Transition-State Vibrational Analysis and Isotope Effects for COMT-Catalyzed Methyl Transfer.

Isotopic partition-function ratios (IPFRs) computed for transition structures (TSs) of the methyl-transfer reaction catalyzed by catechol O-methyltransferase and modeled by hybrid QM/MM methods are analyzed. The ability of smaller Hessians to reproduce trends in α-3H3 and 14Cα IPFRs as obtained using the much larger subset QM/MM Hessians from which they are extracted is investigated critically. A 6-atom-extracted Hessian reproduces perfectly the α-T3 IPFR values from the full-subset Hessians of all the TSs but not the α-14CIPFRs. Average AM1/OPLS-AA harmonic frequencies and mean-square amplitudes are presented for the 12 normal modes of the α-CH3 moiety within the active site of several enzymic transition structures, together with QM/MM potential energy scans along each of these modes to assess the degree of anharmonicity. A novel investigation of ponderal effects upon IPFRs suggests that the value for α-14C tends toward a limiting minimum whereas that for α-T3 tends toward a limiting maximum as the mass of the rest of the system increases. The transition vector is dominated by motions of atoms within the donor and acceptor moieties and is very well described as a simple combination of Walden-inversion "umbrella" bending and asymmetric stretching of the SCα and CαO bonds. The contribution of atoms of the protein residues Met40, Tyr68, and Asp141 to the transition vector is extremely small. Average valence force constants for the COMT TS show significant differences from early BEBOVIB estimates which were used in support of the compression hypothesis for catalysis. There is no correlation between TS IPFRs and the nonbonded distances for close contacts between the S atom of SAM and Tyr68 or between any of the H atoms of the transferring methyl group and either Met40 or Asp141.

Influence of Dielectric Environment upon Isotope Effects on Glycoside Heterolysis: Computational Evaluation and Atomic Hessian Analysis.

Isotope effects depend upon the polarity of the bulk medium in which a chemical process occurs. Implicit solvent calculations with molecule-shaped cavities show that the equilibrium isotope effect (EIE) for heterolysis of the glycosidic bonds in 5'-methylthioadenosine and in 2-(p-nitrophenoxy)tetrahydropyran, both in water, are very sensitive in the range 2 ≤ ε ≤ 10 to the relative permittivity of the continuum surrounding the oxacarbenium ion. However, different implementations of nominally the same PCM method can lead to opposite trends being predicted for the same molecule. Computational modeling of the influence of the inhomogeneous effective dielectric surrounding a substrate within the protein environment of an enzymic reaction requires an explicit treatment. The EIE (KH/KD) for transfer of cyclopentyl, cyclohexyl, tetrahydrofuranyl and tetrahydropyranyl cations from water to cyclohexane is predicted by B3LYP/6-31+G(d) calculations with implicit solvation and confirmed by B3LYP/6-31+G(d)/OPLS-AA calculations with averaging over many explicit solvation configurations. Atomic Hessian analysis, whereby the full Hessian is reduced to the elements belonging to a single atom at the site of isotopic substitution, reveals a remarkable result for both implicit and explicit solvation: the influence of the solvent environment on these EIEs is essentially captured completely by only a 3 × 3 block of the Hessian, although these values must correctly reflect the influence of the whole environment. QM/MM simulation with ensemble averaging has an important role to play in assisting the meaningful interpretation of observed isotope effects for chemical reactions both in solution and catalyzed by enzymes.

Critical evaluation of anharmonicity and configurational averaging in QM/MM modelling of equilibrium isotope effects.

Anharmonic effects upon vibrational frequencies and isotopic partition function ratios are modelled computationally by means of quantum mechanics/molecular mechanics (QM/MM) methods for two systems. First, the methyl cation in explicit water is considered using a B3LYP/6-31+G(d)/TIP3P method in order to check the previous prediction of an inverse equilibrium isotope effect (EIE) KH3/KD3 for transfer from vacuum to water at 298 K. A full QM/MM treatment including Lennard-Jones interactions predicts significantly inverse contributions from both internal (0.843 ± 0.001) and external (0.894 ± 0.001) modes of the solute. This treatment yields a much larger harmonic EIE (0.753 ± 0.002, averaged over 928 independent solvent configurations) than is obtained either by projecting out the translational and rotational contributions (0.853) or by treating the solvent by a point-charge representation (0.9360 ± 0.0006, harmonic; 0.9366 ± 0.0006, anharmonic). The contribution of anharmonicity to the EIE affects the value only in the 3rd significant figure. Second, anharmonicity is investigated by means of QM/MM potential-energy scans along 12 normal modes for internal and external vibrations of methyl cation in water and for three modes (one stretching and two bending) for the Hα atom at the carbenium-ion centre in cyclopentyl, cyclohexyl, tetrahydrofuranyl and tetrahydropyranyl cations in explicit water and cyclohexane solvents, as obtained by means of atomic Hessian analysis.

Insights on the Origin of Catalysis on Glycine N-Methyltransferase from Computational Modeling.

The origin of enzyme catalysis remains a question of debate despite much intense study. We report a QM/MM theoretical study of the SN2 methyl transfer reaction catalyzed by a glycine N-methyltransferase (GNMT) and three mutants to test whether recent experimental observations of rate-constant reductions and variations in inverse secondary α-3H kinetic isotope effects (KIEs) should be attributed to changes in the methyl donor-acceptor distance (DAD): Is catalysis due to a compression effect? Semiempirical (AM1) and DFT (M06-2X) methods were used to describe the QM subset of atoms, while OPLS-AA and TIP3P classical force fields were used for the protein and water molecules, respectively. The computed activation free energies and KIEs are in good agreement with experimental data, but the mutations do not meaningfully affect the DAD: Compression cannot explain the experimental variations on KIEs. On the contrary, electrostatic properties in the active site correlate with the catalytic activity of wild type and mutants. The plasticity of the enzyme moderates the effects of the mutations, explaining the rather small degree of variation in KIEs and reactivities.

Computational Modeling of a Caged Methyl Cation: Structure, Energetics, and Vibrational Analysis.

DFT calculations for CH3+ within a constrained cage of water molecules permit the controlled manipulation of distances rax and req to "axial" and "equatorial" waters. Equatorial CH···O interactions catalyze methyl transfer (MT) between axial waters. Variation in rax has a greater effect on CH bond lengths and stretching force constants in the symmetric SN2-like transition structures than variation in req. In-plane bending frequencies are insensitive to these variations in cage dimensions, but axial interactions loosen the out-of-plane bending mode (OP) whereas equatorial interactions stiffen it. Frequencies for rotational and translational motions of CH3+ within the cage are influenced by rax and req. In particular, translation of CH3+ in the axial direction is always coupled to cage motion. With longer rax, CH3+ translation is coupled with asymmetric CO bond stretching, but with shorter rax, it is also coupled with OP (equivalent to the umbrella mode of trigonal bipyramidal O···CH3+···O); the magnitude of the imaginary MT frequency increases steeply as rax diminishes. This coupling between CH3+ and its cage is removed by eliminating the rows and columns associated with cage atoms from the full Hessian to obtain a reduced Hessian for CH3+ alone. Within a certain range of cage dimensions, the reduced Hessian yields a real frequency for MT. The importance of using a Hessian large enough to describe the reaction coordinate mode correctly is emphasized for modeling chemical reactions and particularly for kinetic isotope effects in enzymic MT.

A computational study of the influence of methyl substituents on competitive ring closure to α- and ß-lactones.

Ring-closure of substituted 2-chlorosuccinates to α- or ß-lactones has been studied by means of MP2/6-311+G(d,p)//MP2/6-31+G(d) calculations in water treated as a polarised continuum (PCM) and in vacuum. Optimised geometries have been obtained for 2-chlorosuccinate and its 2-methyl, 3,3-dimethyl, and 2,3,3-trimethyl derivatives, along with the transition structures and products for intramolecular nucleophilic displacement leading to the 3- or 4-membered rings. Relative enthalpies and Gibbs free energies of activation and reaction are presented, along with key geometrical parameters, and changes in electrostatic-potential-derived atomic charges. The difference in free-energy barriers for α- and ß-lactone formation from the 2-methyl substrate at 298 K is less than 1 kJ mol-1. Primary 14C kinetic isotope effects calculated for substitution at C2 are significantly smaller for α-lactone formation than for ß, suggesting a possible way to distinguish between the competing pathways of reaction. The B3LYP method without dispersion corrections predicts the wrong relative stability order for methyl-substituted succinate dianions in PCM water.

Influence of Equatorial CH⋠⋠⋠O Interactions on Secondary Kinetic Isotope Effects for Methyl Transfer.

DFT calculations for methyl cation complexed within a constrained cage of water molecules permit the controlled manipulation of the "axial" donor/acceptor distance and the "equatorial" distance to hydrogen-bond acceptors. The kinetic isotope effect k(CH3)/k(CT3) for methyl transfer within a cage with a short axial distance becomes less inverse for shorter equatorial C⋅⋅⋅O distances: a decrease of 0.5 Šresults in a 3 % increase at 298 K. Kinetic isotope effects in AdoMet-dependent methyltransferases may be m∧odulated by CH⋅⋅⋅O hydrogen bonding, and factors other than axial compression may contribute, at least partially, to recently reported isotope-effect variations for catechol-O-methyltransferase and its mutant structures.

A critical survey of average distances between catalytic carboxyl groups in glycoside hydrolases.

Published X-ray crystallographic structures for glycoside hydrolases (GHs) from 39 different families are surveyed according to some rigorous selection criteria, and the distances separating 208 pairs of catalytic carboxyl groups (20 α-retaining, 87 ß-retaining, 38 α-inverting, and 63 ß-inverting) are analyzed. First, the average of all four inter-carboxyl O…O distances for each pair is determined; second, the mean of all the pair-averages within each GH family is determined; third, means are determined for groups of GH families. No significant differences are found for free structures compared with those complexed with a ligand in the active site of the enzyme, nor for α-GHs as compared with ß-GHs. The mean and standard deviation (1σ) of the unimodal distribution of average O…O distances for all families of inverting GHs is 8 ± 2Å, with a very wide range from 5Å (GH82) to nearly 13Å (GH46). The distribution of average O…O distances for all families of retaining GHs appears to be bimodal: the means and standard deviations of the two groups are 4.8 ± 0.3Å and 6.4 ± 0.6Å. These average values are more representative, and more likely to be meaningful, than the often-quoted literature values, which are based on a very small sample of structures. The newly-updated average values proposed here may alter perceptions about what separations between catalytic residues are "normal" or "abnormal" for GHs.

C-H functionalization of sp(3) centers with aluminum: a computational and mechanistic study of the Baddeley reaction of decalin.

Decalin undergoes reaction with aluminum trichloride and acetyl chloride to form a tricyclic enol ether in good yield, as first reported by Baddeley. This eye-catching transformation, which may be considered to be an aliphatic Friedel-Crafts reaction, has not previously been studied mechanistically. Here we report experimental and computational studies to elucidate the mechanism of this reaction. We give supporting evidence for the proposition that, in the absence of unsaturation, an acylium ion acts as a hydride acceptor, forming a tertiary carbocation. Loss of a proton introduces an alkene, which reacts with a further acylium ion. A concerted 1,2-hydride shift/oxonium formation, followed by elimination, leads to formation of the observed product.

Ensemble-averaged QM/MM kinetic isotope effects for the S(N)2 reaction of cyanide anions with chloroethane in DMSO solution.

The existence of solvent fluctuations leads to populations of reactant-state (RS) and transition-state (TS) configurations and implies that property calculations must include appropriate averaging over distributions of values for individual configurations. Average kinetic isotope effects 〈KIE〉 for NC(-) + EtCl → NCEt + Cl(-) in DMSO solution at 30 °C are best obtained as the ratio 〈f(RS)〉/〈f(TS)〉 of isotopic partition function ratios separately averaged over all RS and TS configurations. In this way the hybrid AM1/OPLS-AA potential yields 〈KIE〉 values for all six isotopic substitutions (2° α-(2)H(2), 2° ß-(2)H(3), α-(11)C/(14)C, leaving group (37)Cl, and nucleophile (13)C and (15)N) for this reaction in the correct direction as measured experimentally. These thermally-averaged calculated KIEs may be compared meaningfully with experiment, and only one of them differs in magnitude from the experimental value by more than one standard deviation from the mean. This success contrasts with previous KIE calculations based upon traditional methods without averaging. The isotopic partition function ratios are best evaluated using all (internal) vibrational and (external) librational frequencies obtained from Hessians determined for subsets of atoms, relaxed to local minima or saddle points, within frozen solvent environments of structures sampled along molecular dynamics trajectories for RS and TS. The current method may perfectly well be implemented with other QM or QM/MM methods, and thus provides a useful tool for investigating KIEs in relation to studies of chemical reaction mechanisms in solution or catalyzed by enzymes.

Catalysis: transition-state molecular recognition?

THE KEY TO UNDERSTANDING THE FUNDAMENTAL PROCESSES OF CATALYSIS IS THE TRANSITION STATE (TS): indeed, catalysis is a transition-state molecular recognition event. Practical objectives, such as the design of TS analogues as potential drugs, or the design of synthetic catalysts (including catalytic antibodies), require prior knowledge of the TS structure to be mimicked. Examples, both old and new, of computational modelling studies are discussed, which illustrate this fundamental concept. It is shown that reactant binding is intrinsically inhibitory, and that attempts to design catalysts that focus simply upon attractive interactions in a binding site may fail. Free-energy changes along the reaction coordinate for S(N)2 methyl transfer catalysed by the enzyme catechol-O-methyl transferase are described and compared with those for a model reaction in water, as computed by hybrid quantum-mechanical/molecular-mechanical molecular dynamics simulations. The case is discussed of molecular recognition in a xylanase enzyme that stabilises its sugar substrate in a (normally unfavourable) boat conformation and in which a single-atom mutation affects the free-energy of activation dramatically.

G-Protein coupled receptors: structure and function in drug discovery.

The G-protein coupled receptors (GPCRs) superfamily comprise similar proteins arranged into families or classes thus making it one of the largest in the mammalian genome. GPCRs take part in many vital physiological functions making them targets for numerous novel drugs. GPCRs share some distinctive features, such as the seven transmembrane domains, they also differ in the number of conserved residues in their transmembrane domain. Here we provide an introductory and accessible review detailing the computational advances in GPCR pharmacology and drug discovery. An overview is provided on family A-C GPCRs; their structural differences, GPCR signalling, allosteric binding and cooperativity. The dielectric constant (relative permittivity) of proteins is also discussed in the context of site-specific environmental effects.

Probing synergy between two catalytic strategies in the glycoside hydrolase O-GlcNAcase using multiple linear free energy relationships.

Human O-GlcNAcase plays an important role in regulating the post-translational modification of serine and threonine residues with beta-O-linked N-acetylglucosamine monosaccharide unit (O-GlcNAc). The mechanism of O-GlcNAcase involves nucleophilic participation of the 2-acetamido group of the substrate to displace a glycosidically linked leaving group. The tolerance of this enzyme for variation in substrate structure has enabled us to characterize O-GlcNAcase transition states using several series of substrates to generate multiple simultaneous free-energy relationships. Patterns revealing changes in mechanism, transition state, and rate-determining step upon concomitant variation of both nucleophilic strength and leaving group abilities are observed. The observed changes in mechanism reflect the roles played by the enzymic general acid and the catalytic nucleophile. Significantly, these results illustrate how the enzyme synergistically harnesses both modes of catalysis; a feature that eludes many small molecule models of catalysis. These studies also suggest the kinetic significance of an oxocarbenium ion intermediate in the O-GlcNAcase-catalyzed hydrolysis of glucosaminides, probing the limits of what may be learned using nonatomistic investigations of enzymic transition-state structure and offering general insights into how the superfamily of retaining glycoside hydrolases act as efficient catalysts.

Mechanism of glycoside hydrolysis: A comparative QM/MM molecular dynamics analysis for wild type and Y69F mutant retaining xylanases.

Computational simulations have been performed using hybrid quantum-mechanical/molecular-mechanical potentials to investigate the catalytic mechanism of the retaining endo-beta-1, 4-xylanase (BCX) from B. circulans. Two-dimensional potential-of-mean-force calculations based upon molecular dynamics with the AM1/OPLS method for wild-type BCX with a p-nitrophenyl xylobioside substrate in water clearly indicates a stepwise mechanism for glycosylation: the rate-determining step is nucleophilic substitution by Glu78 to form the covalently bonded enzyme-substrate intermediate without protonation of the leaving group by Glu172. The geometrical configuration of the transition state for the enzymic reaction is essentially the same as found for a gas-phase model involving only the substrate and a propionate/propionic acid pair to represent the catalytic glutamate/glutamic acid groups. In addition to stabilizing the (2,5)B boat conformation of the proximal xylose in the non-covalent reactant complex of the substrate with BCX, Tyr69 lowers the free-energy barrier for glycosylation by 42 kJ mol(-1) relative to that calculated for the Y69F mutant, which lacks the oxygen atom O(Y). B3LYP/6-31+G* energy corrections reduce the absolute height of the barrier to reaction. In the oxacarbenium ion-like transition state O(Y) approaches closer to the endocyclic oxygen O(ring) of the sugar ring but donates its hydrogen bond not to O(ring) but rather to the nucleophilic oxygen of Glu78. Comparison of the average atomic charge distributions for the wild-type and mutant indicates that charge separation along the bond between the anomeric carbon and O(ring) is matched in the former by a complementary separation of charge along the O(Y)-H(Y) bond, corresponding to a pair of roughly antiparallel bond dipoles, which is not present in the latter.

Computational mutagenesis reveals the role of active-site tyrosine in stabilising a boat conformation for the substrate: QM/MM molecular dynamics studies of wild-type and mutant xylanases.

Molecular dynamics simulations have been performed for non-covalent complexes of phenyl beta-xylobioside with the retaining endo-beta-1,4-xylanase from B. circulans (BCX) and its Tyr69Phe mutant using a hybrid QM/MM methodology. A trajectory initiated for the wild-type enzyme-substrate complex with the proximal xylose ring bound at the -1 subsite (adjacent to the scissile glycosidic bond) in the (4)C(1) chair conformation shows spontaneous transformation to the (2,5)B boat conformation, and potential of mean force calculations indicate that the boat is approximately 30 kJ mol(-1) lower in free energy than the chair. Analogous simulations for the mutant lacking one oxygen atom confirm the key role of Tyr69 in stabilizing the boat in preference to the (4)C(1) chair conformation, with a relative free energy difference of about 20 kJ mol(-1), by donating a hydrogen bond to the endocyclic oxygen of the proximal xylose ring. QM/MM MD simulations for phenyl beta-xyloside in water, with and without a propionate/propionic acid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the (4)C(1) chair to be stable, although a hydrogen bond between the OH group at C2 of xylose and the propionate moiety seems to provide some stabilization for the (2,5)B conformation.

QM/MM simulations for methyl transfer in solution and catalysed by COMT: ensemble-averaging of kinetic isotope effects.

Sampling of structures from QM/MM molecular dynamics reveals distinct families of reactant-state conformers and yields kinetic isotope effects for reactions in enzyme active sites and in solution, averaged over thermal fluctuations of the environment, that allows meaningful comparison of computed with experimental values.

Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase.

Molecular simulations based on the use of hybrid quantum mechanics/molecular mechanics methods are able to provide detailed information about the complex enzymatic reactions and the consequences of specific mutations on the activity of the enzyme. In this work, the reduction of pyruvate to lactate catalysed by wild-type and Asp168Ala mutant lactate dehydrogenase (LDH) has been studied by means of simulations using a very flexible molecular model consisting of the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules. Our results indicate that the Asp168Ala mutation provokes a shift in the pKa value of Glu199 that becomes unprotonated at neutral pH in the mutant enzyme. This change compensates the loss of the negative charge of Asp168, rendering a still active enzyme. Thus, our methodology gives a calculated barrier height for the Asp168Ala mutant 3 kcal mol-1 higher than that for wild-type LDH, which is in very good agreement with the experiment. The computed potential energy surfaces reveal the reaction pathways and transition structures for the wild-type and mutant enzymes. Hydride transfer is less advanced and the proton transfer is more advanced in the Asp168Ala mutant than in the wild type. This approach provides a very powerful tool for the analysis of the roles of key active-site residues.

Glycosidase inhibitors as conformational transition state analogues.

A method for estimating the conformational similarity between hexopyranose rings is presented and used to probe the behaviour of various glycosyl hydrolase inhibitors as conformational transition state analogues.

Links between de novo fatty acid synthesis and leptin secretion in bovine adipocytes.

Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants.

The Walden cycle revisited: a computational study of competitive ring closure to alpha- and beta-lactones.

The text-book Walden cycle which interconverts the stereochemical configurations of chlorosuccinic and malic acids involves a beta-lactone intermediate in preference to an alpha-lactone intermediate because the O(nuc) C Cl angle in the transition structure for the former (174 degrees) is more favourable than that for the latter (139 degrees), as determined by PCM(epsilon = 78.4)/B3LYP/6-31+G* calculations; the smaller ring-strain energy of the beta-lactone contributes little to the reactivity difference.