Metadynamics Technique Research Articles

Unlocking the specificity of antimicrobial peptide interactions for membrane-targeted therapies

Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

Design of Novel Imidazopyrazine Derivative against Breast Cancer via Targeted NPY1R Antagonist.

Breast cancer is the most frequent malignancy in women with more than one in ten new cancer diagnoses each year. Synthetic products are a key source for the identification of new anticancer medicines and drug leads. Imidazopyrazine is a highly favored skeleton for the design of new anticancer drugs. In silico designed derivatives were screened using computer aided drug design techniques and validated using MTT assay. A template-based methodology was used in the current work to create novel Imidazopyrazine derivatives, targeting the NPY1R protein. Molecular docking, Diffusion docking, MD simulation, MM-GBSA and meta-dynamics techniques were followed. MTT assay was performed to validate the activity of principal compound. A docking score of -6.660 and MMGBSA value of -108.008 (+/-) 9.14 kcal/mol was obtained from the investigations conducted. In addition, molecular dynamics simulation was carried out for 500 ns, yielding a stable RMSD and value of 5.6 Å, thus providing insights on the stability of the protein conformation on interaction with the principal compound. Furthermore, the in vivo validation studies conducted via MTT assay showed an IC50 value of 73.45 (+/-) 0.45 μg /mL. The research has produced encouraging findings and can be applied as a model for precise enumerations in the future. It also encourages the study of novel synthetic compounds with potential anti-cancer properties.

Facet Selectivity of Cetyltrimethyl Ammonium Bromide Surfactants on Gold Nanoparticles Studied Using Molecular Simulations.

We have studied facet selectivity of cetyltrimethyl ammonium bromide (CTAB) surfactants of varying alkyl tail lengths (C17TAB and C10TAB) during their adsorption on a spherical gold metal nanoparticle (MNP) using umbrella sampling and well-tempered metadynamics techniques in molecular simulations. We show that the surfactants strongly adsorb with their alkyl tails wrapped around the MNP. The adsorption morphologies are dictated by the strong preference of the polar head group of the surfactants to adsorb on to the atoms that lie between the facets of the MNP, that is, in the vicinity of low-coordinated gold atoms. The alkyl tails do not display any strong facet preference. Owing to the longer alkyl tails, C17TAB molecules pack together better than the C10TAB molecules in the adsorbed state on the MNP. These findings suggest that the regions near the edges of the facets and low-coordinated atoms are expected to be preferentially covered with the adsorbed surfactants.

Assessing models of force-dependent unbinding rates via infrequent metadynamics.

Protein-ligand interactions are crucial for a wide range of physiological processes. Many cellular functions result in these non-covalent "bonds" being mechanically strained, and this can be integral to proper cellular function. Broadly, two classes of force dependence have been observed-slip bonds, where the unbinding rate increases, and catch bonds, where the unbinding rate decreases. Despite much theoretical work, we cannot predict for which protein-ligand pairs, pulling coordinates, and forces a particular rate dependence will appear. Here, we assess the ability of MD simulations combined with enhanced sampling techniques to probe the force dependence of unbinding rates. We show that the infrequent metadynamics technique correctly produces both catch and slip bonding kinetics for model potentials. We then apply it to the well-studied case of a buckyball in a hydrophobic cavity, which appears to exhibit an ideal slip bond. Finally, we compute the force-dependent unbinding rate of biotin-streptavidin. Here, the complex nature of the unbinding process causes the infrequent metadynamics method to begin to break down due to the presence of unbinding intermediates, despite the use of a previously optimized sampling coordinate. Allowing for this limitation, a combination of kinetic and free energy computations predicts an overall slip bond for larger forces consistent with prior experimental results although there are substantial deviations at small forces that require further investigation. This work demonstrates the promise of predicting force-dependent unbinding rates using enhanced sampling MD techniques while also revealing the methodological barriers that must be overcome to tackle more complex targets in the future.

A Computational Study of RNA Tetraloop Thermodynamics, Including Misfolded States.

An important characteristic of RNA folding is the adoption of alternative configurations of similar stability, often referred to as misfolded configurations. These configurations are considered to compete with correctly folded configurations, although their rigorous thermodynamic and structural characterization remains elusive. Tetraloop motifs found in large ribozymes are ideal systems for an atomistically detailed computational quantification of folding free energy landscapes and the structural characterization of their constituent free energy basins, including nonnative states. In this work, we studied a group of closely related 10-mer tetraloops using a combined parallel tempering and metadynamics technique that allows a reliable sampling of the free energy landscapes, requiring only knowledge that the stem folds into a canonical A-RNA configuration. We isolated and analyzed unfolded, folded, and misfolded populations that correspond to different free energy basins. We identified a distinct misfolded state that has a stability very close to that of the correctly folded state. This misfolded state contains a predominant population that shares the same structural features across all tetraloops studied here and lacks the noncanonical A-G base pair in its loop portion. Further analysis performed with biased trajectories showed that although this competitive misfolded state is not an essential intermediate, it is visited in most of the transitions from unfolded to correctly folded states. Moreover, the tetraloops can transition from this misfolded state to the correctly folded state without requiring extensive unfolding.

Molecular dynamics simulation of the adsorption of per- and polyfluoroalkyl substances (PFASs) on smectite clay

Molecular dynamics (MD) simulations are used to predict the partitioning of per- and polyfluoroalkyl substances (PFASs) to smectite clay, a high surface area adsorbent ubiquitous in temperate soils. Simulated systems model a stack of flexible smectite lamellae in contact with a bulk aqueous reservoir containing PFAS molecules. Perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonic acid (PFOS) are simulated at various aqueous chemistry conditions to examine the effect of PFAS size, salinity, and coordinating cation type (K+, Na+, and Ca2+) on adsorption. The metadynamics technique is employed to facilitate the exploration of the simulation cell and to reconstruct the underlying free energy landscape. Adsorption is favorable on the hydrophobic domains of the external basal surfaces with the fluorinated chain adopting a flat orientation on the surface. Analysis of the adsorption energetics reveals large favorable entropic contributions to adsorption. The enthalpy of adsorption is unfavorable, though much less so in the presence of Ca2+ due to stabilizing ‘lateral cation bridging’ interactions between divalent cations and PFAS sulfonate head groups. Overall, this research advances the mechanistic understanding of PFAS-smectite interactions and provides new insights that could help inform fate and transport models and the development of adsorbents and remediation techniques.

Voltage-Dependent Transport of Neutral Solutes through Nanopores: A Molecular View.

The permeation of (neutral) molecules through nanopores in the presence of external voltages depends on several factors including pore electrostatics, electrophoretic force, and electro-osmotic drag. In earlier single-channel electrophysiology experiments, voltage-dependent asymmetric transport of neutral α-cyclodextrin (α-CD) molecules through the biological nanopore ΔCymA was observed. The voltage-dependent ion-associated flow of water, the so-called electro-osmotic flow, has been suggested to be the key factor behind the observed asymmetric behavior. The influence of pore electrostatics and electrophoretic force and their interplay with the electro-osmotic drag with varying buffers and voltages has not yet been analyzed at the molecular level. Hence, the detailed physical mechanism behind this intriguing permeation process is in part still unclear. In the present study, we have performed 36 μs all-atom free energy calculations by combining applied-field molecular dynamics simulations with metadynamics techniques. The influence of several ionic conditions as well as external voltages on the permeation of α-CD molecules across the ΔCymA pore has been investigated. To decipher the thermodynamic and kinetic details, the lowest energy paths and the permeation times for α-CD translocation have been estimated. In the presence of KCl or MgCl2 salts, the charge of the cations is found to control the direction and magnitude of the electro-osmotic flow, which in turn strongly affects α-CD permeation. Overall, the present findings significantly improve the fundamental understanding of the voltage-dependent transport of neutral solutes across nanopores.

Accessing slow diffusion in solids by employing metadynamics simulation.

Molecular dynamics (MD) is a powerful tool to investigate microscopic transport of atoms and molecules in condensed matter. However, there lies a large class of systems wherein atomic diffusion is too slow a process relative to the feasible time scales of typical atomistic simulations. Here, we demonstrate that with judicial implementation of a metadynamics (MTD) technique, the microscopic mechanism of atomic transport in solids can be accessed within a reasonable computational time. The calculations are carried out on the two end members of the true NASICON solid solutions, namely NaZr2(PO4)3 and Na4Zr2(SiO4)3, wherein Na+ diffusion is too slow to be accessed through standard MD simulations. The study also provides fresh insights on correlated ion hops and their implications on the effective diffusion barrier. The results are compared with climbing image nudged elastic band (CI-NEB) calculation, and available experimental results.

Dcdftbmd: Divide-and-Conquer Density Functional Tight-Binding Program for Huge-System Quantum Mechanical Molecular Dynamics Simulations.

Dcdftbmd is a Fortran 90/95 program that enables efficient quantum mechanical molecular dynamics (MD) simulations using divide-and-conquer density functional tight-binding (DC-DFTB) method. Based on the remarkable performance of previous massively parallel DC-DFTB energy and gradient calculations for huge systems, the code has been specialized to MD simulations. Recent implementations and modifications including DFTB extensions, improved computational speed in the DC-DFTB computational steps, algorithms for efficient initial guess charge prediction, and free energy calculations via metadynamics technique have enhanced the capability to obtain atomistic insights in novel applications to nanomaterials and biomolecules. The energy, structure, and other molecular properties are also accessible through the single-point calculation, geometry optimization, and vibrational frequency analysis. The available functionalities are outlined together with efficiency tests and simulation examples. © 2019 Wiley Periodicals, Inc.

HNCO-based synthesis of formamide in planetary atmospheres

Time-resolved Fourier transform infrared emission spectroscopy, Fourier transform absorption infrared spectroscopy, and high-resolution UV–ViS emission spectroscopy have been used to characterize the chemistry of isocyanic acid (HNCO) under glow discharge conditions in planetary atmospheres. HNCO mixtures (i.e., composed of di-hydrogen or ammonia) have been investigated in order to unveil the possible reaction pathways leading to the synthesis of the key prebiotic molecule formamide (HCONH2), upon planetary atmospheres containing isocyanic acid in presence of di-hydrogen and, separately, of ammonia. In addition, ab initio molecular dynamics simulations coupled with a modern metadynamics technique have been performed in order to identify the most likely chemical pathways connecting HNCO to formamide. It turned out that the direct hydrogenation of HNCO is thermodynamically favored. Incidentally, the experimental results – supplied by a simplified kinetic model – also proved the favorability of the reaction HNCO + H2→ HCONH2which, moreover, spontaneously takes place in unbiased ab initio molecular dynamics simulations carried out under the effect of intense electric fields.

Free Energy Calculations of Membrane Permeation: Challenges Due to Strong Headgroup-Solute Interactions.

Understanding how different classes of molecules move across biological membranes is a prerequisite to predicting a solute's permeation rate, which is a critical factor in the fields of drug design and pharmacology. We use biased molecular dynamics computer simulations to calculate and compare the free energy profiles of translocation of several small molecules across 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) lipid bilayers as a first step toward determining the most efficient method for free energy calculations. We study the translocation of arginine, a sodium ion, alanine, and a single water molecule using the metadynamics, umbrella sampling, and replica exchange umbrella sampling techniques. Within the fixed lengths of our simulations, we find that all methods produce similar results for charge-neutral permeants, but not for polar or positively charged molecules. We identify the long relaxation time scale of electrostatic interactions between lipid headgroups and the solute to be the principal cause of this difference and show that this slow process can lead to an erroneous dependence of computed free energy profiles on the initial system configuration. We demonstrate the use of committor analysis to validate the proper sampling of the presumed transition state, which in our simulations is achieved only in replica exchange calculations. On the basis of these results we provide some useful guidance to perform and evaluate free energy calculations of membrane permeation.

Synthesis of (d)-erythrose from glycolaldehyde aqueous solutions under electric field.

The formation of the first C-C bonds from formaldehyde represents the rate-limiting step of the formose reaction. However, the free-energy surface associated with such a process has never been determined in condensed phase. By means of ab initio molecular dynamics and metadynamics techniques here we report on the free-energy landscape underlying the synthesis of glycolaldehyde from a formaldehyde aqueous solution. Moreover, numerical samples of formaldehyde (both neat and in water solution) and of glycolaldehyde (both neat and in aqueous solution) have been exposed to intense electric fields. The application of electrostatic gradients strongly prevents the formaldehyde umpolung and catalyzes the formation of C-O-bonded polymers in formaldehyde-containing samples. However, when the field is applied on glycolaldehyde aqueous solutions, new C-C bonds are formed and (d)-erythrose is synthesized. This way, a numerical Miller-like experiment led to the formation of a prebiotically relevant (d)-tetrose from ubiquitarious molecules such as glycolaldehyde and water.

Exploring Key Descriptors of Solid Electrolyte Interphase Formation in Lithium Ion Batteries through Atomistic Simulations

Lithium ion batteries (LIBs) have allowed for a technological revolution in portable electronics, power tools, and electric vehicles. Future improvements, however, require an advancement of existing battery technology to provide for faster charging and larger capacities, while maintaining safety and longevity. This fundamentally represents a knowledge and materials challenge that needs to develop a deeper understanding of electrode and electrolyte materials as well as their interfaces. Electrolyte and electrode materials used in LIBs have been studied separately to a great extent, however the structural and dynamical properties of the electrode/electrolyte interface still remain largely unexplored despite its critical role in governing battery performance. In particular, a key issue restraining the utilization of high-energy density anode materials, such as silicon (Si), is their inability to form a stable interphase during charging which results in continuous capacity fade or even cell failure. The design of better storage devices may be improved through a greater fundamental understanding of the formation of the solid electrolyte interphase (SEI) at the anode/electrolyte interface through atomistic computer simulations. We have utilized molecular dynamics (MD) simulations to correlate the bulk composition of the electrolyte to the interfacial structure and its effect on the transport behavior of key intermediates in the reductive decomposition of this electrolyte at a graphitic carbon electrode. Our study clearly demonstrates that the interfacial structure is sensitive to the molecular geometry and polarity of each solvent molecule as well as the surface structure and charge distribution of the negative electrode. Such quantitative analysis of the molecular arrangements at the electrode/electrolyte interface improves the understanding and description of electrolyte decomposition and SEI formation. Using MD and metadynamics techniques, we have systematically studied how factors such as the bulk composition of mixed carbonate electrolytes, precipitation of lithium fluoride (LiF, a result of the unstable PF6- anion species) onto the anode, and some commonly utilized additives (shown to improve the quality of the SEI layer) influence the transport and accumulation of decomposition products/intermediates. In this talk, we will present some of our recent findings including what we believe to be key descriptors in to predicting the decomposition products arising from the electrochemical reduction of the electrolyte. This improved understanding provides design principles for future investigation into forming a stable SEI on high-energy anodes such as Si.

Simulating Protein Mediated Hydrolysis of ATP and Other Nucleoside Triphosphates by Combining QM/MM Molecular Dynamics with Advances in Metadynamics.

The protein mediated hydrolysis of nucleoside triphosphates such as ATP or GTP is one of the most important and challenging biochemical reactions in nature. The chemical environment (water structure, catalytic metal, and amino acid residues) adjacent to the hydrolysis site contains hundreds of atoms, usually greatly limiting the amount of the free energy sampling that one can achieve from computationally demanding electronic structure calculations such as QM/MM simulations. Therefore, the combination of QM/MM molecular dynamics with the recently developed transition-tempered metadynamics (TTMetaD), an enhanced sampling method that can provide a high-quality free energy estimate at an early stage in a simulation, is an ideal approach to address the biomolecular nucleoside triphosphate hydrolysis problem. In this work the ATP hydrolysis process in monomeric and filamentous actin is studied as an example application of the combined methodology. The performance of TTMetaD in these demanding QM/MM simulations is compared with that of the more conventional well-tempered metadynamics (WTMetaD). Our results show that TTMetaD exhibits much better exploration of the hydrolysis reaction free energy surface in two key collective variables (CVs) during the early stages of the QM/MM simulation than does WTMetaD. The TTMetaD simulations also reveal that a key third degree of freedom, the O–H bond-breaking and proton transfer from the lytic water, must be biased for TTMetaD to converge fully. To perturb the NTP hydrolysis dynamics to the least extent and to properly focus the MetaD free energy sampling, we also adopt here the recently developed metabasin metadynamics (MBMetaD) to construct a self-limiting bias potential that only applies to the lytic water after its nucleophilic attack of the phosphate of ATP. With these new, state-of-the-art enhanced sampling metadynamics techniques, we present an effective and accurate computational strategy for combining QM/MM molecular dynamics simulation with free energy sampling methodology, including a means to analyze the convergence of the calculations through robust numerical criteria.

Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase.

Atomistic molecular dynamics (MD) simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.2Mg from its binding site has been observed. The swinging motion of the Asp side chain (D134/D145) in the binding pocket facilitates the detachment of IMP, which triggers the opening of flexible loop II, the gateway to the bulk solvent. In PfHGXPRT, PPi.2Mg and IMP are seen to be released via the same path in all of the biased MD simulations. In HsHGPRT too, the product molecules follow similar routes from the active site; however, an alternate but minor escape route for PPi.2Mg has been observed in the human enzyme. Tyr 104 and Phe 186 in HsHGPRT and Tyr 116 and Phe 197 in PfHGXPRT are the key residues that mediate the release of IMP, whereas the motion of PPi.2Mg away from the reaction center is guided by the negatively charged Asp and Glu and a few positively charged residues (Lys and Arg) that line the product release channels. Mutations of a few key residues present in loop II of Trypanosoma cruzi (Tc) HGPRT have been shown to reduce the catalytic efficiency of the enzyme. Herein, in silico mutation of corresponding residues in loop II of HsHGPRT and PfHGXPRT resulted in partial opening of the flexible loop (loop II), thus exposing the active site to bulk water, which offers a rationale for the reduced catalytic activity of these two mutant enzymes. Investigations of the product release from these HsHGPRT and PfHGXPRT mutants delineate the role of these important residues in the enzymatic turnover.

Cationic Methylene–Pyrene Isomers and Isomerization Pathways: Finite Temperature Theoretical Studies

This paper provides spectral characterizations of the two isomers of the 1-methylenepyrene cation, namely, the 1-pyrenemethylium and a pyrene-like isomer owing a tropylium cycle. Both are possible photodissociation products of the 1-methylpyrene cation and were proposed as potential contributors to the diffuse interstellar bands. In that respect, vibrational and electronic spectra are computed for the optimized structures at the density functional theory (DFT) and time-dependent (TD-)DFT levels. Finite temperature effects on these spectra are estimated from molecular dynamics simulations within the density functional-based tight-binding (DFTB) and TD-DFTB frameworks, these methods being first benchmarked against DFT and TD-DFT calculations. The computed spectra allow discrimination of the two isomers. When the temperature increases, bands are observed to redshift and merge. The isomerization mechanism is investigated with the metadynamics technique, a biased dynamics scheme allowing to probe reaction mechanisms with high energy barriers by investigating the free energy surface at various temperatures. Four pathways with similar barrier heights (3.5-4 eV) are found, showing that the interconversion process would only occur in interstellar clouds under photoactivation. The present study opens the way to simulations on larger methyl- and methylenePAHs of astrophysical interest and their experimental investigation.

Quantum free energy landscapes from ab initio path integral metadynamics: Double proton transfer in the formic acid dimer is concerted but not correlated.

With the goal of computing quantum free energy landscapes of reactive (bio)chemical systems in multi-dimensional space, we combine the metadynamics technique for sampling potential energy surfaces with the ab initio path integral approach to treating nuclear quantum motion. This unified method is applied to the double proton transfer process in the formic acid dimer (FAD), in order to study the nuclear quantum effects at finite temperatures without imposing a one-dimensional reaction coordinate or reducing the dimensionality. Importantly, the ab initio path integral metadynamics technique allows one to treat the hydrogen bonds and concomitant proton transfers in FAD strictly independently and thus provides direct access to the much discussed issue of whether the double proton transfer proceeds via a stepwise or concerted mechanism. The quantum free energy landscape we compute for this H-bonded molecular complex reveals that the two protons move in a concerted fashion from initial to product state, yet world-line analysis of the quantum correlations demonstrates that the protons are as quantum-uncorrelated at the transition state as they are when close to the equilibrium structure.

Capture and Release of CO₂ in Monoethanolamine Aqueous Solutions: New Insights from First-Principles Reaction Dynamics.

Aqueous monoethanolamine (MEA) solution is commonly used for post-combustion carbon capture via chemical absorption. Extensive research has been carried out to characterize both uptake and release of carbon dioxide (CO2), with the aim of improving process performance. However, an intensive research is still needed on fundamental aspects of the key chemical reactions, to achieve a comprehensive understanding of the cyclic process at the microscopic level and a quantitative assessment. We present several ab initio simulations of MEA solutions at a concentration of 30 wt %-the current standard in the industry-and study the dynamics of key multistep chemical reactions, using the metadynamics technique. Pathways for the entire cycle are investigated and characterized in terms of related free-energy and enthalpy barriers, and of the accompanying variations in both structural and electronic properties. The results of this study lead us to propose, among competing processes, an unforeseen scenario in which the zwitterion acts as sn intermediate not only of CO2 uptake, in the form of carbamate, but also of its release. Rate-limiting steps are the formation of the zwitterion for the former and MEAH(+) deprotonation for the latter. Water is shown to play a multifaceted role, which is crucial in determining the development and the energetics of each step of the reactions. The level of comprehension here achieved for MEA should help defining a strategy for solvent optimization.

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

Molecular mechanism of a hotdog-fold acyl-CoA thioesterase.

Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzyme A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone.