Active site dynamics and catalytic mechanism in arabinan hydrolysis catalyzed by GH43 endo-arabinanase from QM/MM molecular dynamics simulation and potential energy surface.

The endo-1,5-α-L-arabinanases, belonging to glycoside hydrolase family 43 (GH43), catalyse the hydrolysis of α-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. These enzymes are proposed targets for industrial and medical applications. Here, molecular dynamics (MD), potential energy surface and free energy (potential of mean force) simulations are undertaken using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to understand the active site dynamics, catalytic mechanism and the electrostatic influence of active site residues of the GH43 endo-arabinanase from G. stearothermophilus. The calculated results give support to the single-displacement mechanism proposed for the inverting GH43 enzymes: first a proton is transferred from the general acid E201 to the substrate, followed by a nucleophilic attack by water, activated by the general base D27, on the anomer carbon. A conformational change (2E ↔E3 ↔ 4E) in the -1 sugar ring is observed involving a transition state featuring an oxocarbenium ion character. Residues D87, K106, H271 are highlighted as potential targets for future mutation experiments in order to increase the efficiency of the reaction. To our knowledge, this is the first QM/MM study providing molecular insights into the glycosidic bond hydrolysis of a furanoside substrate by an inverting GH in solution.Communicated by Ramaswamy H. Sarma.

Theoretical characterization of Al(III) binding to KSPVPKSPVEEKG: Insights into the propensity of aluminum to interact with key sequences for neurofilament formation.

Classical molecular dynamic simulations and density functional theory are used to unveil the interaction of aluminum with various phosphorylated derivatives of the fragment KSPVPKSPVEEKG (NF13), a major multiphosphorylation domain of human neurofilament medium (NFM). Our calculations reveal the rich coordination chemistry of the resultant structures with a clear tendency of aluminum to form multidentate structures, acting as a bridging agent between different sidechains and altering the local secondary structure around the binding site. Our evaluation of binding energies allows us to determine that phosphorylation has an increase in the affinity of these peptides towards aluminum, although the interaction is not as strong as well-known chelators of aluminum in biological systems. Finally, the presence of hydroxides in the first solvation layer has a clear damping effect on the binding affinities. Our results help in elucidating the potential structures than can be formed between this exogenous neurotoxic metal and key sequences for the formation of neurofilament tangles, which are behind of some of the most important degenerative diseases.

Toxic and Physiological Metal Uptake and Release by Human Serum Transferrin.

An atomistic understanding of metal transport in the human body is critical to anticipate the side effects of metal-based therapeutics and holds promise for new drugs and drug delivery designs. Human serum transferrin (hTF) is a central part of the transport processes because of its ubiquitous ferrying of physiological Fe(III) and other transition metals to tightly controlled parts of the body. There is an atomistic mechanism for the uptake process with Fe(III), but not for the release process, or for other metals. This study provides initial insight into these processes for a range of transition metals-Ti(IV), Co(III), Fe(III), Ga(III), Cr(III), Fe(II), Zn(II)-through fully atomistic, extensive quantum mechanical/discrete molecular dynamics sampling and provides, to our knowledge, a new technique we developed to calculate relative binding affinities between metal cations and the protein. It identifies protonation of Tyr188 as a trigger for metal release rather than protonation of Lys206 or Lys296. The study identifies the difficulty of metal release from hTF as potentially related to cytotoxicity. Simulations identify a few critical interactions that stabilize the metal binding site in a flexible, nuanced manner.

The interaction of aluminum with catecholamine-based neurotransmitters: can the formation of these species be considered a potential risk factor for neurodegenerative diseases?

The potential neurotoxic role of Al(iii) and its proposed link with the insurgence of Alzheimer's Disease (AD) have attracted increasing interest towards the determination of the nature of bioligands that are propitious to interact with aluminum. Among them, catecholamine-based neurotransmitters have been proposed to be sensitive to the presence of this non-essential metal ion in the brain. In the present work, we characterize several aluminum-catecholamine complexes in various stoichiometries, determining their structure and thermodynamics of formation. For this purpose, we apply a recently validated computational protocol with results that show a remarkably good agreement with the available experimental data. In particular, we employ Density Functional Theory (DFT) in conjunction with continuum solvation models to calculate complexation energies of aluminum for a set of four important catecholamines: l-DOPA, dopamine, noradrenaline and adrenaline. In addition, by means of the Quantum Theory of Atoms in Molecules (QTAIM) and Energy Decomposition Analysis (EDA) we assessed the nature of the Al-ligand interactions, finding mainly ionic bonds with an important degree of covalent character. Our results point at the possibility of the formation of aluminum-catecholamine complexes with favorable formation energies, even when proton/aluminum competition is taken into account. Indeed, we found that these catecholamines are better aluminum binders than catechol at physiological pH, because of the electron withdrawing effect of the positively-charged amine that decreases their deprotonation penalty with respect to catechol. However, overall, our results show that, in an open biological environment, the formation of Al-catecholamine complexes is not thermodynamically competitive when compared with the formation of other aluminum species in solution such as Al-hydroxide, or when considering other endogenous/exogenous Al(iii) ligands such as citrate, deferiprone and EDTA. In summary, we rule out the possibility, suggested by some authors, that the formation of Al-catecholamine complexes in solution might be behind some of the toxic roles attributed to aluminum in the brain. An up-to-date view of the catecholamine biosynthesis pathway with sites of aluminum interference (according to the current literature) is presented. Alternative mechanisms that might explain the deleterious effects of this metal on the catecholamine route are thoroughly discussed, and new hypotheses that should be investigated in future are proposed.

Tuning the affinity of catechols and salicylic acids towards Al(iii): characterization of Al-chelator interactions.

Due to aluminum's controversial role in neurotoxicity, the goal of chelation therapy, the removal of the toxic metal ion or attenuation of its toxicity by transforming it into less toxic compounds, has attracted considerable interest in the past years. In the present paper we present, validate and apply a state-of-the-art theoretical protocol suitable for the characterization of the interactions between a chelating agent and Al(iii). In particular, we employ a cluster-continuum approach based on Density Functional Theory calculations to evaluate the binding affinity of aluminum for a set of two important families of aromatic chelators: salicylic acids and catechols. Our protocol shows very good qualitative agreement between the computed binding affinities and available experimental stability constants (log ß) values for 1 : 1, 1 : 2 and 1 : 3 complexes. Then, we have investigated the nature of the Al-O bond in an enlarged dataset of 27 complexes of 1 : 1 stoichiometry, by means of the QTAIM and Energy Decomposition Analysis (EDA). Quite interestingly, we have found that although the Al-O interaction is mainly electrostatic, there is a small but significant degree of covalency that explains the modulation of binding affinities in both families of compounds by the addition of electron donating (CH3, OCH3) or withdrawing (NO2, CF3) substituents. The role of aromaticity and the mechanisms of action of the different functional groups were also evaluated. Finally, we have analyzed the competition between Al(iii) and proton toward the binding of these chelators, giving a rationalization of the different trends found experimentally between log ß and the amount of free aluminum in solution in the presence of a given ligand (p[Al]). In summary, we propose a validated and comprehensive computational protocol that can provide a valuable help toward the design and tuning of new efficient aluminum chelators.

Aluminum and Fenton reaction: how can the reaction be modulated by speciation? A computational study using citrate as a test case.

The pro-oxidant ability of aluminum is behind many of the potential toxic effects of this exogenous element in the human organism. Although the overall process is still far from being understood at the molecular level, the well known ability of aluminum to promote the Fenton reaction is mediated through the formation of stable aluminum-superoxide radical complexes. However, the properties of metal complexes are highly influenced by the speciation of the metal. In this paper, we investigate the effect that speciation could have on the pro-oxidant activity of aluminum. We choose citrate as a test case, because it is the main low-molecular-mass chelator of aluminum in blood serum, forming very stable aluminum-citrate complexes. The influence of citrate in the interaction of aluminum with the superoxide radical is investigated, determining how the formation of aluminum-citrate complexes affects the promotion of the Fenton reaction. The results indicate that citrate increases the stability of the aluminum-superoxide complexes through the formation of ternary compounds, and that the Fenton reaction is even more favorable when aluminum is chelated to citrate. Nevertheless, our results demonstrate that overall, citrate may prevent the pro-oxidant activity of aluminum: on one hand, in an excess of citrate, the formation of 1 : 2 aluminum-citrate complexes is expected. On the other hand, the chelation of iron by citrate makes the reduction of iron thermodynamically unfavorable. In summary, the results suggest that citrate can have both a promotion and protective role, depending on subtle factors, such as initial concentration, non-equilibrium behavior and the exchange rate of ligands in the first shell of the metals.

Oxidation of Acid, Base, and Amide Side-Chain Amino Acid Derivatives via Hydroxyl Radical.

Hydroxyl radical (•OH) is known to be highly reactive. Herein, we analyze the oxidation of acid (Asp and Glu), base (Arg and Lys), and amide (Asn and Gln) containing amino acid derivatives by the consecutive attack of two •OH. In this work, we study the reaction pathway by means of density functional theory. The oxidation mechanism is divided into two steps: (1) the first •OH can abstract a H atom or an electron, leading to a radical amino acid derivative, which is the intermediate of the reaction and (2) the second •OH can abstract another H atom or add itself to the formed radical, rendering the final oxidized products. The studied second attack of •OH is applicable to situations where high concentration of •OH is found, e.g., in vitro. Carbonyls are the best known oxidation products for these reactions. This work includes solvent dielectric and confirmation's effects of the reaction, showing that both are negligible. Overall, the most favored intermediates of the oxidation process at the side chain correspond to the secondary radicals stabilized by hyperconjugation. Intermediates show to be more stable in those cases where the spin density of the unpaired electron is lowered. Alcohols formed at the side chains are the most favored products, followed by the double-bond-containing ones. Interestingly, Arg and Lys side-chain scission leads to the most favored carbonyl-containing oxidation products, in line with experimental results.

Quantum Mechanics/Molecular Mechanics Simulations Identify the Ring-Opening Mechanism of Creatininase.

Creatininase catalyzes the conversion of creatinine (a biosensor for kidney function) to creatine via a two-step mechanism: water addition followed by ring opening. Water addition is common to other known cyclic amidohydrolases, but the precise mechanism for ring opening is still under debate. The proton donor in this step is either His178 or a water molecule bound to one of the metal ions, and the roles of His178 and Glu122 are unclear. Here, the two possible reaction pathways have been fully examined by means of combined quantum mechanics/molecular mechanics simulations at the SCC-DFTB/CHARMM22 level of theory. The results indicate that His178 is the main catalytic residue for the whole reaction and explain its role as proton shuttle during the ring-opening step. In the first step, His178 provides electrostatic stabilization to the gem-diolate tetrahedral intermediate. In the second step, His178 abstracts the hydroxyl proton of the intermediate and delivers it to the cyclic amide nitrogen, leading to ring opening. The latter is the rate-limiting step with a free energy barrier of 18.5 kcal/mol, in agreement with the experiment. We find that Glu122 must be protonated during the enzyme reaction, so that it can form a stable hydrogen bond with its neighboring water molecule. Simulations of the E122Q mutant showed that this replacement disrupts the H-bond network formed by three conserved residues (Glu34, Ser78, and Glu122) and water, increasing the energy barrier. Our computational studies provide a comprehensive explanation for previous structural and kinetic observations, including why the H178A mutation causes a complete loss of activity but the E122Q mutation does not.

Elucidating the 3D structures of Al(iii)-Aß complexes: a template free strategy based on the pre-organization hypothesis.

Senile plaques are extracellular deposits found in patients with Alzheimer's Disease (AD) and are mainly formed by insoluble fibrils of ß-amyloid (Aß) peptides. The mechanistic details about how AD develops are not fully understood yet, but metals such as Cu, Zn, or Fe are proposed to have a non-innocent role. Many studies have also linked the non biological metal aluminum with AD, a species whose concentration in the environment and food has been constantly increasing since the industrial revolution. Gaining a molecular picture of how Al(iii) interacts with an Aß peptide is of fundamental interest to improve understanding of the many variables in the evolution of AD. So far, no consensus has been reached on how this metal interacts with Aß, partially due to the experimental complexity of detecting and quantifying the resulting Al(iii)-Aß complexes. Computational chemistry arises as a powerful alternative to investigate how Al(iii) can interact with Aß peptides, as suitable strategies could shed light on the metal-peptide description at the molecular level. However, the absence of any reliable template that could be used for the modeling of the metallopeptide structure makes computational insight extremely difficult. Here, we present a novel strategy to generate accurate 3D models of the Al(iii)-Aß complexes, which still circumvents first principles simulations of metal binding to peptides of Aß. The key to this approach lies in the identification of experimental structures of the isolated peptide that are favourably pre-organized for the binding of a given metal in configurations of the first coordination sphere that were previously identified as the most stable with amino acid models. This approach solves the problem of the absence of clear structural templates for novel metallopeptide constructs. The posterior refinement of the structures via QM/MM and MD calculations allows us to provide, for the first time, physically sound models for Al(iii)-Aß complexes with a 1 : 1 stoichiometry, where up to three carboxylic groups are involved in the metal binding, with a clear preference towards Glu3, Asp7, and Glu11.

A computational study of radical initiated protein backbone homolytic dissociation on all natural amino acids.

Hydroxyl radical (˙OH) is known to be one of the most reactive species. In this work, the hydrogen abstraction by ˙OH from Cα and Cß atoms of all amino acids is studied in the framework of density functional theory as this is the most favorable reaction mechanism when this kind of radical attacks a protein. From the myriad routes that the oxidation of a protein by a ˙OH radical may follow, fragmentation of the protein is one of the most damaging ones as it hampers the normal function of the protein. Therefore, cleavages of the Cα-C and Cα-N backbone bonds have been investigated as the second step of the mechanism. To the best of our knowledge, this is the first time that this reaction pathway has been systematically studied for all natural amino acids. The study includes the effects that the solvent dielectrics or the conformation of the peptide model employed has on the reaction. Interestingly, the results indicate that the nature of the side chain has little effect on the H abstraction reaction, and that for most of amino acids the attack at the Cα atom is favored over the attack at the Cß atom. The origin of this preference relies on the larger capability of the formed radical intermediate to delocalize the unpaired electron, thus maximizing the captodative effect. Moreover, the reaction is more favorable when the reactant presents a ß-sheet conformation, with a completely planar peptide backbone. With respect to the homolytic splitting of the Cα-C and Cα-N bonds, the former is favorable for almost all amino acids, whereas Ser and Thr are the only amino acids favoring the latter. These results agree with previous investigations but an accurate description of the electronic density analysis performed indicates that the origin of the different reaction pathway preferences relies on a large stabilization of the product rather than bond weakening at the radical intermediate.

What is the mechanism of formation of hydroxyaluminosilicates?

The formation of hydroxyaluminosilicates is integral to the biogeochemical cycles of aluminium and silicon. The unique inorganic chemistry which underlies their formation explains the non-essentiality in biota of both of these elements. However, the first steps in the formation of hydroxyaluminosilicates were hitherto only theoretical and plausibly only accessible in silico. Herein we have used computational chemistry to identify and define for the first time these unique and ultimately critically important reaction steps. We have used density-functional theory combined with solvent continuum models to confirm first, the nature of the reactants, an aluminium hydroxide dimer and silicic acid, second, the reaction products, two distinct hydroxyaluminosilicates A and B and finally, how these are the precursors to highly insoluble hydroxyaluminosilicates the role of which has been and continues to be to keep inimical aluminium out of biota.

Phosphorylation promotes Al(iii) binding to proteins: GEGEGSGG as a case study.

Aluminum, the third most abundant element in the Earth's crust and one of the key industrial components of our everyday life, has been associated with several neurodegenerative diseases due to its ability to promote neurofilament tangles and ß-amyloid peptide aggregation. However, the experimental characterization of aluminum speciation in vivo is a difficult task. In the present study, we develop a theoretical protocol that combines molecular dynamics simulations, clustering of structures, and density functional theory for the characterization of the binding of aluminum to the synthetic neurofilament analogue octapeptide GEGEGSGG and its phosphorylated variant. Our protocol is tested with respect to previous NMR experimental data, which allows for a full interpretation of the experimental information available and its relationship with key thermodynamic quantities. Our results demonstrate the importance of phosphorylation in the ability of a peptide to bind to aluminum. Thus, phosphorylation: (i) changes the binding pattern of aluminum to GEGEGSGG, shifting the preferential binding site from the C-terminal to S6(P); (ii) increases the binding affinity by a factor of around 15 kcal mol(-1) in free energy; and (iii) may cause significant changes in the secondary structure and stiffness of the polypeptide chain, specially in the case of bidentate binding modes. Our results shed light on the possibility of aluminum to induce aggregation of ß-amyloid proteins and neurofilament tangles.

·OH Oxidation Toward S- and OH-Containing Amino Acids.

The hydroxyl radical is the most reactive oxygen species, and it is able to attack macromolecules such as proteins. Such oxidation processes are the cause of a number of diseases. Several oxidized products have been experimentally characterized, but the reaction pathways remain unclear. Herein, we present a theoretical study on the attack of hydroxyl radicals on hydroxyl- and sulfur-containing amino acid side chains. Several reaction mechanisms, such as hydrogen abstraction, electron transfer, or ·OH addition have been considered to investigate several reaction mechanisms. Two different dielectric values (4 and 80) have been used to model the effect of different protein environments. In addition, different alternative conformations of the amino acid backbone have been considered. Overall, the results indicate that the thermodynamics is the main factor driving the reaction pathway preference and, to a great extent, explains the formation of the experimental oxidized produts. Sulfur-containing amino acids would be oxidized more easily than OH-containing amino acids, which confirms the experimental evidence. This is determined by the stability of the sulfur radical intermediates. These results are not dramatically affected by either different dielectrics or backbone conformations.

Aluminum and its effect in the equilibrium between folded/unfolded conformation of NADH.

Nicotinamide adenine dinucleotide (NADH) is one of the most abundant cofactor employed by proteins and enzymes. The molecule is formed by two nucleotides that can lead to two main conformations: folded/closed and unfolded/open. Experimentally, it has been determined that the closed form is about 2 kcal/mol more stable than the open formed. Computationally, a correct description of the NADH unfolding process is challenging due to different reasons: 1) The unfolding process shows a very low energy difference between the two conformations 2) The molecule can form a high number of internal hydrogen bond interactions 3) Subtle effects such as dispersion may be important. In order to tackle all these effects, we have employed a number of different state of the art computational techniques, including: a) well-tempered metadynamics, b) geometry optimizations, and c) Quantum Theory of Atoms in Molecules (QTAIM) calculations, to investigate the conformational change of NADH in solution and interacting with aluminum. All the results indicate that aluminum indeed favors the closed conformation of NADH, due mainly to the formation of a more rigid structure through key hydrogen bond interactions.

Mapping the affinity of aluminum(III) for biophosphates: interaction mode and binding affinity in 1 : 1 complexes.

The interaction of aluminum with biomolecular building blocks is a topic of interest as a first step to understand the potential toxic effects of aluminum in biosystems. Among the different molecules that aluminum can bind in a biological environment, phosphates are the most likely ones, due to their negatively charged nature. In the present paper, we combined DFT quantum mechanical calculations with the implicit solvent effect in order to characterize the interaction of Al(III) with these molecules. An extended set composed of a total of 59 structures was investigated, which includes various types of phosphates (monoester, diester, triester-phosphates) and various phosphate units (mono-, di- and tri-phosphate), considering various charge and protonation states, and different binding modes. The goal is to unveil the preferential interaction mode of Al(III) with phosphates in 1 : 1 complexes. Our results reveal that Al(III) prefers to form dicoordinated complexes with two phosphates, in which the interaction with each of the phosphates is of monodentate character. Our results also suggest a high affinity for binding basic phosphate groups, pointing to ATP, phosphorylated peptides, and basic diphosphates (such as 2,3-DPG) as strong aluminum chelators.

Aluminium in biological environments: a computational approach.

The increased availability of aluminium in biological environments, due to human intervention in the last century, raises concerns on the effects that this so far "excluded from biology" metal might have on living organisms. Consequently, the bioinorganic chemistry of aluminium has emerged as a very active field of research. This review will focus on our contributions to this field, based on computational studies that can yield an understanding of the aluminum biochemistry at a molecular level. Aluminium can interact and be stabilized in biological environments by complexing with both low molecular mass chelants and high molecular mass peptides. The speciation of the metal is, nonetheless, dictated by the hydrolytic species dominant in each case and which vary according to the pH condition of the medium. In blood, citrate and serum transferrin are identified as the main low molecular mass and high molecular mass molecules interacting with aluminium. The complexation of aluminium to citrate and the subsequent changes exerted on the deprotonation pathways of its tritable groups will be discussed along with the mechanisms for the intake and release of aluminium in serum transferrin at two pH conditions, physiological neutral and endosomatic acidic. Aluminium can substitute other metals, in particular magnesium, in protein buried sites and trigger conformational disorder and alteration of the protonation states of the protein's sidechains. A detailed account of the interaction of aluminium with proteic sidechains will be given. Finally, it will be described how alumnium can exert oxidative stress by stabilizing superoxide radicals either as mononuclear aluminium or clustered in boehmite. The possibility of promotion of Fenton reaction, and production of hydroxyl radicals will also be discussed.

Theoretical study of the pH-dependent antioxidant properties of vitamin C.

Molecules acting as antioxidants capable of scavenging reactive oxygen species (ROS) are of utmost importance in the living cell. Vitamin C is known to be one of these molecules. In this study we have analyzed the reactivity of vitamin C toward the [Formula: see text] and [Formula: see text] ROS species, in all acidic, neutral and basic media. In order to do so, density functional theory (DFT) have been used. More concretely, the meta-GGA functional MPW1B95 have been used. Two reaction types have been studied in each case: addition to the ring atoms, and hydrogen/proton abstraction. Our results show that [Formula: see text] is the most reactive species, while [Formula: see text] displays low reactivity. In all three media, vitamin C reactions with two hydroxyl radicals show a wide variety of possible products.

Mechanism of C-terminal intein cleavage in protein splicing from QM/MM molecular dynamics simulations.

Protein splicing is a post-translational process in which a biologically inactive protein is activated by the release of a segment denoted as an intein. The process involves four steps. In the third, the scission of the intein takes place after the cyclization of the last amino acid of the segment, an asparagine. Little is known about the chemical reaction necessary for this cyclization. Experiments demonstrate that two histidines (the penultimate amino acid of the intein, and a histidine located 10 amino acids upstream) are relevant in the cyclization of the asparagine. We have investigated the mechanism and determinants of reaction in the GyrA intein focusing on the requirements for asparagine activation for its cyclization. First, the influence that the protonation states of these two histidines have on the orientation of the asparagine side chain is investigated by means of molecular dynamics simulation. Molecular dynamics simulations using the CHARMM27 force field were carried out on the three possible protonation states for each of these two histidines. The results indicate that the only protonation state in which the conformation of the system is suitable for cyclization is when the penultimate histidine is fully protonated (positively charged), and the upstream histidine is in the His(ε) neutral tautomeric form. The free energy profile for the reaction in which the asparagine is activated by a proton transfer to the upstream histidine is presented, computed by hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics at the SCCDFTB/CHARMM27 level of theory. The calculated free energy barrier for the reaction is 19.0 kcal mol(-1). B3LYP/6-31+G(d) QM/MM single-point calculations give a qualitatively a similar energy profile, although with somewhat higher energy barriers, in good agreement with the value derived from experiment of 25 kcal mol(-1) at 60 °C. QM/MM molecular dynamics simulations of the reactant, activated reactant and intermediate states highlight the importance of the Arg181-Val182-Asp183 segment in catalysing the reaction. Overall, the results indicate that nucleophilic activation of the asparagine for its cyclization by the upstream histidine acting as the base is a plausible mechanism for the C-terminal cleavage in protein splicing.

Modeling protein splicing: reaction pathway for C-terminal splice and intein scission.

Protein splicing is a post-translational process where a biologically inactive protein is activated after the release of a so-called intein domain. In spite of the importance of this type of process, the specific molecular mechanism for the catalysis is still uncertain. In this work, we present a computational study of one of the key steps in protein splicing: the release of the intein due to the cyclization of an asparagine, the last amino acid of the intein. Density functional theory (DFT) calculations using the B3LYP functional in conjunction with the polarizable continuum model (PCM) were used to study the main stationary points along various possible reaction pathways. The results are compared with other DFT functionals and the MP2 ab initio method. In the first part of this work, the Asn-Thr dipeptide is analyzed with the aim of determining the specific requirements for the activation of the intrinsically slow Asn cyclization. The results show that the nucleophilic activation of the Asn side chain by removing one of its proton decreases the free energy barrier by approximately 20 kcal/mol. A full pathway of the reaction was also characterized in a larger model, including two imidazole molecules and two water molecules. The proposed reaction mechanism consists of two main steps: Asn side chain activation by a proton transfer to one of the imidazole groups, and cleavage of the peptide bond upon protonation of its nitrogen atom by the other imidazole. The overall free energy barrier in solution was determined to be 29.3 kcal/mol, in reasonable agreement with the apparent experimental barrier in the enzyme. The proposed mechanism suggests that the penultimate histidine stabilizes the tetrahedral intermediate and protonates the nitrogen of the scissile peptide bond, while a second histidine (located 10 amino acids upstream) activates the Asn side chain by deprotonating it.