve as environmental, Marcus-type inner-sphere reorganization coordinates. The classical free energy surface is obtained from multidimensional quantum mechanics–molecular mechanics (QM/MM) potential of mean force (PMF) simulations in conjunction with a general and efficient multidimensional weighted histogram method implementation. The minimum free energy path, or the collective reaction coordinate, connecting the dividing hypersurface to reactants and products, is obtained using an iterative scheme. In this approach, the string method is used to find the minimum free energy path. This path guides the multidimensional sampling, while the path is adaptively refined until convergence is achieved. As a model system, we choose the hydride transfer reaction in Escherichia coli dihydrofolate reductase (<i>ec</i>DHFR) using a recently developed accurate semiempirical potential energy surface. To estimate the advantages of the collective reaction coordinate, we perform activated dynamics simulations to obtain the reaction transmission coefficient. The results show that the combination of a distinguished reaction coordinate and an inner-sphere reorganization coordinate considerably reduces the dividing surface recrossing

Substrate Activation in Flavin-Dependent Thymidylate Synthase

Thymidylate is a critical DNA nucleotide that has to be synthesized in cells <i>de novo</i> by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this <i>de novo</i> production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors

QM/MM Calculations Suggest a Novel Intermediate Following the Proton Abstraction Catalyzed by Thymidylate Synthase

The cleavage of covalent C–H bonds is one of the most energetically demanding, yet biologically essential, chemical transformations. Two C–H bond cleavages are involved in the reaction catalyzed by thymidylate synthase (TSase), which provides the sole <i>de novo</i> source of thymidylate (i.e., the DNA base T) for most organisms. Our QM/MM free energy calculations show that the C–H → O proton transfer has three transition states that are energetically similar but structurally diverse. These characteristics are different from our previous calculation results on the C–H → C hydride transfer, providing an explanation for differences in temperature dependences of KIEs on these two C–H bond activation steps. The calculations also suggest that the traditionally proposed covalent bond between the protein and substrate (the C6–S bond) is very labile during the multistep catalytic reaction. Collective protein motions not only assist cleavage of the C6–S bond to stabilize the transition state of the proton transfer step but also rearrange the H-bond network at the end of this step to prepare the active site for subsequent chemical steps. These computational results illustrate functionalities of specific protein residues that reconcile many previous experimental observations and provide guidance for future experiments to examine the proposed mechanisms. The synchronized conformational changes in the protein and ligands observed in our simulations demonstrate participation of protein motions in the reaction coordinate of enzymatic reactions. Our computational findings suggest the existence of new reaction intermediates not covalently bound to TSase, which may lead to a new class of drugs targeting DNA biosynthesis

Extension and Limits of the Network of Coupled Motions Correlated to Hydride Transfer in Dihydrofolate Reductase

Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a “global dynamic network” of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts

How Accurate Are Transition States from Simulations of Enzymatic Reactions?

The rate expression of traditional transition state theory (TST) assumes no recrossing of the transition state (TS) and thermal quasi-equilibrium between the ground state and the TS. Currently, it is not well understood to what extent these assumptions influence the nature of the activated complex obtained in traditional TST-based simulations of processes in the condensed phase in general and in enzymes in particular. Here we scrutinize these assumptions by characterizing the TSs for hydride transfer catalyzed by the enzyme Escherichia coli dihydrofolate reductase obtained using various simulation approaches. Specifically, we compare the TSs obtained with common TST-based methods and a dynamics-based method. Using a recently developed accurate hybrid quantum mechanics/molecular mechanics potential, we find that the TST-based and dynamics-based methods give considerably different TS ensembles. This discrepancy, which could be due equilibrium solvation effects and the nature of the reaction coordinate employed and its motion, raises major questions about how to interpret the TSs determined by common simulation methods. We conclude that further investigation is needed to characterize the impact of various TST assumptions on the TS phase-space ensemble and on the reaction kinetics