Mechanical Free Energy Research Articles

Engineered Bi-Histidine Metal Chelation Sites Map the Structure of the Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The ϕM2+U-value is defined as ΔΔG‡-N/ΔΔGU-N, which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG‡-N) relative to its thermodynamic stability (ΔGU-N) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify ϕM2+U-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1’s mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.

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.

QM/MM MD and free energy simulations of G9a-like protein (GLP) and its mutants: understanding the factors that determine the product specificity.

Certain lysine residues on histone tails could be methylated by protein lysine methyltransferases (PKMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Since the methylation states of the target lysines play a fundamental role in the regulation of chromatin structure and gene expression, it is important to study the property of PKMTs that allows a specific number of methyl groups (one, two or three) to be added (termed as product specificity). It has been shown that the product specificity of PKMTs may be controlled in part by the existence of specific residues at the active site. One of the best examples is a Phe/Tyr switch found in many PKMTs. Here quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed on wild type G9a-like protein (GLP) and its F1209Y and Y1124F mutants for understanding the energetic origin of the product specificity and the reasons for the change of product specificity as a result of single-residue mutations at the Phe/Tyr switch as well as other positions. The free energy barriers of the methyl transfer processes calculated from our simulations are consistent with experimental data, supporting the suggestion that the relative free energy barriers may determine, at least in part, the product specificity of PKMTs. The changes of the free energy barriers as a result of the mutations are also discussed based on the structural information obtained from the simulations. The results suggest that the space and active-site interactions around the ε-amino group of the target lysine available for methyl addition appear to among the key structural factors in controlling the product specificity and activity of PKMTs.

Accelerating the Convergence and Reducing the Variance of Path Integral Calculations of Quantum Mechanical Free Energies by Using Local Reference Potentials

We present two new methods to accelerate the convergence of Feynman path integral calculations of thermodynamic partition functions. The first enhancement uses information from instantaneous normal mode (INM) calculations to decrease the number of discretized points necessary to represent the Feynman paths and is denoted the local generalized Pitzer-Gwinn (LGPG) scheme. The second enhancement, denoted harmonically guided variance reduction (HGVR), reduces the variance in Monte Carlo (MC) calculations by exploiting the correlation between the sampling error associated with the sum over paths at a particular centroid location for the accurate potential and for the INM approximation of a model potential, the latter of which can be exactly calculated. The LGPG scheme can reduce the number of quadrature points required along the paths by nearly an order of magnitude, and the HGVR scheme can reduce the number of MC samples needed to achieve a target accuracy by more than an order of magnitude. Numerical calculations are presented for H2O2, a very anharmonic system where torsional motion is important, and H2O, a system more amenable to harmonic reference treatment.

Variational calculation of quantum mechanical/molecular mechanical free energy with electronic polarization of solvent

Quantum mechanical/molecular mechanical (QM/MM) free energy calculation presents a significant challenge due to an excessive number of QM calculations. A useful approach for reducing the computational cost is that based on the mean field approximation to the QM subsystem. Here, we describe such a mean-field QM/MM theory for electronically polarizable systems by starting from the Hartree product ansatz for the total system and invoking a variational principle of free energy. The MM part is then recast to a classical polarizable model by introducing the charge response kernel. Numerical test shows that the potential of mean force (PMF) thus obtained agrees quantitatively with that obtained from a direct QM/MM calculation, indicating the utility of self-consistent mean-field approximation. Next, we apply the obtained method to prototypical reactions in several qualitatively different solvents and make a systematic comparison of polarization effects. The results show that in aqueous solution the PMF does not depend very much on the water models employed, while in nonaqueous solutions the PMF is significantly affected by explicit polarization. For example, the free energy barrier for a phosphoryl dissociation reaction in acetone and cyclohexane is found to increase by more than 10 kcal/mol when switching the solvent model from an empirical to explicitly polarizable one. The reason for this is discussed based on the parametrization of empirical nonpolarizable models.

Understanding Product Specificity of Protein Lysine Methyltransferases from QM/MM Molecular Dynamics and Free Energy Simulations: The Effects of Mutation on SET7/9 beyond the Tyr/Phe Switch

The results of hybrid quantum mechanical/molecular mechanical (QM/MM) free energy (potential of mean force) simulations for methyl-transfer processes in SET7/9 and its Y245A mutant are compared to address the question concerning the change of the product specificity as well as catalytic efficiency due to the mutation. One of the key questions is whether or not the free energy profiles of methyl transfers may be used to predict the change of the product specificity as a result of the mutations for the residues that are not located at the Tyr/Phe switch position. The simulations show that while the wild-type SET7/9 is a monomethylase, the Y245→A mutation increases the ability of the enzyme to add more methyl groups on the target lysine (i.e., acting as a trimethylase). However, the first methyl-transfer process seems to become less efficient in the mutant compared to that in wild-type. All these results are consistent with experimental observations concerning the effects of the mutation on the product specificity and catalytic efficiency. Thus, the previous suggestion that the energetics of the methyl-transfer reactions may determine the product specificity, at least in some cases, is confirmed by the present work. Moreover, the dynamic information of the reactant complexes obtained from the QM/MM molecular dynamics simulations shows that the ability of the reactant complexes to form the reactive transition-state-like configurations may be used as an important indicator for the prediction of the product specificity of PKMTs, consistent with previous computational studies.

Reaction Pathway and Free Energy Profiles for Butyrylcholinesterase-Catalyzed Hydrolysis of Acetylthiocholine

The catalytic mechanism for butyrylcholineserase (BChE)-catalyzed hydrolysis of acetylthiocholine (ATCh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy (QM/MM-FE) calculations on both acylation and deacylation of BChE. Additional quantum mechanical (QM) calculations have been carried out, along with the QM/MM-FE calculations, to understand the known substrate activation effect on the enzymatic hydrolysis of ATCh. It has been shown that the acylation of BChE with ATCh consists of two reaction steps including the nucleophilic attack on the carbonyl carbon of ATCh and the dissociation of thiocholine ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of substrate and dissociation between the carboxyl carbon of substrate and hydroxyl oxygen of Ser198 side chain. QM/MM-FE calculation results reveal that the acylation of BChE is rate-determining. It has also been demonstrated that an additional substrate molecule binding to the peripheral anionic site (PAS) of BChE is responsible for the substrate activation effect. In the presence of this additional substrate molecule at PAS, the calculated free energy barrier for the acylation stage (rate-determining step) is decreased by ~1.7 kcal/mol. All of our computational predictions are consistent with available experimental kinetic data. The overall free energy barriers calculated for BChE-catalyzed hydrolysis of ATCh at regular hydrolysis phase and substrate activation phase are ~13.6 and ~11.9 kcal/mol, respectively, which are in reasonable agreement with the corresponding experimentally derived activation free energies of 14.0 kcal/mol (for regular hydrolysis phase) and 13.5 kcal/mol (for substrate activation phase).

QM/MM Reweighting Free Energy SCF for Geometry Optimization on Extensive Free Energy Surface of Enzymatic Reaction.

We developed a quantum mechanical/molecular mechanical (QM/MM) free energy geometry optimization method by which the geometry of a quantum chemically treated (QM) molecule is optimized on a free energy surface defined with thermal distribution of the surrounding molecular environment obtained by molecular dynamics simulation with a molecular mechanics (MM) force field. The method called QM/MM reweighting free energy self-consistent field combines a mean field theory of QM/MM free energy geometry optimization developed by Yamamoto (Yamamoto, T. J. Chem. Phys.2008, 129, 244104) with a reweighting scheme for updating the MM distribution introduced by Hu et al. (Hu, H., et al. J. Chem. Phys.2008, 128, 034105) and features high computational efficiency suitable for exploring the reaction free energy surface of extensive protein conformational space. The computational efficiency with improved treatment of a long-range electrostatic (ES) interaction using the Ewald summation technique permits one to take into account global conformational relaxation of an entire protein of an enzyme in the free energy geometry optimization of its reaction center. We applied the method to an enzymatic reaction of a substrate complex of psychrophilic α-amylase from Antarctic bacterium Pseudoalteromonas haloplanktis and succeeded in geometry optimizations of the reactant and the product of the catalytic reaction that involve large conformational changes of protein loops adjacent to the reaction center on time scales reaching sub-microseconds. We found that the adjacent loops in the reactant and the product form in different conformations and produce catalytic ES potentials on the reaction center.

Fundamental reaction pathway and free energy profile for hydrolysis of intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) catalyzed by phosphodiesterase-4.

As important drug targets for a variety of human diseases, cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes sharing a similar catalytic site. We have performed pseudobond first-principles quantum mechanical/molecular mechanical-free energy perturbation (QM/MM-FE) and QM/MM-Poisson-Boltzmann surface area (PBSA) calculations to uncover the detailed reaction mechanism for PDE4-catalyzed hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP). This is the first report on QM/MM reaction-coordinate calculations including the protein environment of any PDE-catalyzed reaction system, demonstrating a unique catalytic reaction mechanism. The QM/MM-FE and QM/MM-PBSA calculations revealed that the PDE4-catalyzed hydrolysis of cAMP consists of two reaction stages: cAMP hydrolysis (stage 1) and bridging hydroxide ion regeneration (stage 2). The stage 1 includes the binding of cAMP in the active site, nucleophilic attack of the bridging hydroxide ion on the phosphorus atom of cAMP, cleavage of O3'-P phosphoesteric bond of cAMP, protonation of the departing O3' atom, and dissociation of hydrolysis product (AMP). The stage 2 includes the binding of solvent water molecules with the metal ions in the active site and regeneration of the bridging hydroxide ion. The dissociation of the hydrolysis product is found to be rate-determining for the enzymatic reaction process. The calculated activation Gibbs free energy of ≥16.0 and reaction free energy of -11.1 kcal/mol are in good agreement with the experimentally derived activation free energy of 16.6 kcal/mol and reaction free energy of -11.5 kcal/mol, suggesting that the catalytic mechanism obtained from this study is reliable and provides a solid base for future rational drug design.

Van der Waals interactions: Evaluations by use of a statistical mechanical method

In this work the induced van der Waals interaction between a pair of neutral atoms or molecules is considered by use of a statistical mechanical method. With use of the Schrödinger equation this interaction can be obtained by standard quantum mechanical perturbation theory to second order. However, the latter is restricted to electrostatic interactions between dipole moments. So with radiating dipole-dipole interaction where retardation effects are important for large separations of the particles, other methods are needed, and the resulting induced interaction is the Casimir-Polder interaction usually obtained by field theory. It can also be evaluated, however, by a statistical mechanical method that utilizes the path integral representation. We here show explicitly by use of this method the equivalence of the Casimir-Polder interaction and the van der Waals interaction based upon the Schrödinger equation. The equivalence is to leading order for short separations where retardation effects can be neglected. In recent works [J. S. Høye, Physica A 389, 1380 (2010); Phys. Rev. E 81, 061114 (2010)], the Casimir-Polder or Casimir energy was added as a correction to calculations of systems like the electron clouds of molecules. The equivalence to van der Waals interactions indicates that the added Casimir energy will improve the accuracy of calculated molecular energies. Thus, we give numerical estimates of this energy including analysis and estimates for the uniform electron gas.

Optimal scaling factors for CM1 and CM3 atomic charges in RM1‐based aqueous simulations

Scaling factors for atomic charges derived from the RM1 semiempirical quantum mechanical wavefunction in conjunction with CM1 and CM3 charge models have been optimized by minimizing errors in absolute free energies of hydration, ΔG(hyd) , for a set of 40 molecules. Monte Carlo statistical mechanics simulations and free energy perturbation theory were used to annihilate the solutes in gas and in a box of TIP4P water molecules. Lennard-Jones parameters from the optimized potentials for liquid simulations-all atom (OPLS-AA) force field were utilized for the organic compounds. Optimal charge scaling factors have been determined as 1.11 and 1.14 for the CM1R and CM3R methods, respectively, and the corresponding unsigned average errors in ΔG(hyd) relative to experiment were 2.05 and 1.89 kcal/mol. Computed errors in aniline and two derivatives were particularly large for RM1 and their removal from the data set lowered the overall errors to 1.61 and 1.75 kcal/mol for CM1R and CM3R. Comparisons are made to the AM1 method which yielded total errors in ΔG(hyd) of 1.50 and 1.64 kcal/mol for CM1A*1.14 and CM3A*1.15, respectively. This work is motivated by the need for a highly efficient yet accurate quantum mechanical (QM) method to study condensed-phase and enzymatic chemical reactions via mixed QM and molecular mechanical (QM/MM) simulations. As an initial test, the Menshutkin reaction between NH(3) and CH(3) Cl in water was computed using a RM1/TIP4P-Ew/CM3R procedure and the resultant ΔG(‡) , ΔG(rxn) , and geometries were in reasonable accord with other computational methods; however, some potentially serious shortcomings in RM1 are discussed.

Development of the CHARMM Force Field for Lipids

The development of the CHARMM additive all-atom lipid force field (FF) is traced from the early 1990's to the most recent version (C36) published in 2010. Though simulations with early versions yielded useful results, they failed to reproduce two important quantities: a zero surface tension at the experimental bilayer surface area, and the signature splitting of the deuterium order parameters in the glycerol and upper chain carbons. Systematic optimization of parameters based on high level quantum mechanical data and free energy simulations have resolved these issues, and bilayers with a wide range of lipids can be simulated in tensionless ensembles using C36. Issues associated with other all-atom lipid FFs, success and limitations in the C36 FF and ongoing developments are also discussed.

Catalytic Mechanism of Cytochrome P450 for 5′-Hydroxylation of Nicotine: Fundamental Reaction Pathways and Stereoselectivity

A series of computational methods were used to study how cytochrome P450 2A6 (CYP2A6) interacts with (S)-(-)-nicotine, demonstrating that the dominant molecular species of (S)-(-)-nicotine in CYP2A6 active site exists in the free base state (with two conformations, SR(t) and SR(c)), despite the fact that the protonated state is dominant for the free ligand in solution. The computational results reveal that the dominant pathway of nicotine metabolism in CYP2A6 is through nicotine free base oxidation. Further, first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations were carried out to uncover the detailed reaction pathways for the CYP2A6-catalyzed nicotine 5'-hydroxylation reaction. In the determined CYP2A6-(S)-(-)-nicotine binding structures, the oxygen of Compound I (Cpd I) can abstract a hydrogen from either the trans-5'- or the cis-5'-position of (S)-(-)-nicotine. CYP2A6-catalyzed (S)-(-)-nicotine 5'-hydroxylation consists of two reaction steps, that is, the hydrogen transfer from the 5'-position of (S)-(-)-nicotine to the oxygen of Cpd I (the H-transfer step), followed by the recombination of the (S)-(-)-nicotine moiety with the iron-bound hydroxyl group to generate the 5'-hydroxynicotine product (the O-rebound step). The H-transfer step is rate-determining. The 5'-hydroxylation proceeds mainly with the stereoselective loss of the trans-5'-hydrogen, that is, the 5'-hydrogen trans to the pyridine ring. The calculated overall stereoselectivity of ∼97% favoring the trans-5'-hydroxylation is close to the observed stereoselectivity of 89-94%. This is the first time it has been demonstrated that a CYP substrate exists dominantly in one protonation state (cationic species) in solution, but uses its less-favorable protonation state (neutral free base) to perform the enzymatic reaction.

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.

Quantum mechanical binding free energy calculation for phosphopeptide inhibitors of the Lck SH2 domain

The accurate and efficient calculation of binding free energies is essential in computational biophysics. We present a linear-scaling quantum mechanical (QM)-based end-point method termed MM/QM-COSMO to calculate binding free energies in biomolecular systems, with an improved description of entropic changes. Molecular dynamics trajectories are re-evaluated using a semiempirical Hamiltonian and a continuum solvent model; translational and rotational entropies are calculated using configurational integrals, and internal entropy is calculated using the harmonic oscillator approximation. As an application, we studied the binding of a series of phosphotyrosine tetrapeptides to the human Lck SH2 domain, a key component in intracellular signal transduction, modulation of which can have therapeutic relevance in the treatment of cancer, osteoporosis, and autoimmune diseases. Calculations with molecular mechanics Poisson-Boltzmann, and generalized Born surface area methods showed large discrepancies with experimental data stemming from the enthalpic component, in agreement with an earlier report. The empirical force field-based solvent interaction energy scoring function yielded improved results, with average unsigned error of 3.6 kcal/mol, and a better ligand ranking. The MM/QM-COSMO method exhibited the best agreement both for absolute (average unsigned error = 0.7 kcal/mol) and relative binding free energy calculations. These results show the feasibility and promise of a full QM-based end-point method with an adequate balance of accuracy and computational efficiency.

Clarification of the Mechanism of Acylation Reaction and Origin of Substrate Specificity of the Serine-Carboxyl Peptidase Sedolisin through QM/MM Free Energy Simulations

Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzyme-catalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P(1) site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers as well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P(1) site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P(1) site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N-H of P(1')-Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P(1)-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P(1)-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P(1)-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton transfers during acylation are also given.

Hysteresis and interfacial energies in smooth‐walled microfluidic channels

Hysteresis in the capillary pressure‐saturation relationship (Pc–Sw) for a porous medium has contributions from the complex geometry of the pore network as well as the physical chemistry of the grain surfaces. To isolate the role of wettability on hysteresis, we fabricated microfluidic cells that contain a single wedge‐shaped channel that simulates a single pore throat. Using confocal microscopy of the three‐dimensional interfaces under imbibition and drainage, we demonstrate an accurate balance between mechanical work and surface free energy that was evaluated using measured advancing and receding contact angles. The closed‐loop mechanical work per surface water molecule is 95 kJ/mol, which is consistent with physisorption. Therefore, the hysteresis in the Pc–Sw relationship for a single pore throat is defined by advancing and receding contact angles that are controlled by dissipative surface adsorption chemistry.

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.

Active Participation of the Mg2+ Ion in the Reaction Coordinate of RNA Self-Cleavage Catalyzed by the Hammerhead Ribozyme

We report results from combined quantum mechanical/molecular mechanical (QM/MM) free energy simulations to explore metal-assisted phosphoryl transfer and general acid catalysis in the extended hammerhead ribozyme. The mechanisms considered here assume that the 2′OH group of C17 has already been activated (i.e., is deprotonated) and acts as a nucleophile to go on an in-line attack to the adjacent scissile phosphate, passing through a pentavalent phosphorane intermediate/transition state, followed by acid-catalyzed departure of the O5′ leaving group of C1.1. A series of six two-dimensional potential of mean force profiles are reported in this study, requiring an aggregate of over 100 ns of QM/MM simulation. The simulations employ the AM1/d-PhoT semiempirical quantum model and linear-scaling QM/MM-Ewald method and explore mechanistic pathways for the self-cleavage. Results support the plausibility of a cleavage mechanism where phosphoryl transfer and general acid catalysis are stepwise, and where the catalytic divalent metal ion plays an active role in the chemical steps of catalysis.

Understanding the Mechanism of Deacylation Reaction Catalyzed by the Serine Carboxyl Peptidase Kumamolisin-As: Insights from QM/MM Free Energy Simulations

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics and free energy simulations are performed to study the process of the deacylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism. The results given here suggest that Asp-164 acts as a general acid/base catalyst not only for the acylation reaction but also for the deacylation reaction. It is shown that the electrostatic oxyanion hole interactions may be less effective in transition state stabilization for the kumamolisin-As catalyzed reaction compared to the general acid/base mechanism involving the proton transfer from or to Asp-164. The dynamic substrate-assisted catalysis (DSAC) involving His at the P1 site of the substrate is found to be less important for the deacylation reaction than for the acylation reaction in the kumamolisin-As catalyzed reaction. The proton transfer processes during the enzyme-catalyzed process are examined and their role in the catalysis is discussed.