Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis: an ab initio QM/MM study

The initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE) has been studied by a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach. The reaction proceeds through the nucleophilic addition of the Ser203 O to the carbonyl C of acetylcholine, and the reaction is facilitated by simultaneous proton transfer from Ser203 to His447. The calculated potential energy barrier at the MP2(6-31+G) QM/MM level is 10.5 kcal/mol, consistent with the experimental reaction rate. The third residue of the catalytic triad, Glu334, is found to be essential in stabilizing the transition state through electrostatic interactions. The oxyanion hole, formed by peptidic NH groups from Gly121, Gly122, and Ala204, is also found to play an important role in catalysis. Our calculations indicate that, in the AChE-ACh Michaelis complex, only two hydrogen bonds are formed between the carbonyl oxygen of ACh and the peptidic NH groups of Gly121 and Gly122. As the reaction proceeds, the distance between the carbonyl oxygen of ACh and NH group of Ala204 becomes smaller, and the third hydrogen bond is formed both in the transition state and in the tetrahedral intermediate.     

Ab initio and density functional theory studies of the catalytic mechanism for ester hydrolysis in serine hydrolases

We present results from ab initio and density functional theory studies of the mechanism for serine hydrolase catalyzed ester hydrolysis. A model system containing both the catalytic triad and the oxyanion hole was studied. The catalytic triad was represented by formate anion, imidazole, and methanol. The oxyanion hole was represented by two water molecules. Methyl formate was used as the substrate. In the acylation step, our computations show that the cooperation of the Asp group and oxyanion hydrogen bonds is capable of lowering the activation barrier by about 15 kcal/mol. The transition state leading to the first tetrahedral intermediate in the acylation step is rate limiting with an activation barrier (ΔE₀) of 13.4 kcal/mol. The activation barrier in the deacylation step is smaller. The double-proton-transfer mechanism is energetically unfavorable by about 2 kcal/mol. The bonds between the Asp group and the His group, and the hydrogen bonds in the oxyanion hole, increase in strength going from the Michaelis complex toward the transition state and the tetrahedral intermediate. In the acylation step, the tetrahedral intermediate is a very shallow minimum on the energy surface and is not viable when molecular vibrations are included. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 69: 89–103, 1998     

Mechanisms of antibiotic resistance: QM/MM modeling of the acylation reaction of a class A beta-lactamase with benzylpenicillin

Understanding the mechanisms by which beta-lactamases destroy beta-lactam antibiotics is potentially vital in developing effective therapies to overcome bacterial antibiotic resistance. Class A beta-lactamases are the most important and common type of these enzymes. A key process in the reaction mechanism of class A beta-lactamases is the acylation of the active site serine by the antibiotic. We have modeled the complete mechanism of acylation with benzylpenicillin, using a combined quantum mechanical and molecular mechanical (QM/MM) method (B3LYP/6-31G+(d)//AM1-CHARMM22). All active site residues directly involved in the reaction, and the substrate, were treated at the QM level, with reaction energies calculated at the hybrid density functional (B3LYP/6-31+Gd) level. Structures and interactions with the protein were modeled by the AM1-CHARMM22 QM/MM approach. Alternative reaction coordinates and mechanisms have been tested by calculating a number of potential energy surfaces for each step of the acylation mechanism. The results support a mechanism in which Glu166 acts as the general base. Glu166 deprotonates an intervening conserved water molecule, which in turn activates Ser70 for nucleophilic attack on the antibiotic. This formation of the tetrahedral intermediate is calculated to have the highest barrier of the chemical steps in acylation. Subsequently, the acylenzyme is formed with Ser130 as the proton donor to the antibiotic thiazolidine ring, and Lys73 as a proton shuttle residue. The presented mechanism is both structurally and energetically consistent with experimental data. The QM/MM energy barrier (B3LYP/ 6-31G+(d)//AM1-CHARMM22) for the enzymatic reaction of 9 kcal mol(-1) is consistent with the experimental activation energy of about 12 kcal mol(-1). The effects of essential catalytic residues have been investigated by decomposition analysis. The results demonstrate the importance of the "oxyanion hole" in stabilizing the transition state and the tetrahedral intermediate. In addition, Asn132 and a number of charged residues in the active site have been identified as being central to the stabilizing effect of the enzyme. These results will be potentially useful in the development of stable beta-lactam antibiotics and for the design of new inhibitors.     

Theoretical studies on the deacylation step of serine protease catalysis in the gas phase, in solution, and in elastase

The deacylation step of serine protease catalysis is studied using DFT and ab initio QM/MM calculations combined with MD/umbrella sampling calculations. Free energies of the entire reaction are calculated in the gas phase, in a continuum solvent, and in the enzyme elastase. The calculations show that a concerted mechanism in the gas phase is replaced by a stepwise mechanism when solvent effects or an acetate ion are added to the reference system, with the tetrahedral intermediate being a shallow minimum on the free energy surface. In the enzyme, the tetrahedral intermediate is a relatively stable species ( approximately 7 kcal/mol lower in energy than the transition state), mainly due to the electrostatic effects of the oxyanion hole and Asp102. It is formed in the first step of the reaction, as a result of a proton transfer from the nucleophilic water to His57 and of an attack of the remaining hydroxyl on the ester carbonyl. This is the rate-determining step of the reaction, which requires approximately 22 kcal/mol for activation, approximately 5 kcal/mol less than the reference reaction in water. In the second stage of the reaction, only small energy barriers are detected to facilitate the proton transfer from His57 to Ser195 and the breakdown of the tetrahedral intermediate. Those are attributed mainly to a movement of Ser195 and to a rotation of the His57 side chain. During the rotation, the imidazolium ion is stabilized by a strong H-bond with Asp102, and the C(epsilon)(1)-H...O H-bond with Ser214 is replaced by one with Thr213, suggesting that a "ring-flip mechanism" is not necessary as a driving force for the reaction. The movements of His57 and Ser195 are highly correlated with rearrangements of the binding site, suggesting that product release may be implicated in the deacylation process.     

Ab initio QM/MM study of class A beta-lactamase acylation: dual participation of Glu166 and Lys73 in a concerted base promotion of Ser70

Beta-lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics. The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation, destroying the beta-lactam. The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations, augmented by extensive molecular dynamics simulations reported herein, describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate. Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme. A remarkable duality of mechanism for tetrahedral formation is implicated. Following substrate binding, the pathway initiates by a low energy barrier (5 kcal mol(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166, through the catalytic water molecule and Ser70. This gives unprotonated Lys73 and protonated Glu166. Tetrahedral formation ensues in a concerted general base process, with Lys73 promoting Ser70 addition to the beta-lactam carbonyl. Moreover, the three-dimensional potential energy surface also shows that the previously proposed pathway, involving Glu166 as the general base promoting Ser70 through a conserved water molecule, exists in competition with the Lys73 process. The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.     

Modeling effects of oxyanion hole on the ester hydrolysis catalyzed by human cholinesterases

Molecular dynamics (MD) simulations and hydrogen bonding energy (HBE) calculations have been performed on the prereactive enzyme-substrate complexes (ES), transition states (TS1), and intermediates (INT1) for acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine (ACh), butyrylcholinesterase (BChE)-catalyzed hydrolysis of ACh, and BChE-catalyzed hydrolysis of (+)/(-)-cocaine to examine the protein environmental effects on the catalytic reactions. The hydrogen bonding of cocaine with the oxyanion hole of BChE is found to be remarkably different from that of ACh with AChE/BChE. Whereas G121/G116, G122/G117, and A204/A199 of AChE/BChE all can form hydrogen bonds with ACh to stabilize the transition state during the ACh hydrolysis, BChE only uses G117 and A199 to form hydrogen bonds with cocaine. The change of the estimated total HBE from ES to TS1 is ca. -5.4/-4.4 kcal/mol for AChE/BChE-catalyzed hydrolysis of ACh and ca. -1.7/-0.8 kcal/mol for BChE-catalyzed hydrolysis of (+)/(-)-cocaine. The remarkable difference of approximately 3 to 5 kcal/mol reveals that the oxyanion hole of AChE/BChE can lower the energy barrier of the ACh hydrolysis significantly more than that of BChE for the cocaine hydrolysis. These results help to understand why the catalytic activity of AChE against ACh is considerably higher than that of BChE against cocaine and provides valuable clues on how to improve the catalytic activity of BChE against cocaine.     

Theoretical perspectives on the reaction mechanism of serine proteases: the reaction free energy profiles of the acylation process

The reaction mechanism of serine proteases (trypsin), which catalyze peptide hydrolysis, is studied theoretically by ab initio QM/MM electronic structure calculations combined with Molecular Dynamics-Free Energy Perturbation calculations. We have calculated the entire reaction free energy profiles of the first reaction step of this enzyme (acylation process). The present calculations show that the rate-determining step of the acylation is the formation of the tetrahedral intermediate, and the breakdown of this intermediate has a small energy barrier. The calculated activation free energy for the acylation is approximately 17.8 kcal/mol at QM/MM MP2/(aug)-cc-pVDZ//HF/6-31(+)G/AMBER level, and this reaction is an exothermic process. MD simulations of the enzyme-substrate (ES) complex and the free enzyme in aqueous phase show that the substrate binding induces slight conformational changes around the active site, which favor the alignment of the reactive fragments (His57, Asp102, and Ser195) together in a reactive orientation. It is also shown that the proton transfer from Ser195 to His57 and the nucleophilic attack of Ser195 to the carbonyl carbon of the scissile bond of the substrate occur in a concerted manner. In this reaction, protein environment plays a crucial role to lowering the activation free energy by stabilizing the tetrahedral intermediate compared to the ES complex. The polarization energy calculations show that the enzyme active site is in a very polar environment because of the polar main chain contributions of protein. Also, the ground-state destabilization effect (steric strain) is not a major catalytic factor. The most important catalytic factor of stabilizing the tetrahedral intermediate is the electrostatic interaction between the active site and particular regions of protein: the main chain NH groups in Gly193 and Ser195 (so-called oxyanion hole region) stabilize negative charge generated on the carbonyl oxygen of the scissile bond, and the main chain carbonyl groups in Ile212 approximately Ser214 stabilize a positive charge generated on the imidazole ring of His57.     

Semiempirical Molecular Orbital Studies of the Acylation Step in the Lipase‐Catalyzed Ester Hydrolysis

In this study, we present the results from the semiempirical molecular orbital calculations for the acylation step in the lipase‐catalyzed ester hydrolysis. The results reveal that the lowest energy path for the formation of the tetrahedral intermediate is for the serine residue of the catalytic triad to attack the substrate, followed by coupling heavy atom movement and proton transfer. The calculations of four active site models show that the cooperation of the aspartate group and the oxyanion hole is capable of lowering the activation energy by about 16 kcalmol^?1. Our results further suggest that the lipase‐catalyzed ester hydrolysis adopts the single proton transfer mechanism.     

Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 A and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.     

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two‐layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn‐bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate‐determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn‐bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate‐determining step is the proton transfer from Zn‐OH to 6‐NH₂ of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

An Unsual Cys-Glu-Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2

Nitrilase 2 (Nit2) is a representative member of the nitrilase superfamily that catalyzes the hydrolysis of α-ketosuccinamate into oxaloacetate. It has been associated with the metabolism of rapidly dividing cells like cancer cells. The catalytic mechanism of Nit2 employs a catalytic triad formed by Cys191, Glu81 and Lys150. The Cys191 and Glu81 play an active role during the catalytic process while the Lys150 is shown to play only a secondary role. The results demonstrate that the catalytic mechanism of Nit2 involves four steps. The nucleophilic attack of Cys191 to the α-ketosuccinamate, the formation of two tetrahedral enzyme adducts and the hydrolysis of a thioacyl-enzyme intermediate, from which results the formation of oxaloacetate and enzymatic turnover. The rate limiting step of the catalytic process is the formation of the first tetrahedral intermediate with a calculated activation free energy of 18.4 kcal/mol, which agrees very well with the experimental k_cat (17.67 kcal/mol).     

Investigation of the mechanism of the cell wall DD-carboxypeptidase reaction of penicillin-binding protein 5 of Escherichia coli by quantum mechanics/molecular mechanics calculations

Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem peptides of the cell wall peptidoglycan. The mechanism of PBP 5 catalysis of amide bond hydrolysis is initial acylation of an active site serine by the peptide substrate, followed by hydrolytic deacylation of this acyl-enzyme intermediate to complete the turnover. The microscopic events of both the acylation and deacylation half-reactions have not been studied. This absence is addressed here by the use of explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations. The potential-energy surface for the acylation reaction, based on MP2/6-31+G(d) calculations, reveals that Lys47 acts as the general base for proton abstraction from Ser44 in the serine acylation step. A discrete potential-energy minimum for the tetrahedral species is not found. The absence of such a minimum implies a conformational change in the transition state, concomitant with serine addition to the amide carbonyl, so as to enable the nitrogen atom of the scissile bond to accept the proton that is necessary for progression to the acyl-enzyme intermediate. Molecular dynamics simulations indicate that transiently protonated Lys47 is the proton donor in tetrahedral intermediate collapse to the acyl-enzyme species. Two pathways for this proton transfer are observed. One is the direct migration of a proton from Lys47. The second pathway is proton transfer via an intermediary water molecule. Although the energy barriers for the two pathways are similar, more conformers sample the latter pathway. The same water molecule that mediates the Lys47 proton transfer to the nitrogen of the departing D-Ala is well positioned, with respect to the Lys47 amine, to act as the hydrolytic water in the deacylation step. Deacylation occurs with the formation of a tetrahedral intermediate over a 24 kcal x mol(-1) barrier. This barrier is approximately 2 kcal x mol(-1) greater than the barrier (22 kcal x mol(-1)) for the formation of the tetrahedral species in acylation. The potential-energy surface for the collapse of the deacylation tetrahedral species gives a 24 kcal x mol(-1) higher energy species for the product, signifying that the complex would readily reorganize and pave the way for the expulsion of the product of the reaction from the active site and the regeneration of the catalyst. These computational data dovetail with the knowledge on the reaction from experimental approaches.     

New insight into the gas-phase bimolecular self-reaction of the HOO radical

The singlet and triplet potential energy surfaces (PESs) for the gas-phase bimolecular self-reaction of HOO*, a key reaction in atmospheric environments, have been investigated by means of quantum-mechanical electronic structure methods (CASSCF and CASPT2). All the reaction pathways on both PESs consist of a first step involving the barrierless formation of a prereactive doubly hydrogen-bonded complex, which is a diradical species lying about 8 kcal/mol below the energy of the reactants at 0 K. The lowest energy reaction pathway on both PESs is the degenerate double hydrogen exchange between the HOO* moieties of the prereactive complex via a double proton transfer mechanism involving an energy barrier of only 1.1 kcal/mol for the singlet and 3.3 kcal/mol for the triplet at 0 K. The single H-atom transfer between the two HOO* moieties of the prereactive complex (yielding HOOH + O2) through a pathway keeping a planar arrangement of the six atoms involves a conical intersection between either two singlet or two triplet states of A' and A" symmetries. Thus, the lowest energy reaction pathway occurs via a nonplanar cisoid transition structure with an energy barrier of 5.8 kcal/mol for the triplet and 17.5 kcal/mol for the singlet at 0 K. The simple addition between the terminal oxygen atoms of the two HOO* moieties of the prereactive complex, leading to the straight chain H2O4 intermediate on the singlet PES, involves an energy barrier of 7.3 kcal/mol at 0 K. Because the decomposition of such an intermediate into HOOH + O2 entails an energy barrier of 45.2 kcal/mol at 0 K, it is concluded that the single H-atom transfer on the triplet PES is the dominant pathway leading to HOOH + O2. Finally, the strong negative temperature dependence of the rate constant observed for this reaction is attributed to the reversible formation of the prereactive complex in the entrance channel rather than to a short-lived tetraoxide intermediate.     

Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD)

Extensive combined quantum mechanical (B3LYP/6‐31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton‐transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2‐pyrimidinone. The rate‐determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc.     

Peptide hydrolysis catalyzed by matrix metalloproteinase 2: a computational study

The MMP-2 reaction mechanism is investigated by using different computational methodologies. First, quantum mechanical (QM) calculations are carried out on a cluster model of the active site bound to an Ace-Gly approximately Ile-Nme peptide. Along the QM reaction path, a Zn-bound water molecule attacks the Gly carbonyl group to give a tetrahedral intermediate. The breaking of the C-N bond is completed thanks to the Glu 404 residue that shuttles a proton from the water molecule to Ile-N atom. The gas-phase QM energy barrier is quite low ( approximately 14 kcal/mol), thus suggesting that the essential catalytic machinery is included in the cluster model. A similar reaction path occurs in the MMP-2 catalytic domain bound to an octapeptide substrate according to hybrid QM and molecular mechanical (QM/MM) geometry optimizations. However, the rupture of the Gly( P 1) approximately Ile( P 1') amide bond is destabilized in the static QM/MM calculations, owing to the positioning of the Ile( P 1') side chain inside the MMP-2 S 1' pocket and to the inability of simple energy miminization methodologies to properly relax complex systems. Molecular dynamics simulations show that these steric limitations are overcome easily through structural fluctuations. The energetic effect of structural fluctuations is taken into account by combining QM energies with average MM Poisson-Boltzmann free energies, resulting in a total free energy barrier of 14.8 kcal/mol in good agreement with experimental data. The rate-determining event in the MMP-2 mechanism corresponds to a H-bond rearrangement involving the Glu 404 residue and/or the Glu 404-COOH --> N-Ile( P 1') proton transfer. Overall, the present computational results and previous experimental data complement each other well in order to provide a detailed view of the MMPs catalytic mechanism.     

Direct ab‐initio molecular dynamics study on the radiation effects on catalytic triad composed of Ser‐His‐Glu residues

High energy irradiation to the hydrogen bonded system is important in relevance with the initial process of DNA and enzyme damages. In the present study, the effects of radiation to catalytic triad have been investigated by means of direct ab‐initio molecular dynamics (AIMD) calculation. As a model of the catalytic triad, Ser‐His‐Glu residue, which is one of the important enzymes in the acylation reaction, was examined. The ionization and electron attachment processes in Ser‐His‐Glu were investigated as the radiation effects. The direct AIMD calculation showed that a proton of His is spontaneously transferred to carbonyl oxygen of Glu after the ionization. However, the whole structure of catalytic triad was essentially kept after the ionization. On the other hand, in the case of the electron capture in the model catalytic triad Ser‐His‐Glu, the dissociation of Glu residue from [Ser‐His]^? was found as a product channel. The mechanism of ionization and electron capture process in the catalytic triad was discussed on the basis of theoretical results. © 2015 Wiley Periodicals, Inc.     

Catalytic mechanism of class A beta-lactamase. I. The role of Glu166 and Serl30 in the deacylation reaction

The tetrahedral intermediate formation process, which is the first step in the deacylation reaction by class A beta-lactamase, was investigated by the ab initio molecular orbital method. In this study, benzyl penicillin was used as the substrate. From the results of our molecular dynamics study of the structure of beta-lactam antibiotics-beta-lactamase complex, the substrate, Ser70, Lys73, Ser130, Glu166 and a water molecule for the deacylation reaction were considered for construction of a model for calculation. The calculation results indicated that Glu166 plays a role in holding a water molecule, which is necessary for the deacylation reaction, and that the hydrogen bond network among Lys73Nzeta, Ser130Ogamma, and the carboxyl group of the beta-lactam antibiotics was formed by the uptake of beta-lactam antibiotics by beta-lactamase. The activation energy for this reaction was 33.3 kcal/mol, and it is very likely that the reaction occurred at body temperature. Subsequent calculation results obtained by using the model excluding Ser130 and the carboxyl group of the substrate indicated that the activation energy for this reaction was 40.8 kcal/mol, which is 7.5 kcal/mol higher than that of the previous reaction. It was found that the hydrogen bond network plays an important role in decreasing the activation energy for the tetrahedral intermediate formation reaction. Lys73Nzeta, which is located at the edge of the hydrogen bond network, played a role in forming a hydrogen bond with Glu166Oepsilon in order to help the deacylation reaction. The role of amino acid residues around the active site of class A beta-lactamase was also discussed.     

Acetylcholinesterase: mechanisms of covalent inhibition of wild-type and H447I mutant determined by computational analyses

The reaction mechanisms of two inhibitors TFK+ and TFK0 binding to both the wild-type and H447I mutant mouse acetylcholinesterase (mAChE) have been investigated by using a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach and classical molecular dynamics (MD) simulations. In the wild-type mAChE, the binding reactions of TFK+ and TFK0 are both spontaneous processes, which proceed through the nucleophilic addition of the Ser203-Ogamma to the carbonyl-C of TFK+ or TFK0, accompanied with a simultaneous proton transfer from Ser203 to His447. No barrier is found along the reaction paths, consistent with the experimental reaction rates approaching the diffusion-controlled limit. By contrast, TFK+ binding to the H447I mutant may proceed with a different reaction mechanism. A water molecule takes over the role of His447 and participates in the bond breaking and forming as a "charge relayer". Unlike in the wild-type mAChE case, Glu334, a conserved residue from the catalytic triad, acts as a catalytic base in the reaction. The calculated energy barrier for this reaction is about 8 kcal/mol. These predictions await experimental verification. In the case of the neutral ligand TFK0, however, multiple MD simulations on the TFK0/H447I complex reveal that none of the water molecules can be retained in the active site as a "catalytic" water. Furthermore, our alchemical free energy calculation also suggests that the binding of TFK0 to H447I is much weaker than that of TFK+. Taken together, our computational studies confirm that TFK0 is almost inactive in the H447I mutant and also provide detailed mechanistic insights into the experimental observations.     

Divergent function in the crotonase superfamily: an anhydride intermediate in the reaction catalyzed by 3-hydroxyisobutyryl-CoA hydrolase

3-Hydroxyisobutyryl-CoA hydrolase (HICH), a member of the enoyl-CoA (crotonase) superfamily, catalyzes the hydrolysis of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate. Like other members of the superfamily, the sequence of HICH contains conserved sequences for an oxyanion hole that stabilizes anionic intermediates. In contrast to most members of the superfamily, the reaction catalyzed by HICH does not proceed via formation of a thioester enolate anion; instead, evidence based on substrate deuterium isotope effects, the reactivity of substrate analogues that cannot form thioester enolate anions, single-turnover experiments in H218O, and the kinetic phenotypes of site-directed mutants provide evidence for a mechanism involving the formation of an anhydride intermediate involving Glu143 in the active site. In the reactions catalyzed by many members of the superfamily, homologues of Glu143 abstract the alpha proton of the thioester substrate to generate the thioester enolate anion intermediate. Presumably, one or more of the anionic tetrahedral intermediates on the HICH reaction coordinate are stabilized by the oxyanion hole. Thus, we conclude that the conserved oxyanion hole in this superfamily can be used to stabilize a variety of anionic intermediates.     

Ab initio QM/MM dynamics simulation of the tetrahedral intermediate of serine proteases: insights into the active site hydrogen-bonding network

Ab initio QM/MM dynamics simulation is employed to examine the stability of the tetrahedral intermediate during the deacylation step in elastase-catalyzed hydrolysis of a simple peptide. An extended quantum region includes the catalytic triad, the tetrahedral structure, and the oxyanion hole. The calculations indicate that the tetrahedral intermediate of serine proteases is a stable species on the picosecond time scale. On the basis of geometrical and dynamical properties, and in agreement with many experimental and theoretical studies, it is suggested that the crucial hydrogen bonds involved in stabilizing this intermediate are between Asp-102 and His-57 and between the charged oxygen of the intermediate and the backbone N-H group of Gly-193 in the oxyanion hole. The mobility of the imidazolium ring between O(w) and O(gamma), two of the oxygens of the tetrahedral structure, shows how the intermediate could proceed toward the product state without a "ring-flip mechanism", proposed earlier on the basis of NMR data. In addition to the proposed C(epsilon)(1)-H.O hydrogen bond between the imidazolium ring and the backbone carbonyl of Ser-214, we observe an alternative C(epsilon)(1)-H.O hydrogen bond with the backbone carbonyl of Thr-213, that can stabilize the intermediate during the imidazolium movement. Proton hopping occurs between Asp-102 and His-57 during the simulation. The proton is, however, largely localized on the nitrogen, and hence it does not participate in a low-barrier hydrogen bond. The study also suggests factors that may be implicated in product release: breaking the hydrogen bond of the charged oxygen with the backbone of Ser-195 in the oxyanion hole and a loop opening between residues 216-225 that enables the breaking of a hydrogen bond in subsite S(3).