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We present a detailed theoretical analysis of the reaction mechanism of proteolysis catalyzed by the main protease of SARS-CoV-2. Using multiscale simulation methods, we have characterized the interactions established by a peptidic substrate in the active site, and then we have explored the free energy landscape associated with the acylation and deacylation steps of the proteolysis reaction, characterizing the transition states of the process. Our mechanistic proposals can explain most of the experimental observations made on the highly similar ortholog protease of SARS-CoV. We point to some key interactions that may facilitate the acylation process and thus can be crucial in the design of more specific and efficient inhibitors of the main protease activity. In particular, from our results, the P1' residue can be a key factor to improve the thermodynamics and kinetics of the inhibition process., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

An unsolved mystery in biology concerns the link between enzyme catalysis and protein motions. Comparison between isotopically labelled "heavy" dihydrofolate reductases and their natural-abundance counterparts has suggested that the coupling of protein motions to the chemistry of the catalysed reaction is minimised in the case of hydride transfer. In alcohol dehydrogenases, unnatural, bulky substrates that induce additional electrostatic rearrangements of the active site enhance coupled motions. This finding could provide a new route to engineering enzymes with altered substrate specificity, because amino acid residues responsible for dynamic coupling with a given substrate present as hotspots for mutagenesis. Detailed understanding of the biophysics of enzyme catalysis based on insights gained from analysis of "heavy" enzymes might eventually allow routine engineering of enzymes to catalyse reactions of choice., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Photodynamic therapy is a promising approach to treat a variety of superficial tumors and other diseases. One of its major limitations arises from its dependence on molecular oxygen, which decreases the efficiency of the therapy in hypoxia conditions commonly developed by solid tumors. The present contribution reveals the molecular mechanism of a modified thymine bearing a nitroimidazole substituent, a photosensitizer able to produce highly harmful interstrand cross-links in the DNA double strand after irradiation selectively in absence of oxygen. The mechanism is resolved at a fully atomistic and electronic level relying on quantum mechanics (CASPT2, coupled-cluster, DFT, and TD-DFT methods), classical molecular dynamics, and advanced biased QM/MM simulations, revealing an energy penalty of ∼8 kcal/mol for the anionic nitromidazole release. Our findings indicate that the global interstrand cross-link production is driven by a combination of multiple factors, namely, the reverse energy penalty, the diffusion of the nitroimidazole anion, and the further reactivity of the formed thymine radical. On the basis of these results, we also suggest some possible strategies to improve the efficiency of interstrand cross-link production.

Hydride transfer is widespread in nature and has an essential role in applied research. However, the mechanisms of how this transformation occurs in living organisms remain a matter of vigorous debate. Here, we examined dihydrofolate reductase (DHFR), an enzyme that catalyzes hydride from C4' of NADPH to C6 of 7,8-dihydrofolate (H 2 F). Despite many investigations of the mechanism of this reaction, the contribution of polarization of the π-bond of H 2 F in driving hydride transfer remains unclear. H 2 F was stereospecifically labeled with deuterium β to the reacting center, and β-deuterium kinetic isotope effects were measured. Our experimental results combined with analysis derived from QM/MM simulations reveal that hydride transfer is triggered by polarization at the C6 of H 2 F. The σ C β -H bonds contribute to the buildup of the cationic character during the chemical transformation, and hyperconjugation influences the formation of the transition state. Our findings provide key insights into the hydride transfer mechanism of the DHFR-catalyzed reaction, which is a target for antiproliferative drugs and a paradigmatic model in mechanistic enzymology., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Dihydrofolate Reductase from Thermotoga maritima (TmDFHFR) is a dimeric thermophilic enzyme that catalyzes the hydride transfer from the cofactor NADPH to dihydrofolate less efficiently than other DHFR enzymes, such as the mesophilic analogue Escherichia coli DHFR (EcDHFR). Using QM/MM potentials we show that the reduced catalytic efficiency of TmDHFR is most likely due to differences in the amino acid sequence that stabilize the M20 loop in an open conformation, which prevents the formation of some interactions in the transition state and increases the number of water molecules in the active site. However, dimerization provides two advantages to the thermophilic enzyme; it protects its structure against denaturation by reducing thermal fluctuations and it provides a less negative activation entropy, toning down the increase of the activation free energy with temperature. Our molecular picture is confirmed by the analysis of the temperature dependence of enzyme kinetic isotope effects in different DHFR enzymes.

We estimate the time- and temperature-evolution of spin energy levels in a metallopeptide by combining molecular dynamics with crystal field analysis. Fluctuations of tens of cm -1 for spin energy levels at fs times gradually average out at longer times. We confirm that local vibrations are key in spin dynamics.

Hydride transfer is one of the most common reactions catalyzed by enzymatic systems and it has become an object of study due to possible significant quantum tunneling effects. In the present work, we provide a combination of theoretical QM/MM simulations and experimental measurements of the rate constants and kinetic isotopic effects (KIEs) for the hydride transfer reaction catalyzed by morphinone reductase, MR. Quantum mechanical tunneling coefficients, computed in the framework of variational transition-state theory, play a significant role in this reaction, reaching values of 23.8 ± 5.5 for the lightest isotopologue; one of the largest values reported for enzymatic systems. This prediction is supported by the agreement between the theoretically predicted rate constants and the corresponding experimental values. Simulations indicate that the role of protein motions can be satisfactorily described as equilibrium fluctuations along the reaction coordinate, in line with a high degree of preorganization displayed by this enzyme.

Enzymatic catalysis is of great importance to the chemical industry. However, we are still scratching the surface of the potential of biocatalysis due to the limited operating range of enzymes in harsh environments or their low recyclability. The role of Metal-Organic Frameworks (MOFs) as active supports to help overcome these limitations, mainly by immobilization and stabilization of enzymes, is rapidly expanding. Here we make use of mild heating and a non-polar medium during incubation to induce the translocation of a small enzyme like protease in the mesoporous MOF MIL-101(Al)-NH 2 . Our proteolytic tests demonstrate that protease@MIL-101(Al)-NH 2 displays higher activity than the free enzyme under all the conditions explored and, more importantly, its usability can be extended to extreme conditions of pH and high temperatures. MOF immobilization is also effective in providing the biocomposite with long-term stability, recyclability and excellent compatibility with competing enzymes. This simple, one-step infiltration strategy might accelerate the discovery of new MOF-enzyme biocatalysts that meet the requirements for biotechnological applications.

Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli , which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg 2+ . The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.

Catechol- O-methyltransferase is an enzyme that catalyzes the methylation reaction of dopamine by S-adenosylmethionine, increasing the reaction rate by almost 16 orders of magnitude compared to the reaction in aqueous solution. Here, we combine the recently introduced adaptive string method and the mean reaction force method, in combination with the structural and electronic descriptors to characterize the reaction mechanism. The catalytic effect of the enzyme is addressed by the comparison of the reaction in the human wild-type enzyme, in the less effective Y68A mutant, and in aqueous solution. The influence of these different environments at different stages of the chemical process and the significance of the key collective variables describing the reaction were quantified. Our results show that the native enzyme limits the access of water molecules to the active site, enhancing the interaction between the reactants and providing a more favorable electrostatic environment to assist the S N 2 methyl transfer reaction.

The intrinsic photostability of nucleic acids is intimately related to evolution of life, while its understanding at the molecular and electronic levels remains a challenge for modern science. Among the different decay pathways proposed in the last two decades, the excited-state hydrogen transfer between guanine-cytosine base pairs has been identified as an efficient non-reactive channel to dissipate the excess of energy provided by light absorption. The present work studies the dynamics of such phenomena taking place in a (dG)·(dC) B-DNA homopolymer in water solution using state-of-the-art molecular modelling and simulation methods. A dynamic effect that boosts the photostability of the inter-strand hydrogen atom transfers, inherent to the Watson-Crick base pairing, is unveiled and ascribed to the energy released during the proton transfer step. Our results also reveal a novel mechanism of DNA decay named four proton transfer (FPT), in which two protons of two adjacent G-C base pairs are transferred to form a biradical zwitterionic intermediate. Decay of the latter intermediate to the ground state triggers the transfer of the protons back to the guanine molecules recovering the Watson-Crick structure of the tetramer. This FPT process is activated by the close interaction of a nearby Na + counterion with the oxygen atoms of the guanine nucleobases and hence represents a photostable channel operative in natural nucleic acids.

The origin of enzyme catalysis remains a question of debate despite much intense study. We report a QM/MM theoretical study of the S N 2 methyl transfer reaction catalyzed by a glycine N-methyltransferase (GNMT) and three mutants to test whether recent experimental observations of rate-constant reductions and variations in inverse secondary α- 3 H kinetic isotope effects (KIEs) should be attributed to changes in the methyl donor-acceptor distance (DAD): Is catalysis due to a compression effect? Semiempirical (AM1) and DFT (M06-2X) methods were used to describe the QM subset of atoms, while OPLS-AA and TIP3P classical force fields were used for the protein and water molecules, respectively. The computed activation free energies and KIEs are in good agreement with experimental data, but the mutations do not meaningfully affect the DAD: Compression cannot explain the experimental variations on KIEs. On the contrary, electrostatic properties in the active site correlate with the catalytic activity of wild type and mutants. The plasticity of the enzyme moderates the effects of the mutations, explaining the rather small degree of variation in KIEs and reactivities.

Here we present a modified version of the on-the-fly string method for the localization of the minimum free energy path in a space of arbitrary collective variables. In the proposed approach the shape of the biasing potential is controlled by only two force constants, defining the width of the potential along the string and orthogonal to it. The force constants and the distribution of the string nodes are optimized during the simulation, improving the convergence. The optimized parameters can be used for umbrella sampling with a path CV along the converged string as the reaction coordinate. We test the new method with three fundamentally different processes: chloride attack to chloromethane in bulk water, alanine dipeptide isomerization, and the enzymatic conversion of isochorismate to piruvate. In each case the same set of parameters resulted in a rapidly converging simulation and a precise estimation of the potential of mean force. Therefore, the default settings can be used for a wide range of processes, making the method essentially parameter free and more user-friendly.

While being one of the most popular reaction rate theories, the applicability of transition state theory to the study of enzymatic reactions has been often challenged. The complex dynamic nature of the protein environment raised the question about the validity of the nonrecrossing hypothesis, a cornerstone in this theory. We present a computational strategy to quantify the error associated to transition state theory from the number of recrossings observed at the equicommittor, which is the best possible dividing surface. Application of a direct multidimensional transition state optimization to the hydride transfer step in human dihydrofolate reductase shows that both the participation of the protein degrees of freedom in the reaction coordinate and the error associated to the nonrecrossing hypothesis are small. Thus, the use of transition state theory, even with simplified reaction coordinates, provides a good theoretical framework for the study of enzymatic catalysis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Oxidation of nucleic acids is ubiquitous in living beings under metabolic impairments and/or exposed to external agents such as radiation, pollutants, or drugs, playing a central role in the development of many diseases mediated by DNA/RNA degeneration. Great efforts have been devoted to unveil the molecular mechanisms behind the OH radical additions to the double bonds of nucleobases; however, the specific role of the biological environment remains relatively unexplored. The present contribution tackles the study of the OH radical addition to uracil from the gas phase to a full RNA macromolecule by means of quantum-chemistry methods combined with molecular dynamics simulations. It is shown that, in addition to the intrinsic reactivity of each position driven by the electronic effects, the presence of bridge water molecules intercalated into the RNA structure favors the addition to the C5 position of uracil in biological conditions. The results also suggest that diffusion of the OH radical does not play a relevant role in the regioselectivity of the reaction, which is mainly controlled at the chemical stage of the addition process.

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein isotope labeling is a powerful technique to probe functionally important motions in enzyme catalysis and can be applied to investigate the conformational dynamics of proteins. Previous investigations have indicated that dynamic coupling is detrimental to catalysis by dihydrofolate reductase (DHFR) from the mesophile Escherichia coli (EcDHFR). Comparison of DHFRs from organisms adapted to survive at a wide range of temperatures suggests that dynamic coupling in DHFR catalysis has been minimized during evolution; it arises from reorganizational motions needed to facilitate charge transfer events. Contrary to the behaviour observed for the DHFR from the moderate thermophile Geobacillus stearothermophilus (BsDHFR), the chemical transformation catalyzed by the cold-adapted bacterium Moritella profunda (MpDHFR) is only weakly affected by protein isotope substitutions at low temperatures, but the isotopically substituted enzyme is a substantially inferior catalyst at higher, non-physiological temperatures. QM/MM studies revealed that this behaviour is caused by the enzyme's structural sensitivity to temperature changes, which enhances unfavorable dynamic coupling at higher temperatures by promoting additional recrossing trajectories on the transition state dividing surface. We postulate that these motions are minimized by fine-tuning DHFR flexibility through optimization of the free energy surface of the reaction, such that a nearly static reaction-ready configuration with optimal electrostatic properties is maintained under physiological conditions.

In the present work, we use Variational Transition State Theory (VTST) to develop a practical method for transition state ensemble optimization by looking for an optimal hyperplanar dividing surface in a space of meaningful trial collective variables. These might be interatomic distances, angles, electrostatic potentials, etc. Restrained molecular dynamics simulations are used to obtain on-the-fly estimates of ensemble averages that guide the variations of the hyperplane maximizing the transmission coefficient. A central result of our work is an expression that quantitatively estimates the importance of the coordinates used for the localization of the transition state ensemble. Starting from an arbitrarily large set of trial coordinates, one can distinguish those that are indeed essential for the advance of the reaction. This facilitates the use of VTST as a practical theory to study reaction mechanisms of complex processes. The technique was applied to the reaction catalyzed by an isochorismate pyruvate lyase. This reaction involves two simultaneous chemical steps and has a shallow transition state region, making it challenging to define a good reaction coordinate. Nevertheless, the hyperplanar transition state optimized in the space of 18 geometrical coordinates provides a transmission coefficient of 0.8 and a committor histogram well-peaked about 0.5, proving the strength of the method. We have also tested the approach with the study of the NaCl dissociation in aqueous solution, a stringest test for a method based on transition state theory. We were able to find essential degrees of freedom consistent with the previous studies and to improve the transmission coefficient with respect to the value obtained using solely the NaCl distance as the reaction coordinate.

We offer some thoughts on the much debated issue of dynamical effects in enzyme catalysis, and more specifically on their potential role in the acceleration of the chemical step. Since the term 'dynamics' has been used with different meanings, we find it useful to first return to the Transition State Theory rate constant, its assumptions and the choices it involves, and detail the various sources of deviations from it due to dynamics (or not). We suggest that much can be learned about the key current questions for enzyme catalysis from prior extensive studies of dynamical and other effects in the case of reactions in solution. We analyze dynamical effects both in the neighborhood of the transition state and far from it, together with the situation when quantum nuclear motion is central to the reaction, and we illustrate our discussion with various examples of enzymatic reactions., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A theoretical study on the alkaline hydrolysis of paraoxon, one of the most popular organophosphorus pesticides, in aqueous solution and in the active site of Pseudomonas diminuta phosphotriesterase (PTE) is presented. Simulations by means of hybrid quantum mechanics/molecular mechanics (QM/MM) potentials show that the hydrolysis of paraoxon takes place through an A(N)D(N) or associative mechanism both in solution and in the active site of PTE. The results correctly reproduce the magnitude of the activation free energies and can be used to rationalize the observed kinetic isotope effects (KIEs) for the hydrolysis of paraoxon in both media. Enzymatic hydrolysis of O,O-diethyl p-chlorophenyl phosphate, a phosphotriester having a leaving group with higher pK(a) than paraoxon, was also simulated. Hydrolysis of this phosphotriester by PTE follows a A(N)+D(N) mechanism with a pentacoordinate intermediate. Moreover, the leaving group of this new substrate coordinates to one of the zinc ions of the bimetallic active site in order to stabilize the large negative charge developed on the oxygen atom of the leaving group when the P-O bond is broken in the products state. To accommodate this new ligand in the coordination shell, carbamylated Lys169 must be displaced from one zinc ion to the other, which in turn affects the acidity of Asp301, a residue originally bound to the second zinc ion. This ability to displace some of the ligands of the coordination shell of the zinc centers would explain the promiscuity of this enzyme, which is capable of catalyzing hydrolysis of different substrate by means of different mechanisms., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of characterization techniques, advanced synthesis methods, as well as molecular modeling has transformed the study of systems in a well-established research field. The current research challenges in biocatalysis and biotransformation evolve around enzyme discovery, design, and optimization. How can we find or create enzymes that catalyze important synthetic reactions, even reactions that may not exist in nature? What is the source of enzyme catalytic power? To answer these and other related questions, the standard strategies have evolved from trial-and-error methodologies based on chemical knowledge, accumulated experience, and common sense into a clearly multidisciplinary science that allows one to reach the molecular design of tailor-made enzyme catalysts. This is even more so when one refers to enzyme catalysts, for which the detailed structure and composition are known and can be manipulated to introduce well-defined residues which can be implicated in the chemical rearrangements taking place in the active site. The methods and techniques of theoretical and computational chemistry are becoming more and more important in both understanding the fundamental biological roles of enzymes and facilitating their utilization in biotechnology. Improvement of the catalytic function of enzymes is important from scientific and industrial viewpoints, and to put this fact in the actual perspective as well as the potentialities, we recommend the very recent report of Sanderson [Sanderson, K. (2011). Chemistry: enzyme expertise. Nature 471, 397.]. Great fundamental advances have been made toward the ab initio design of enzyme catalysts based on molecular modeling. This has been based on the molecular mechanistic knowledge of the reactions to be catalyzed, together with the development of advanced synthesis and characterization techniques. The corresponding molecular mechanism can be studied by means of powerful quantum chemical calculations. The catalytic active site can be optimized to improve the transition state analogues (TSA) and to enhance the catalytic activity, even improve the active site to favor a desired direction of some promiscuous enzymes. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. Current computational tools to assist experimentalists for the design and engineering of proteins with desired catalytic properties are described. The interplay between enzyme design, molecular simulations, and experiments will be presented to emphasize the interdisciplinary nature of this research field. This text highlights the recent advances and examples selected from our laboratory are shown, of how the applications of these tools are a first attempt to de novo design of protein active sites. Identification of neutral/advantageous/deleterious mutation platforms can be exploited to penetrate some of Nature's closely guarded secrets of chemical reactivity. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. The first part describes briefly how the molecular modeling is carried out. Then, we discuss the requirements of hybrid quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) simulations, analyzing what are the basis of these theoretical methodologies, how we can use them with a view to its application in the study of enzyme catalysis, and what are the best methodologies for assessing its catalytic potential. In the second part, we focus on some selected examples, taking as a common guide the chorismate to prephenate rearrangement, studying the corresponding molecular mechanism in vacuo, in solution and in an enzyme environment. In addition, examples involving catalytic antibodies (CAs) and promiscuous enzymes will be presented. Finally, a special emphasis is made to provide some hints about the logical evolution that can be anticipated in this research field. Moreover, it helps in understanding the open directions in this area of knowledge and highlights the importance of computational approaches in discovering specific drugs and the impact on the rational design of tailor-made enzymes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A theoretical study of dynamic effects on the rate-limiting step of the thymidylate synthase catalyzed reaction has been carried out by means of Grote-Hynes theory, successfully predicting the values of the recrossing effects for a chemical reaction that involves the transfer of a classical light particle. The transmission coefficients, obtained at 278, 293, 303, and 313 K, are almost invariant and in all cases far from unity, revealing a significant coupling of the environment motions and the reaction coordinate. Nevertheless, their energetic contribution to the activation free energy represents less than 0.50 kcal/mol for each of the four tested temperatures. Calculation of the transmission coefficient for the isotopically labeled hydride transfer has rendered almost the same values, in agreement with the experimentally observed temperature-independent KIEs. Fourier transform of the time-dependent friction kernel at these four temperatures has allowed obtaining the transition-state friction spectra, which present very small dependence with temperature. Their analysis has led to the identification of some key vibrational modes governing the coupling between the reaction coordinate and the protein environment, thus identifying the relevant motions in the active site and obtaining a full picture of the role of each amino acid.

A theoretical study of the protein dynamic effects on the hydride transfer between the formate anion and nicotinamide adenine dinucleotide (NAD(+)), catalyzed by formate dehydrogenase (FDH), is presented in this paper. The analysis of free downhill molecular dynamic trajectories, performed in the enzyme and compared with the reaction in aqueous solution, has allowed the study of the dynamic coupling between the reacting fragments and the protein or the solvent water molecules, as well as an estimation of the dynamic effect contribution to the catalytic effect from calculation of the transmission coefficient in the enzyme and in solution. The obtained transmission coefficients for the enzyme and in solution were 0.46±0.04 and 0.20±0.03, respectively. These values represent a contribution to catalysis of 0.5 kcal mol(-1), which, although small, is not negligible keeping in mind the low efficiency of FDH. The analysis of the reactive trajectories also reveals how the relative movements of some amino acids, mainly His332 and Arg284, precede and promote the chemical reaction. In spite of these movements, the time-dependent evolution of the electric field created by the enzyme on the key atoms of the reaction reveals a permanent field, which reduces the work required to reach the transition state, with a concomitant polarization of the cofactor. Finally, application of Grote-Hynes theory has allowed the identification of the modes responsible for the substrate-environment coupling, showing how some protein motions take place simultaneously with the reaction. Thus, the equilibrium approach would provide, in this case, an overestimation of the catalyzed rate constant.

The stereospecific 5 beta-reduction of progesterone is a required step for cardiac glycoside biosynthesis in foxglove plants. Recently, we have isolated the gene P5 beta R, and here we investigate the function and regulation of P5 beta R2, a new progesterone 5 beta-reductase gene from Digitalis purpurea. P5 beta R2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. Like P5 beta R, P5 beta R2 catalyzes the 5 beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5 beta R expression does not vary, whereas P5 beta R2 is highly responsive. P5 beta R2 expression is regulated by ethylene and hydrogen peroxide. The correlation between P5 beta R2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide biosynthesis, and therefore in the chemical defense of foxglove plants.

O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.

We report a hybrid quantum mechanics/molecular mechanics theoretical study on the reaction mechanism of mammalian histidine decarboxylase that allows us to obtain valuable insights on the structure of the cofactor-substrate adduct (external aldimine) in the active site of rat histidine decarboxylase. By means of molecular dynamics simulations, we traced the potential of mean force corresponding to the decarboxylation reaction of the adduct both in the active site of the enzyme and in aqueous solution. By comparing this process in both media, we have identified the key electrostatic interactions that explain the lowering of the free energy barrier in the enzyme. Our analysis also offers a validation of Dunathan's hypothesis (Dunathan, H. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 712-716) regarding the role of stereoelectronic effects in the enzymatic decarboxylation process.

Dynamical effects have recently received much attention in the context of the theoretical investigation of enzymatic catalysis. In this paper we use a combination of Grote-Hynes theory with quantum mechanical/molecular mechanical modeling that is a powerful tool to understand and quantify these dynamical effects in a particular enzyme, the glycine N-methyltransferase (GNMT). Comparison of the results obtained for this enzyme with another methyltransferase (catechol O-methyltransferase, COMT) allows us to understand the different nature of the coupling of the environment to the reaction coordinate as a function of the electrostatic interaction established by the reactive subsystem. The transmission coefficients obtained using Grote-Hynes theory are in excellent agreement with molecular dynamics estimations and show that the coupling is higher in GNMT than in COMT. The larger friction observed in GNMT is explained on the basis of the interaction established by the substrate in the active site. The larger value of the friction leads to a smaller value of the reaction frequency and thus also to a larger disagreement with the estimation of the transmission coefficient based on the frozen environment approach.

The Diels-Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39 A-11, that catalyze a Diels-Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate-protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate.

Human immunodeficiency virus type-1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Diketo acids such as L-731,988 and S-1360 are potent and selective inhibitors of HIV-1 IN. In this study, we used molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy between HIV-1 IN and L-731,988 and 10 of its derivatives and analogues. This hybrid methodology has the advantage that it includes quantum effects such as ligand polarisation upon binding, which can be very important when highly polarisable groups are embedded in anisotropic environments, as for example in metal-containing active sites. Furthermore, an energy decomposition analysis was performed to determine the contributions of individual residues to the enzyme-inhibitor interactions on averaged structures obtained from rather extensive conformational sampling. Analysis of the results reveals first that there is a correlation between protein-ligand interaction energy and experimental strand transfer into human chromosomes and secondly that the Asn-155, Lys-156 and Lys-159 residues and the Mg(2+) ion are crucial to anti-HIV IN activity. These results may explain the available experimental data.

The generalized Langevin equation (GLE)-based Grote-Hynes (GH) theory is used to calculate the transmission coefficients, kappa, for the methyl transfer from S-adenosylmethionine to catecholate both in aqueous solution and in the catechol O-methyltransferase active site. Values of kappa, which measures the deviation of the rate constants from the Transition State Theory (TST) predictions, are obtained by means of rare event molecular dynamics simulations. The results are 0.62 +/- 0.04 and 0.83 +/- 0.03 for the aqueous and enzymatic environments, respectively, while the Grote-Hynes predictions are 0.58 +/- 0.09 and 0.89 +/- 0.03, respectively. The Kramers theory estimates are much smaller, about 0.01 and 0.1, respectively. Thus, the enzymatic transmission coefficient is closer to TST predictions than the value obtained in solution. In addition, our results show that the enzymatic coefficient is also closer to its nonadiabatic (or frozen environment) limit than is the solution coefficient. These findings can be understood considering that, during the passage over the barrier top, there is a smaller coupling between the reactive system and the environment in the enzyme than in solution, as well as a smaller reorganization suffered by the enzyme. Analysis of the transition state friction kernel leads to the identification of some key vibrational modes governing the coupling between the two different environments and the reacting solute in the transition state region and insights on their relevance for the reaction dynamics' influence on the transmission coefficient.

Catalytic antibodies are very interesting not only because of the rate enhancement of the reactions that they catalyze but also because of the selectivities they can achieve that are sometimes not present in natural enzyme processes. We have selected the study of the stereoselectivity of the matured AZ28 that catalyzes an oxy-Cope rearrangement. For this particular case, the presence of a chiral center in the substrate provokes the existence of two different enantiomers, R and S. Furthermore, it is also possible to locate two different orientations for the hydroxyl group in the central ring of the substrate in the transition state, equatorial and axial, rendering two different conformers. In this paper we present the free energy profiles obtained for different substrate isomers in the cavity created by the matured catalytic antibody. Our simulations have reproduced the stereoselectivity of the matured AZ28, differentiating between the axial or equatorial orientations and preferentially stabilizing the S forms, at a qualitative level. Finally, the inclusion of the substrate-CA interactions in a flexible molecular model has allowed us to observe the different pattern of interactions that are related to different interaction energies, which seem to be crucial in the stereoselectivity behavior of the catalytic antibody.

We use quantum mechanics/molecular mechanics (QM/MM) calculations to estimate the activation free energy for the chemical reaction catalyzed by catechol O-methyltransferase. While in many cases the activation free energy of a chemical process is directly determined by the potential of mean force associated with a particular reaction coordinate, here we have included several corrections that have been proposed in the literature. These include the free energy change associated with release of the reaction coordinate motion in the reactant state, consideration of the curvilinear nature of the reaction coordinate, and correction due to the classical treatment of molecular vibrations. In addition, since potentials of mean force are usually obtained from low levels of QM theory to describe the quantum subsystem, we have included an interpolated correction term to improve this description at small additional cost. This last correction term has a dramatic effect, improving the agreement between the theoretical predictions and the experimental value, while the other terms considered make only small contributions to this particular reaction.

The neutral hydrolysis of formamide in water is a suitable reference to quantify the efficiency of proteolytic enzymes. However, experimental data for this reaction has only very recently been obtained and the kinetic constant determined experimentally is significantly higher than that predicted by previous theoretical estimations. In this work, we have investigated in detail the possible mechanisms of this reaction. Several solvent models have been considered that represent a considerable improvement on those used in previous studies. Density functional and ab initio calculations have been carried out on a system which explicitly includes the first solvation shell of the formamide molecule. Its interaction with the bulk has been treated with the aid of a dielectric continuum model. Molecular dynamics simulations at the combined density functional/molecular mechanics level have been carried out in parallel to better understand the structure of the reaction intermediates in aqueous solution. Overall, the most favored mechanism predicted by our study involves two reaction steps. In the first step, the carbonyl group of the formamide molecule is hydrated to form a diol intermediate. The corresponding transition structure involves two water molecules. From this intermediate, a water-assisted proton transfer occurs from one of the hydroxy groups to the amino group. This reaction step may lead either to the formation of a new reaction intermediate with a marked zwitterionic character or to dissociation of the system into ammonia and formic acid. The zwitterionic intermediate dissociates quite easily but its lifetime is not negligible and it could play a role in the hydrolysis of substituted amides or peptides. The predicted pseudo-first-order kinetic constant for the rate-limiting step (the first step) of the hydrolysis reaction at 25 degrees C (3.9x10(-10) s(-1)) is in excellent agreement with experimental data (1.1x10(-10) s(-1)).

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.

We present a theoretical study of a mechanism for the hydrolysis of the acyl-enzyme complex formed by a class A beta-lactamase (TEM1) and an antibiotic (penicillanate), as a part of the process of antibiotic's inactivation by this type of enzymes. In the presented mechanism the carboxylate group of a particular residue (Glu166) activates a water molecule, accepting one of its protons, and afterward transfers this proton directly to the acylated serine residue (Ser70). In our study we employed a quantum mechanics (AM1)-molecular mechanics partition scheme (QM/MM) where all the atoms of the system were allowed to relax. For this purpose we used the GRACE procedure in which part of the system is used to define the Hessian matrix while the rest is relaxed at each step of the stationary structures search. By use of this computational scheme, the hydrolysis of the acyl-enzyme is described as a three-step process: The first step corresponds to the proton transfer from the hydrolytic water molecule to the carboxylate group of Glu166 and the subsequent formation of a tetrahedral adduct as a consequence of the attack of this activated water molecule to the carbonyl carbon atom of the beta-lactam. In the second step, the acyl-enzyme bond is broken, obtaining a negatively charged Ser70. In the last step this residue is protonated by means of a direct proton transfer from Glu166. The large mobility of Glu166, a residue that is placed in a Ohms-loop, is essential to facilitate this mechanism. The geometry of the acyl-enzyme complex shows a large distance between Glu166 and Ser70 and thus, if protein coordinates were kept frozen during the reaction path, it would be difficult to get a direct proton transfer between these two residues. This computational study shows how a flexible treatment suggests the feasibility of a mechanism that could have been discounted on the basis of crystallographic positions.

A theoretical study of the water-assisted alkaline hydrolysis of 2-azetidinone, 3-formylamino-2-azetidinone and 3-formylamino-2-azetidine-1-sulfonate ion is carried out at the B3LYP/6-31+G* level. The effect of bulk solvent is taken into account using the PCM solvation model while specific solvent effects are represented by the inclusion of an ancillary water molecule along the reaction profile. The calculated free energy barriers in solution are in reasonable agreement with experimental values. The observed substituent effects due to the presence of the 3-formylamino and the SO(3) groups attached to the beta-lactam ring are crucial factors determining the hydrolysis of monobactam antibiotics.