Mechanical Free Energy Calculations Research Articles

Proton Transfers to DNA in Native Electrospray Ionization Mass Spectrometry: A Quantum Mechanics/Molecular Mechanics Study.

Native electrospray ionization-ion mobility mass spectrometry (N-ESI/IM-MS) is a powerful approach for low-resolution structural studies of DNAs in the free state and in complex with ligands. Solvent vaporization is coupled with proton transfers from ammonium ions to the DNA, resulting in a reduction of the DNA charge. Here we provide insight into these processes by classical molecular dynamics and quantum mechanics/molecular mechanics free energy calculations on the d(GpCpGpApApGpC) heptamer, for which a wealth of experiments is available. Our multiscale simulations, consistent with experimental data, reveal a highly complex scenario. The proton either sits on one of the molecules or is fully delocalized on both, depending on the level of hydration of the analytes and the size of the droplets formed during the electrospray experiments. This work complements our previous study of the intramolecular proton transfer on the same heptamer occurring after the processes studied here, and together, they provide a first molecular view of proton transfer in N-ESI/IM-MS.

Efficient Electrocatalytic CO2 Reduction to C2+ Alcohols at Defect-Site-Rich Cu Surface

Summary Electrochemical CO2 reduction is a promising approach for upgrading excessive CO2 into value-added chemicals, while the exquisite control of the catalyst atomic structures to obtain high C2+ alcohol selectivity has remained challenging due to the intrinsically favored ethylene pathways at Cu surface. Herein, we demonstrate a rational strategy to achieve ∼70% faradaic efficiency toward C2+ alcohols. We utilized a CO-rich environment to construct Cu catalysts with stepped sites that enabled high surface coverages of ∗CO intermediates and the bridge-bound ∗CO adsorption, which allowed to trigger CO2 reduction pathways toward the formation alcohols. Using this defect-site-rich Cu catalyst, we achieved C2+ alcohols with partial current densities of > 100 mA·cm−2 in both a flow-cell electrolyzer and a membrane electrode assembly (MEA) electrolyzer. A stable alcohol faradaic efficiency of ∼60% was also obtained, with ∼500 mg C2+ alcohol production per cm2 catalyst during a continuous 30-h operation.

Mechanism of the Nitric Oxide Dioxygenase Reaction of Mycobacterium tuberculosis Hemoglobin N.

Many globins convert •NO to innocuous NO3- through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O2•- and •NO species and their OONO- and •NO2 derivatives) through the action of truncated hemoglobin N (trHbN), which catalyzes the NOD reaction with one of the highest rates among globins. The general NOD mechanism comprises the following steps: binding of O2 to the heme, diffusion of •NO into the heme pocket and formation of peroxynitrite (OONO-), isomerization of OONO-, and release of NO3-. Using quantum mechanics/molecular mechanics free-energy calculations, we show that the NOD reaction in trHbN follows a mechanism in which heme-bound OONO- undergoes homolytic cleavage to give FeIV═O2- and the •NO2 radical but that these potentially harmful intermediates are short-lived and caged by the heme pocket residues. In particular, the simulations show that Tyr33(B10) side chain is shielded from FeIV═O2- and •NO2 (and protected from irreversible oxidation and nitration) by forming stable hydrogen bonds with Gln58(E11) side chain and Leu54(E7) backbone. Aromatic residues Phe46(CD1), Phe32(B9), and Tyr33(B10) promote NO3- dissociation via C-H···O bonding and provide stabilizing interactions for the anion along its egress route.

Reaction Mechanisms for the Electrochemical Reduction of CO2 to CO and Formate on the Cu(100) Surface at 298 K from Quantum Mechanics Free Energy Calculations with Explicit Water.

Copper is the only elemental metal that reduces a significant fraction of CO2 to hydrocarbons and alcohols, but the atomistic reaction mechanism that controls the product distributions is not known because it has not been possible to detect the reaction intermediates on the electrode surface experimentally, or to carry out Quantum Mechanics (QM) calculations with a realistic description of the electrolyte (water). Here, we carry out QM calculations with an explicit description of water on the Cu(100) surface (experimentally shown to be stable under CO2 reduction reaction conditions) to examine the initial reaction pathways to form CO and formate (HCOO-) from CO2 through free energy calculations at 298 K and pH 7. We find that CO formation proceeds from physisorbed CO2 to chemisorbed CO2 (*CO2δ-), with a free energy barrier of ΔG⧧ = 0.43 eV, the rate-determining step (RDS). The subsequent barriers of protonating *CO2δ- to form COOH* and then dissociating COOH* to form *CO are 0.37 and 0.30 eV, respectively. HCOO- formation proceeds through a very different pathway in which physisorbed CO2 reacts directly with a surface H* (along with electron transfer), leading to ΔG⧧ = 0.80 eV. Thus, the competition between CO formation and HCOO- formation occurs in the first electron-transfer step. On Cu(100), the RDS for CO formation is lower, making CO the predominant product. Thus, to alter the product distribution, we need to control this first step of CO2 binding, which might involve controlling pH, alloying, or changing the structure at the nanoscale.

Reaction pathways and free energy profiles for cholinesterase-catalyzed hydrolysis of 6-monoacetylmorphine

As the most active metabolite of heroin, 6-monoacetylmorphine (6-MAM) can penetrate into the brain for the rapid onset of heroin effects. The primary enzymes responsible for the metabolism of 6-MAM to the less potent morphine in humans are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The detailed reaction pathways for AChE- and BChE-catalyzed hydrolysis of 6-MAM to morphine have been explored, for the first time, in the present study by performing first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the two enzymatic reaction processes follow similar catalytic reaction mechanisms, and the whole catalytic reaction pathway for each enzyme consists of four reaction steps. According to the calculated results, the second reaction step associated with the transition state TS2(a)/TS2(b) should be rate-determining for the AChE/BChE-catalyzed hydrolysis, and the free energy barrier calculated for the AChE-catalyzed hydrolysis (18.3 kcal mol(-1)) is 2.5 kcal mol(-1) lower than that for the BChE-catalyzed hydrolysis (20.8 kcal mol(-1)). The free energy barriers calculated for the AChE- and BChE-catalyzed reactions are in good agreement with the experimentally derived activation free energies (17.5 and 20.7 kcal mol(-1) for the AChE- and BChE-catalyzed reactions, respectively). Further structural analysis reveals that the aromatic residues Phe295 and Phe297 in the acyl pocket of AChE (corresponding to Leu286 and Val288 in BChE) contribute to the lower energy of TS2(a) relative to TS2(b). The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse.

Fundamental Reaction Pathway for Peptide Metabolism by Proteasome: Insights from First-Principles Quantum Mechanical/Molecular Mechanical Free Energy Calculations

Proteasome is the major component of the crucial non-lysosomal protein degradation pathway in the cells, but the detailed reaction pathway is unclear. In this study, first-principles quantum mechanical/molecular mechanical free energy calculations have been performed to explore, for the first time, possible reaction pathways for proteasomal proteolysis/hydrolysis of a representative peptide, succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-AMC). The computational results reveal that the most favorable reaction pathway consists of six steps. The first is a water-assisted proton transfer within proteasome, activating Thr1-O(γ). The second is a nucleophilic attack on the carbonyl carbon of a Tyr residue of substrate by the negatively charged Thr1-O(γ), followed by the dissociation of the amine AMC (third step). The fourth step is a nucleophilic attack on the carbonyl carbon of the Tyr residue of substrate by a water molecule, accompanied by a proton transfer from the water molecule to Thr1-N(z). Then, Suc-LLVY is dissociated (fifth step), and Thr1 is regenerated via a direct proton transfer from Thr1-N(z) to Thr1-O(γ). According to the calculated energetic results, the overall reaction energy barrier of the proteasomal hydrolysis is associated with the transition state (TS3(b)) for the third step involving a water-assisted proton transfer. The determined most favorable reaction pathway and the rate-determining step have provided a reasonable interpretation of the reported experimental observations concerning the substituent and isotopic effects on the kinetics. The calculated overall free energy barrier of 18.2 kcal/mol is close to the experimentally derived activation free energy of ∼18.3-19.4 kcal/mol, suggesting that the computational results are reasonable.

Fundamental Reaction Pathway and Free Energy Profile for Butyrylcholinesterase-Catalyzed Hydrolysis of Heroin

The pharmacological function of heroin requires an activation process that transforms heroin into 6-monoacetylmorphine (6-MAM), which is the most active form. The primary enzyme responsible for this activation process in human plasma is butyrylcholinesterase (BChE). The detailed reaction pathway of the activation process via BChE-catalyzed hydrolysis has been explored computationally, for the first time, in this study via molecular dynamics simulation and first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the whole reaction process includes acylation and deacylation stages. The acylation consists of two reaction steps, i.e., the nucleophilic attack on the carbonyl carbon of the 3-acetyl group of heroin by the hydroxyl oxygen of the Ser198 side chain and the dissociation of 6-MAM. The deacylation also consists of two reaction steps, i.e., the nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate by a water molecule and the dissociation of the acetic acid from Ser198. The calculated free energy profile reveals that the second transition state (TS2) should be rate-determining. The structural analysis reveals that the oxyanion hole of BChE plays an important role in the stabilization of rate-determining TS2. The free energy barrier (15.9 ± 0.2 or 16.1 ± 0.2 kcal/mol) calculated for the rate-determining step is in good agreement with the experimentally derived activation free energy (~16.2 kcal/mol), suggesting that the mechanistic insights obtained from this computational study are reliable. The obtained structural and mechanistic insights could be valuable for use in the future rational design of a novel therapeutic treatment of heroin abuse.

Why Does the G117H Mutation Considerably Improve the Activity of Human Butyrylcholinesterase against Sarin? Insights from Quantum Mechanical/Molecular Mechanical Free Energy Calculations

Human butyrylcholinesterase (BChE) is recognized as the most promising bioscavenger for organophosphorus (OP) warfare nerve agents. The G117H mutant of human BChE has been identified as a potential catalytic bioscavenger with a remarkably improved activity against OP nerve agents such as sarin, but it still does not satisfy the clinical use. For further design of the higher-activity mutants against OP nerve agents, it is essential to understand how the G117H mutation improves the activity. The reaction mechanisms and the free energy profiles for spontaneous reactivation of wild-type BChE and its G117H mutant phosphorylated by sarin have been explored, in this study, by performing first-principles quantum mechanical/molecular mechanical free energy calculations, and the remarkable role of the G117H mutation on the activity has been elucidated. For both the wild-type and G117H mutant enzymes, H438 acts as a general base to initiate the spontaneous reactivation that consists of two reaction steps: the nucleophilic attack at the phosphorus by a water molecule and decomposition of the pentacoordinated phosphorus intermediate. The calculated overall free energy barriers, i.e., 30.2 and 23.9 kcal/mol for the wild type and G117H mutant, respectively, are in good agreement with available experimental kinetic data. On the basis of the calculated results, the mutated residue (H117 in the G117H mutant) cannot initiate the spontaneous reactivation as a general base. Instead, it skews the oxyanion hole and makes the phosphorus more open to the nucleophilic water molecule, resulting in a remarkable change in the rate-determining step and significantly improved catalytic activity of human BChE.

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.

Reaction mechanism for cocaine esterase-catalyzed hydrolyses of (+)- and (-)-cocaine: unexpected common rate-determining step.

First-principles quantum mechanical/molecular mechanical free energy calculations have been performed to examine the catalytic mechanism for cocaine esterase (CocE)-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. It has been shown that the acylation of (+)-cocaine consists of nucleophilic attack of the hydroxyl group of Ser117 on the carbonyl carbon of (+)-cocaine benzoyl ester and the dissociation of (+)-cocaine benzoyl ester. The first reaction step of deacylation of (+)-cocaine, which is identical to that of (-)-cocaine, is rate-determining, indicating that CocE-catalyzed hydrolyses of (+)- and (-)-cocaine have a common rate-determining step. The computational results predict that the catalytic rate constant of CocE against (+)-cocaine should be the same as that of CocE against (-)-cocaine, in contrast with the remarkable difference between human butyrylcholinesterase-catalyzed hydrolyses of (+)- and (-)-cocaine. The prediction has been confirmed by experimental kinetic analysis on CocE-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. The determined common rate-determining step indicates that rational design of a high-activity mutant of CocE should be focused on the first reaction step of the deacylation. Furthermore, the obtained mechanistic insights into the detailed differences in the acylation between the (+)- and (-)-cocaine hydrolyses provide indirect clues for rational design of amino acid mutations that could more favorably stabilize the rate-determining transition state in the deacylation and, thus, improve the catalytic activity of CocE. This study provides a valuable mechanistic base for rational design of an improved esterase for therapeutic treatment of cocaine abuse.

Reaction Pathway and Free Energy Profile for Butyrylcholinesterase-Catalyzed Hydrolysis of Acetylcholine

A catalytic mechanism for the butyrylcholinesterase (BChE)-catalyzed hydrolysis of acetylcholine (ACh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy calculations on both acylation and deacylation of BChE. It has been shown that the acylation with ACh includes two reaction steps, including nucleophilic attack on the carbonyl carbon of ACh and dissociation of choline ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of the substrate and dissociation between the carboxyl carbon of the substrate and the hydroxyl oxygen of the Ser198 side chain. Notably, despite the fact that acetylcholinesterase (AChE) and BChE are very similar enzymes, the acylation of BChE with ACh is rate-determining, which is remarkably different from the AChE-catalyzed hydrolysis of ACh, in which the deacylation is rate-determining. The computational prediction is consistent with available experimental kinetic data. The overall free energy barrier calculated for BChE-catalyzed hydrolysis of ACh is 13.8 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 13.3 kcal/mol.

Direct Observation of Protein Folding, Aggregation, and a Prion-like Conformational Conversion

Protein conformational transition from alpha-helices to beta-sheets precedes aggregation of proteins implicated in many diseases, including Alzheimer and prion diseases. Direct characterization of such transitions is often hindered by the complicated nature of the interaction network among amino acids. A recently engineered small protein-like peptide with a simple amino acid composition features a temperature-driven alpha-helix to beta-sheet conformational change. Here we studied the conformational transition of this peptide by molecular dynamics simulations. We observed a critical temperature, below which the peptide folds into an alpha-helical coiled-coil state and above which the peptide misfolds into beta-rich structures with a high propensity to aggregate. The structures adopted by this peptide during low temperature simulations have a backbone root mean square deviation less than 2 A from the crystal structure. At high temperatures, this peptide adopts an amyloid-like structure, which is mainly composed of coiled anti-parallel beta-sheets with the cross-beta-signature of amyloid fibrils. Most strikingly, we observed conformational conversions in which an alpha-helix is converted into a beta-strand by proximate stable beta-sheets with exposed hydrophobic surfaces and unsaturated hydrogen bonds. Our study suggested a possible generic molecular mechanism of the template-mediated aggregation process, originally proposed by Prusiner (Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13363-13383) to account for prion infectivity.

ChemInform Abstract: Elucidating the Nature of Enzyme Catalysis Utilizing a New Twist on an Old Methodology: Quantum Mechanical—Free Energy Calculations on Chemical Reactions in Enzymes and in Aqueous Solution

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Extrapolation and perturbation schemes for accelerating the convergence of quantum mechanical free energy calculations via the Fourier path-integral Monte Carlo method

We present two simple but effective techniques designed to improve the rate of convergence of the Fourier path-integral Monte Carlo method for quantum partition functions with respect to the Fourier space expansion length, K, especially at low temperatures. The first method treats the high Fourier components as a perturbation, and the second method involves an extrapolation of the partition function (or perturbative correction to the partition function) with respect to the parameter K. We perform a sequence of calculations at several values of K such that the statistical errors for the set of results are correlated, and this permits extremely accurate extrapolations. We demonstrate the high accuracy and efficiency of these new approaches by computing partition functions for H2O from 296 to 4000 K and comparing to the accurate results of Partridge and Schwenke.

Combined ab initio and Free Energy Calculations To Study Reactions in Enzymes and Solution: Amide Hydrolysis in Trypsin and Aqueous Solution

We present a new more general way to combine ab initio quantum mechanical calculations with classical mechanical free energy perturbation approach to calculate the energetics of enzyme-catalyzed reactions and the same reaction in solution. This approach, which enables enzyme and solution reactions to be compared without the use of empirical parameters, is applied to the formation of the tetrahedral intermediate in trypsin, but it should be generally applicable to any enzymatic reaction. Critical to the accurate calculation of the reaction energetics in solution is the estimate of the free energy to assemble the reacting groups, where the approach recently published by Hermans and Wang (J. Am. Chem. Soc. 1997, 119, 2707) was used. A central aspect of this new approach is the use of the RESP protocol to calculate the charge distribution of structures along the reaction pathway, which enables us to circumvent problems in partitioning the charge across a residue that is being divided into QM and MM parts. The classical mechanical free energy calculations are implemented with two different approaches, “Cartesian mapping” and “flexible FEP”. The similarity of the results found by using these two approaches supports the robustness of the calculated free energies. The calculated free energies are in quite good agreement with available experimental data for the activation free energies in the enzyme and aqueous phase reactions.