Density functional calculations of ATP systems. 2. ATP hydrolysis at the active site of actin

The hydrolysis of adenosine 5'-triphosphate (ATP) at the active site of actin has been studied using density functional calculations. The active site is modeled by the triphosphate tail of ATP, an Mg cation, surrounding water molecules, and the nearby protein residues. Four reaction paths have been followed by constraining coordinates that allow phosphate stretching, nucleophilic attack of the catalytic water, and OH(-) formation via water deprotonation. The lowest-energy barrier (21.0 kcal/mol) is obtained for a dissociative reaction where the terminal phosphate breaks on approaching the catalytic water, followed by proton release via a proton wire mechanism. A higher barrier (39.6 kcal/mol) results for an associative reaction path where OH(-) is formed first, with a pentacoordinated phosphorus atom (P-O distances 2.1 A). Stretching the terminal bridging P-O bond results in bond rupture at 2.8 A with an energy barrier of 28.8 kcal/mol. The residues Gln137 and His161 are not important in the reactions, but insight into their roles in vivo has been obtained. The favored coordination of the end products H(2)PO(4)(-) and ADP(3-) includes a hydrogen bond and an O-Mg-O bridge between the phosphates as well as a hydrogen bond between H(2)PO(4)(-) and the Ser14 side chain. The total energy is 2.1 kcal/mol lower than in the initial reactants. Classical simulations of ATP- and ADP.P(i)-actin show few hydrolysis-induced differences in the protein structure, indicating that phosphate migration is necessary for a change in conformation.     

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 insights into the mechanism of the Schiff base formation catalyzed by type I dehydroquinate dehydratase from <Emphasis Type="Italic">S. enterica</Emphasis>

The Schiff base formation catalyzed by type I dehydroquinate dehydratase (DHQD) from Salmonella enterica has been studied by molecular docking, molecular dynamics simulation, and quantum chemical calculations. The substrate locates stably a similar position as the Schiff base intermediate observed in the crystal structure and forms strong hydrogen bonds with several active site residues. This binding mode is different from that of several other Schiff base enzymes. Then, the quantum chemical model has been constructed and the fundamental reaction pathways have been explored by performing quantum chemical calculation. The energy barrier of the previously proposed reaction pathway is calculated to be 30.7 kcal/mol, which is much higher than the experimental value of 14.3 kcal/mol of the whole dehydration reaction by type I DHQD from S. enterica. It means that this pathway is not favorable in energy. Therefore, a new and unexpected reaction pathway has been investigated with the favorable and reasonable energy barrier of 12.1 kcal/mol. The complicated role of catalytic His143 residue has also been elucidated that it mediates two proton transfers to facilitate the reaction. Moreover, the similarity and the difference between these two reaction pathways have been analyzed in detail. The new structural and mechanistic insights may direct the design of the inhibitors of type I dehydroquinate dehydratase as non-toxic antimicrobials, antifungals, and herbicides.     

Computational study of phosphatase activity in soluble epoxide hydrolase: high efficiency through a water bridge mediated proton shuttle

Recently, a new branch of fatty acid metabolism has been opened by the novel phosphatase activity found in the N-terminal domain of the, hence bifunctional, soluble epoxide hydrolase (sEH). Importantly, this finding has also provided a new site for drug targeting in sEH's activity regulation. Classical MD and hybrid Car-Parrinello QM/MM calculations have been performed to investigate the reaction mechanism of the phosphoenzyme intermediate formation in the first step of the catalysis. The results support a concerted multi-event reaction mechanism: (1) a dissociative in-line nucleophilic substitution for the phosphoryl transfer reaction; (2) a double proton transfer involved in the formation of a good leaving group in the transition state. The presence of a water bridge in the substrate/enzyme complex allowed an efficient proton shuttle, showing its key role in speeding up the catalysis. The calculated free energy of the favored catalytic pathway is approximately 19 kcal/mol, in excellent agreement with experimental data.     

Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization

The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.     

Self-catalyzed keto-enol tautomerization of malonic acid

We demonstrate through quantum chemical calculations that the keto-enol tautomerization of malonic acid can be catalyzed by the two tautomers of malonic acid itself. This self-catalyzed process proceeds with a relatively low barrier (Gibbs energy ca. 13 kcal/mol in gas phase, 20 kcal/mol in aqueous phase), and involves the concerted transfer of two protons between the substrate and the carboxylic acid functionality of the malonic acid catalyst. This mechanism is expected to compete with the proton relay mechanism currently favored to explain the tautomerization of malonic acid in aqueous media. Malonic acid is an important constituent of secondary organic aerosol where the present chemistry may play a role in determining chemical composition.     

Fundamental reaction pathway and free energy profile for inhibition of proteasome by Epoxomicin

First-principles quantum mechanical/molecular mechanical free energy calculations have been performed to provide the first detailed computational study on the possible mechanisms for reaction of proteasome with a representative peptide inhibitor, Epoxomicin (EPX). The calculated results reveal that the most favorable reaction pathway consists of five steps. The first is a proton transfer process, activating Thr1-O(γ) directly by Thr1-N(z) to form a zwitterionic intermediate. The next step is nucleophilic attack on the carbonyl carbon of EPX by the negatively charged Thr1-O(γ) atom, followed by a proton transfer from Thr1-N(z) to the carbonyl oxygen of EPX (third step). Then, Thr1-N(z) attacks on the carbon of the epoxide group of EPX, accompanied by the epoxide ring-opening (S(N)2 nucleophilic substitution) such that a zwitterionic morpholino ring is formed between residue Thr1 and EPX. Finally, the product of morpholino ring is generated via another proton transfer. Noteworthy, Thr1-O(γ) can be activated directly by Thr1-N(z) to form the zwitterionic intermediate (with a free energy barrier of only 9.9 kcal/mol), and water cannot assist the rate-determining step, which is remarkably different from the previous perception that a water molecule should mediate the activation process. The fourth reaction step has the highest free energy barrier (23.6 kcal/mol) which is reasonably close to the activation free energy (～21-22 kcal/mol) derived from experimental kinetic data. The obtained novel mechanistic insights should be valuable for not only future rational design of more efficient proteasome inhibitors but also understanding the general reaction mechanism of proteasome with a peptide or protein.     

Ab initio study of the mechanism of singlet-dioxygen addition to hydroxyaromatic compounds: negative evidence for the involvement of peroxa and endoperoxide intermediates

In this article we report our study of two possible mechanisms of photooxidation of hydroxyaromatic compounds, involving the intermediacy of zwitterionic peroxa intermediates or 1,4-endoperoxides. To study the pathway of the first of them, as yet unexplored by theoretical methods, a simpler system composed of 1,3-butadiene-1-ol and singlet ((1)Delta(g)) dioxygen was considered first, for which calculations were carried out at the CASSCF/MCQDPT2 ab initio level, mostly with the 6-31G* basis set. The cumulative activation barrier to this reaction was found to be 20 kcal/mol and corresponded to a proton transfer (from the hydroxy oxygen atom to the attached oxygen molecule) in the cyclic zwitterionic peroxacyclopenta-3-ene-2-ol intermediate. This intermediate and the proton-transfer transition state were found to have a closed-shell character, which enabled us to estimate the corresponding activation barrier for the phenol-dioxygen system by carrying out optimization at the RHF level and single-point calculations at the MP2, CASSCF, and MCQDPT2 levels of theory. The energy barrier to the reaction was estimated to at least about 40 kcal/mol, rendering this mechanism for the phenol-oxygen system unlikely for nonpolar solvents. Similarly, calculations of the barrier to proton transfer from the 1,4-endoperoxide of phenol to its hydroperoxide were found to exceed 60 kcal/mol, eliminating such a mechanism too, which leaves only the earlier postulated mechanisms involving an initial charge or hydrogen-atom transfer to dioxygen as probable.     

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.     

Theoretical studies on the hydrolysis of mono-phosphate and tri-phosphate in gas phase and aqueous solution

Phosphate hydrolysis by GTPases plays an important role as a molecular switch in signal transduction and as an initiator of many other biological processes. Despite the centrality of this ubiquitous reaction, the mechanism is still poorly understood. As a first step to understand the mechanisms of this process, the nonenzymatic hydrolysis of mono-phosphate and tri-phosphate esters were systematically studied in gas phase and aqueous solution using hybrid density functional methods. The dielectric effect of the environment on the energetics of these processes was also explored. Theoretical results show that for mono-phosphate ester, the dissociative pathway is much more favorable than the associative pathway. However, the reaction barriers for the dissociative and associative pathways of tri-phosphate hydrolysis are very close in aqueous solution, though the dissociative pathway is more favorable in the gas phase. High dielectric solvents, such as water, significantly lower the activation barrier of the associative pathway due to the greater solvation energy of the associative transition states than that of the reactant complex. By contrast, the barrier of the dissociative pathway, with respect to the gas phase, is less sensitive to the surrounding dielectric. In the associative hydrolysis pathway of the tri-phosphate ester, negative charge is transferred from the gamma-phosphate to beta-phosphate through the bridging ester oxygen and results in Pgamma-O bond dissociation. No analogous charge transfer was observed in the dissociative pathway, where Pgamma-O bond dissociation resulted from proton transfer from the gamma-phosphate to the bridge oxygen. Finally, the active participation of local water molecules can significantly lower the activation energy of the dissociative pathway for both mono-phosphate and tri-phosphate.     

Computer simulation of the chemical catalysis of DNA polymerases: discriminating between alternative nucleotide insertion mechanisms for T7 DNA polymerase

Understanding the chemical step in the catalytic reaction of DNA polymerases is essential for elucidating the molecular basis of the fidelity of DNA replication. The present work evaluates the free energy surface for the nucleotide transfer reaction of T7 polymerase by free energy perturbation/empirical valence bond (FEP/EVB) calculations. A key aspect of the enzyme simulation is a comparison of enzymatic free energy profiles with the corresponding reference reactions in water using the same computational methodology, thereby enabling a quantitative estimate for the free energy of the nucleotide insertion reaction. The reaction is driven by the FEP/EVB methodology between valence bond structures representing the reactant, pentacovalent intermediate, and the product states. This pathway corresponds to three microscopic chemical steps, deprotonation of the attacking group, a nucleophilic attack on the P(alpha) atom of the dNTP substrate, and departure of the leaving group. Three different mechanisms for the first microscopic step, the generation of the RO(-) nucleophile from the 3'-OH hydroxyl of the primer, are examined: (i) proton transfer to the bulk solvent, (ii) proton transfer to one of the ionic oxygens of the P(alpha) phosphate group, and (iii) proton transfer to the ionized Asp654 residue. The most favorable reaction mechanism in T7 pol is predicted to involve the proton transfer to Asp654. This finding sheds light on the long standing issue of the actual role of conserved aspartates. The structural preorganization that helps to catalyze the reaction is also considered and analyzed. The overall calculated mechanism consists of three subsequent steps with a similar activation free energy of about 12 kcal/mol. The similarity of the activation barriers of the three microscopic chemical steps indicates that the T7 polymerase may select against the incorrect dNTP substrate by raising any of these barriers. The relative height of these barriers comparing right and wrong dNTP substrates should therefore be a primary focus of future computational studies of the fidelity of DNA polymerases.     

A quantum mechanical investigation of possible mechanisms for the nucleotidyl transfer reaction catalyzed by DNA polymerase beta

Several quantum mechanical (QM) and hybrid quantum/molecular mechanical (QM/MM) studies have been employed recently to analyze the nucleotidyl transfer reaction in DNA polymerase beta (pol beta). Our examination reveals strong dependence of the reported mechanism on the initial molecular model. Thus, we explore here several model systems by QM methods to investigate pol beta's possible pathway variations. Although our most favorable pathway involves a direct proton transfer from O3'(primer) to O2alpha(Palpha), we also discuss other initial proton-transfer steps--to an adjacent water, to triphosphate, or to aspartic units--and the stabilizing effect of crystallographic water molecules in the active site. Our favored reaction route has an energetically undemanding initial step of less than 1.0 kcal/mol (at the B3LYP/6-31G(d,p) level), and involves a slight rearrangement in the geometry of the active site. This is followed by two major steps: (1) direct proton transfer from O3'(primer) to O2alpha(Palpha) leading to the formation of a pentavalent, trigonal bipyramidal Palpha center, via an associative mechanism, at a cost of about 28 kcal/mol, and (2) breakage of the triphosphate unit (exothermic process, approximately 22 kcal/mol) that results in the full transfer of the nucleotide to the DNA and the formation of pyrophosphate. These energy values are expected to be lower in the physical system when full protein effects are incorporated. We also discuss variations from this dominant pathway, and their impact on the overall repair process. Our calculated barrier for the chemical reaction clearly indicates that chemistry is rate-limiting overall for correct nucleotide insertion in pol beta, in accord with other studies. Protonation studies on relevant intermediates suggest that, although protonation at a single aspartic residue may occur, the addition of a second proton to the system significantly disturbs the active site. We conclude that the active site rearrangement step necessary to attain a reaction-competent geometry is essential and closely related to the "pre-chemistry" avenue described recently as a key step in the overall kinetic cycle of DNA polymerases. Thus, our work emphasizes the many possible ways for DNA polymerase beta's chemical reaction to occur, determined by the active site environment and initial models.     

Reduction mechanism in class A methionine sulfoxide reductases: a theoretical chemistry investigation

Ab initio calculations at the B3LYP/6–311 ++G(2df,2p) and B3LYP/6–31G(d) level have been carried out to investigate the reaction mechanism of methionine sulfoxide reductases of class A. These enzymes reduce oxidized methionine in vivo and therefore play an important role in repairing protein damage caused by the oxidative stress. Our calculations have been carried out for a model reaction in a model active site. Several reaction mechanisms have been explored that can roughly be described as (2H⁺ + 2e⁻) or (H⁺ + e⁻). The results suggest that the actual reaction mechanism is of the (2H⁺ + 2e⁻) type corresponding to a more or less asynchronous-concerted double-proton transfer reaction leading to the formation of methionine (dimethylthioether in our model) and a sulfenic acid Cys-SOH. The Michaelis complex would involve one deprotonated Cys and one protonated Glu residues in the active site, this protonation state being mandatory to stabilize the sulfoxide substrate. Then, proton transfer from Glu to the substrate takes place, followed by proton transfer from one Tyr residue and fast reorganization of the system. The overall activation energy barrier is estimated to fall in the range 7–9 kcal/mol, much lower than the predicted barrier in DMSO solution (29.6 kcal/mol) reported before.     

Selective functionalization of the Si(100) surface by a bifunctional alkynylamine molecule: density functional study of the switching adsorption linkage. 2

The reaction of the bifunctional organic molecule 1-(dimethylamino)-2-propyne (DMAP) on the Si(100) surface has been investigated by density functional calculations employing a two-dimer cluster model. We found that, once in the physisorbed dative bonded well (-20.0 kcal mol(-1)), DMAP can proceed via a number of pathways, involving the formation of Si-C sigma bonds, which lead to thermodynamically more stable configurations. We first considered the cycloaddition of the CC triple bond, leading to a Si-C di-sigma bonded product (-58.7 kcal mol(-1)), for which we computed an energy barrier of only 12.5 kcal mol(-1), consistently with the observed switching of DMAP adsorption linkage at 300 K. We also explored the dissociative pathway involving the methylene C-H bond cleavage on the dative bonded DMAP, leading to three adsorption products with one (-57.3 kcal mol(-1)) and three Si-C sigma bonds (-58.7 and -60.6 kcal mol(-1)). The energy barrier for this pathway is computed 24.7 kcal mol(-1) and may therefore compete at temperature above 300 K with the reaction pathway involving the addition of the alkyne unit.     

DFT study of the glutathione peroxidase-like activity of phenylselenol incorporating solvent-assisted proton exchange

Modeling of the glutathione peroxidase-like activity of phenylselenol has been accomplished using density-functional theory and solvent-assisted proton exchange (SAPE). SAPE is a modeling technique intended to mimic solvent participation in proton transfer associated with chemical reaction. Within this method, explicit water molecules incorporated into the gas-phase model allow relay of a proton through the water molecules from the site of protonation in the reactant to that in the product. The activation barriers obtained by SAPE for the three steps of the GPx-like mechanism of PhSeH fall within the limits expected for a catalytic system at physiological temperatures (DeltaG(1)++ = 19.1 kcal/mol; DeltaG(2)++= 6.6 kcal/mol; G(3)++ = 21.7 kcal/mol) and are significantly lower than studies which require direct proton transfer. The size of the SAPE network is also considered for the model of the reduction of the selenenic acid, step 2 of the GPx-like cycle. Use of a four-water network better accommodates the reaction pathway and reduces the activation barrier by 5 kcal/mol over the two-water model.     

DFT study on the mechanism of water-assisted dihydrogen elimination in group 6 octahedral metal hydride complexes

The reaction of water with octahedral bis-, tris- and tetrakis-(phosphine)tungsten, (phosphine)molybdenum and (phosphine)chromium complexes has been studied using B3LYP/def2-TZVPP level of DFT to elucidate dissociative, associative and hydride migratory insertion mechanisms for hydrogen elimination. In the dissociative mechanism, phosphine dissociation requires 19.3-28.5 kcal mol(-1) of energy. The phosphine-water ligand exchange is endergonic due to poor coordination ability of water to group 6 metals (binding energy 8.8-15.5 kcal mol(-1)). The ligand exchange leads to intermolecular M-HH(2)O dihydrogen interaction and facilitates dihydrogen elimination (G(act) = 6.8-15.5 kcal mol(-1)). In the associative mechanism, a water molecule in the first solvation shell interacts with the M-H bond through a dihydrogen bond (interaction energy 2.7-4.0 kcal mol(-1)) and leads to the elimination of H(2) by forming a hydroxide complex. Compared to the dissociative mechanism, G(act) of associative mechanisms are ～22 kcal mol(-1) higher. In the hydride migratory insertion mechanism, the hydride ligand shifts to the CO ligand (G(act) = 25.4-30.4 kcal mol(-1)) to afford a formyl complex and subsequently the H-H bond coupling occurs between formyl and water ligand (G(act) = 2.8-4.4 kcal mol(-1)). In many cases, the migratory insertion mechanism can simultaneously operate with the dissociative mechanism as a minor pathway, whereas owing to high G(act) value, the associative mechanism can be described as inactive in the reaction. The general argument that dihydrogen elimination is preceded by the formation of a dihydrogen intermediate is not applicable for the systems studied herein as the most favoured dissociative mechanism does not pass through such an intermediate. On the other hand, irrespective of the mechanisms, dihydrogen elimination invariably occurs with the formation of a dihydrogen bonded transition state. Our results also suggest that group 6 octahedral metal hydride complexes are attractive targets for the design of water splitting reactions.     

Zinc-homocysteine binding in cobalamin-dependent methionine synthase and its role in the substrate activation: DFT, ONIOM, and QM/MM molecular dynamics studies

Cobalamin-dependent methionine synthase (MetH) is an important metalloenzyme responsible for the biosynthesis of methionine. It catalyzes methyl transfer from N(5)-methyl-tetrahydrofolate to homocysteine (Hcy) by using a zinc ion to activate the Hcy substrate. Density functional theory (B3LYP) calculations on the active-site model in gas phase and in a polarized continuum model were performed to study the Zn coordination changes from the substrate-unbound state to the substrate-bound state. The protein effect on the Zn(2+) coordination exchange was further investigated by ONIOM (B3LYP:AMBER)-ME and EE calculations. The Zn(2+)-coordination exchange is found to be highly unfavorable in the gas phase with a high barrier and endothermicity. In the water solution, the reaction becomes exothermic and the reaction barrier is drastically decreased to about 10.0 kcal/mol. A considerable protein effect on the coordination exchange was also found; the reaction is even more exothermic and occurs without barrier. The enzyme was suggested to constrain the zinc coordination sphere in the reactant state (Hcy-unbound state) more than that in the product state (Hcy-bound state), which promotes ligation of the Hcy substrate. Molecular dynamics simulations using molecular mechanics (MM) and PM3/MM potentials suggest a correlation between the flexibility of the Zn(2+)-binding site and regulation of the enzyme function. Directed in silico mutations of selected residues in the active site were also performed. Our studies support a dissociative mechanism starting with the Zn-O(Asn234) bond breaking followed by the Zn-S((Hcy)) bond formation; the proposed associative mechanism for the Zn(2+)-coordination exchange is not supported.     

Double proton transfer behavior and one-electron oxidation effect in double H-bonded glycinamide-formic acid complex

The behavior of double proton transfer occurring in a representative glycinamide-formic acid complex has been investigated at the B3LYP/6-311 + + G( * *) level of theory. Thermodynamic and, especially, kinetic parameters, such as tautomeric energy, equilibrium constant, and barrier heights have been discussed, respectively. The relevant quantities involved in the double proton transfer process, such as geometrical changes, interaction energies, and intrinsic reaction coordinate calculations have also been studied. Computational results show that the participation of a formic acid molecule favors the proceeding of the proton transfer for glycinamide compared with that without mediate-assisted case. The double proton transfer process proceeds with a concerted mechanism rather than a stepwise one since no ion-pair complexes have been located during the proton transfer process. The calculated barrier heights are 11.48 and 0.85 kcal/mol for the forward and reverse directions, respectively. However, both of them have been reduced by 2.95 and 2.61 kcal/mol to 8.53 and -1.76 kcal/mol if further inclusion of zero-point vibrational energy corrections, where the negative barrier height implies that the reverse reaction should proceed with barrierless spontaneously, analogous to that occurring between glycinamide and formamide. Furthermore, solvent effects on the thermodynamic and kinetic processes have also been predicted qualitatively employing the isodensity surface polarized continuum model within the framework of the self-consistent reaction field theory. Additionally, the oxidation process for the double H-bonded glycinamide-formic acid complex has also been investigated. Contrary to that neutral form possessing a pair of two parallel intermolecular H bonds, only a single H bond with a comparable strength has been found in its ionized form. The vertical and adiabatic ionization potentials for the neutral complex have been determined to be about 9.40 and 8.69 eV, respectively, where ionization is mainly localized on the glycinamide fragment. Like that ionized glycinamide-formamide complex, the proton transfer in the ionized complex is characterized by a single-well potential, implying that the proton initially attached to amide N4 in the glycinamide fragment cannot be transferred to carbonyl O13 in the formic acid fragment at the geometry of the optimized complex.     

Modeling the catalysis of anti-cocaine catalytic antibody: competing reaction pathways and free energy barriers

The competing reaction pathways and the corresponding free energy barriers for cocaine hydrolysis catalyzed by an anti-cocaine catalytic antibody, mAb15A10, were studied by using a novel computational strategy based on the binding free energy calculations on the antibody binding with cocaine and transition states. The calculated binding free energies were used to evaluate the free energy barrier shift from the cocaine hydrolysis in water to the antibody-catalyzed cocaine hydrolysis for each reaction pathway. The free energy barriers for the antibody-catalyzed cocaine hydrolysis were predicted to be the corresponding free energy barriers for the cocaine hydrolysis in water plus the calculated free energy barrier shifts. The calculated free energy barrier shift of -6.87 kcal/mol from the dominant reaction pathway of the cocaine benzoyl ester hydrolysis in water to the dominant reaction pathway of the antibody-catalyzed cocaine hydrolysis is in good agreement with the experimentally derived free energy barrier shift of -5.93 kcal/mol. The calculated mutation-caused shifts of the free energy barrier are also reasonably close to the available experimental activity data. The good agreement suggests that the protocol for calculating the free energy barrier shift from the cocaine hydrolysis in water to the antibody-catalyzed cocaine hydrolysis may be used in future rational design of possible high-activity mutants of the antibody as anti-cocaine therapeutics. The general strategy of the free energy barrier shift calculation may also be valuable in studying a variety of chemical reactions catalyzed by other antibodies or proteins through noncovalent bonding interactions with the substrates.