QM/MM calculations of kinetic isotope effects in the chorismate mutase active site

Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephenate in aqueous solution and in the active site of chorismate mutase from B. subtilus. These included primary 13C and 18O and secondary 3H effects for substitutions at the bond-making and bond-breaking positions. The initial structures of the putative stationary points on the potential energy surface, required for the calculations of isotope effects using the CAMVIB/CAMISO programs, have been selected from hybrid QM/MM molecular dynamical simulations using the DYNAMO program. Refinement of the reactant complex and transition-state structures has been carried out by means of AM1/CHARMM24/TIP3P calculations using the GRACE program, with full gradient relaxation of the position of > 5200 atoms for the enzymic simulations, and with a box containing 711 water molecules for the corresponding reaction in aqueous solution. Comparison of these results, and of gas phase calculations, with experimental data has shown that the chemical rearrangement is largely rate-determining for the enzyme mechanism. Inclusion of the chorismate conformational pre-equilibrium step in the modelled kinetic scheme leads to better agreement between recent experimental data and theoretical predictions. These results provide new information on an important enzymatic transformation, and the key factors responsible for the kinetics of its molecular mechanism are clarified. Treatment of the enzyme and/or solvent environment by means of a large and flexible model is absolutely essential for prediction of kinetic isotope effects.     

Kinetic isotope effects in the active site of B. subtilis chorismate mutase

Kinetic isotope effects are determined for the enzyme‐catalyzed Claisen rearrangement of chorismate to prephenate using computational methods. The calculated kinetic isotope effects (KIEs) compare reasonably with the few available experimental values with both the theory and experiment obtaining a large KIE for the ether oxygen, indicating large polarization of the transition‐state geometry. Because there is a question of the extent that the experimental rate constants are for chemistry as the rate‐limiting step, the KIEs for all the atoms of the substrate are reported with the exception of the carboxylate groups. A substantial number of large regular and inverse isotope effects are predicted for the hydrogens on the cyclohexadienyl ring related to activation of the reactant and charge reorganization in the transition state. A large KIE is predicted for the hydrogen atom bound to the ether carbon atom because the largest valency change and charge transfer occurs at the ether bond in both the reactant and tansition state. Observation of the overall pattern of predicted KIEs would ensure that conditions are favorable for the rate‐limiting chemistry. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 287–292, 2003     

Conformational effects in enzyme catalysis: QM/MM free energy calculation of the 'NAC' contribution in chorismate mutase

The controversial 'near attack conformation'(NAC) effect in the important model enzyme chorismate mutase is calculated to be 3.8-4.6 kcal mol(-1) by QM/MM free energy perturbation molecular dynamics methods, showing that the NAC effect by itself does not account for catalysis in this enzyme.     

QM/MM determination of kinetic isotope effects for COMT-catalyzed methyl transfer does not support compression hypothesis

Secondary alpha-D3 kinetic isotope effects calculated by the hybrid AM1/TIP3P/CHARMM method for the reaction of S-adenosylmethionine with catecholate anion in aqueous solution and catalyzed by rat liver catechol O-methyltransferase at 298 K are 0.94 and 0.85, respectively, in good accord with experiment. The large inverse effect for the enzymatic reaction is not due to compression but arises from significant increases in the stretching and bending force constants involving the isotopically substituted atoms of the transferring methyl group as between the reactant complex and the transition structure, larger than for the reaction in water.     

Multiple high-level QM/MM reaction paths demonstrate transition-state stabilization in chorismate mutase: correlation of barrier height with transition-state stabilization

Multiple profiles for the reaction from chorismate to prephenate in the enzyme chorismate mutase calculated with hybrid density functional combined quantum mechanics/molecular mechanics methods (B3LYP/6-31G(d)-CHARMM27) agree well with experiment, and provide direct evidence of transition-state stabilization by this important enzyme, which is at the centre of current debates about the nature of enzyme catalysis.     

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° β-(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.     

A new form of kinetic isotope effect. Dynamic effects on isotopic selectivity and regioselectivity

The intramolecular H/D kinetic isotope effect in the ene reaction of singlet oxygen with tetramethylethylene is studied using quasiclassical direct dynamics calculations on a B3LYP/6-31G* potential energy surface. Starting from the area of the energy surface around a valley-ridge inflection point, random trajectories lead to predominantly H abstraction over D abstraction, despite the symmetry of the surface and the absence of a barrier to either reaction. This demonstrates a new form of kinetic isotope effect, unrelated to the usual effect of zero-point energies on barriers. Dynamics calculations on the reaction of cis-2-pentene predict the experimentally observed mixture of regioisomeric products, while the minimum-energy path leads to only one product. For energy surfaces containing two adjacent saddle points, dynamics effects are important for understanding both product and isotopic selectivity, and this should be considered in the interpretation of experimental results.     

A new interpretation of chlorine leaving group kinetic isotope effects; a theoretical approach

The chlorine leaving group kinetic isotope effects (KIEs) for the S(N)2 reactions between methyl chloride and a wide range of anionic, neutral, and radical anion nucleophiles were calculated in the gas phase and, in several cases, using a continuum solvent model. In contrast to the expected linear dependence of the chlorine KIEs on the C(alpha)-Cl bond order in the transition state, the KIEs fell in a very small range (1.0056-1.0091), even though the C(alpha)-Cl transition state bond orders varied widely from approximately 0.32 to 0.78, a range from reactant-like to very product-like. This renders chlorine KIEs, and possibly other leaving-group KIEs, less useful for studies of reaction mechanisms than commonly assumed. A partial explanation for this unexpected relationship between the C(alpha)-Cl transition state bond order and the magnitude of the chlorine KIE is presented.     

Temperature dependence of the kinetic isotope effects in thymidylate synthase. A theoretical study

In recent years, the temperature dependence of primary kinetic isotope effects (KIE) has been used as indicator for the physical nature of enzyme-catalyzed H-transfer reactions. An interactive study where experimental data and calculations examine the same chemical transformation is a critical means to interpret more properly temperature dependence of KIEs. Here, the rate-limiting step of the thymidylate synthase-catalyzed reaction has been studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) simulations in the theoretical framework of the ensemble-averaged variational transition-state theory with multidimensional tunneling (EA-VTST/MT) combined with Grote-Hynes theory. The KIEs were calculated across the same temperature range examined experimentally, revealing a temperature independent behavior, in agreement with experimental findings. The calculations show that the H-transfer proceeds with ～91% by tunneling in the case of protium and ～80% when the transferred protium is replaced by tritium. Dynamic recrossing coefficients are almost invariant with temperature and in all cases far from unity, showing significant coupling between protein motions and the reaction coordinate. In particular, the relative movement of a conserved arginine (Arg166 in Escherichia coli ) promotes the departure of a conserved cysteine (Cys146 in E. coli ) from the dUMP by polarizing the thioether bond thus facilitating this bond breaking that takes place concomitantly with the hydride transfer. These promoting vibrations of the enzyme, which represent some of the dimensions of the real reaction coordinate, would limit the search through configurational space to efficiently find those decreasing both barrier height and width, thereby enhancing the probability of H-transfer by either tunneling (through barrier) or classical (over-the-barrier) mechanisms. In other words, the thermal fluctuations that are coupled to the reaction coordinate, together with transition-state geometries and tunneling, are the same in different bath temperatures (within the limited experimental range examined). All these terms contribute to the observed temperature independent KIEs in thymidylate synthase.     

Combined QM/MM and path integral simulations of kinetic isotope effects in the proton transfer reaction between nitroethane and acetate ion in water

An integrated Feynman path integral-free energy perturbation and umbrella sampling (PI-FEP/UM) method has been used to investigate the kinetic isotope effects (KIEs) in the proton transfer reaction between nitroethane and acetate ion in water. In the present study, both nuclear and electronic quantum effects are explicitly treated for the reacting system. The nuclear quantum effects are represented by bisection sampling centroid path integral simulations, while the potential energy surface is described by a combined quantum mechanical and molecular mechanical (QM/MM) potential. The accuracy essential for computing KIEs is achieved by a FEP technique that transforms the mass of a light isotope into a heavy one, which is equivalent to the perturbation of the coordinates for the path integral quasiparticle in the bisection sampling scheme. The PI-FEP/UM method is applied to the proton abstraction of nitroethane by acetate ion in water through molecular dynamics simulations. The rule of the geometric mean and the Swain-Schaad exponents for various isotopic substitutions at the primary and secondary sites have been examined. The computed total deuterium KIEs are in accord with experiments. It is found that the mixed isotopic Swain-Schaad exponents are very close to the semiclassical limits, suggesting that tunneling effects do not significantly affect this property for the reaction between nitroethane and acetate ion in aqueous solution.     

Unraveling the origin of regioselectivity in rhodium diphosphine catalyzed hydroformylation. A DFT QM/MM study

The origin of regioselectivity in rhodium diphosphine catalyzed hydroformilation was investigated by means of hybrid QM/MM calculations using the IMOMM method. The roles of the diphosphine bite angle and of the nonbonding interactions were analyzed in detail by considering rhodium systems containing xantphos-type ligands, for which a correlation between the natural bite angle and regioselectivity has been recently reported. From the pentacoordinated equatorial--equatorial HRh(CO)(alkene)(diphosphine) key intermediate, eight possible reaction paths were defined and characterized through their respective transition states (TS). We succeeded in reproducing the experimentally observed trends for the studied diphosphines. By performing additional calculations on model systems, in which the steric effects induced by the phenyl substituents of xantphos ligands were canceled, we were able to separate, identify, and evaluate the different contributions to regioselectivity. These additional calculations showed that regioselectivity is governed by the nonbonding interactions between the diphenylphosphino substituents and the substrate, whereas the effects directly associated to the bite angle, what we call orbital effects, seem to have a smaller influence.     

BEBO calculations of kinetic isotope effects for methyl radical reactions with alkanes and ketones

Kinetic isotope effects for hydrogen abstraction reactions of methyl radicals with methane, ethane, propane, acetone, butanone-2, pentanone-3 and biacetyl have been calculated using the BEBO method, considering recent values for the Pauling constant and the noble gas parameters. The results were compared with the experimental data.

---

, , , , -2, -3 BEBO, . .

     

QM/MM calculations with DFT for taking into account protein effects on the EPR and optical spectra of metalloproteins. Plastocyanin as a case study

A detailed study of the influence of the surrounding protein on magnetic and optical spectra of metalloproteins is presented using the quantum-mechanical/molecular mechanical (QM/MM) approach. The well-studied type I copper site in plastocyanin in the cupric oxidation state is taken as a test case because its spectroscopic properties have been extensively studied and are well understood. The calculations have been performed using nonrelativistic and scalar relativistic (at the level of the zeroth order regular approximation, ZORA) calculations (B3LYP functional). Linear response theory has been used to calculate first- and second-order properties, namely the EPR g-tensor, the central metal hyperfine couplings (HFCs), the HFCs of the directly coordinating ligands, as well as superhyperfine couplings (1H, 14N) from remote nuclei, transition energies, and oscillator strengths. Two different model systems have been defined that do not and do include important amino acids from the second coordination sphere, respectively. For comparison, calculations have been carried out in the gas phase and in a dielectric continuum (conductor like screening model, COSMO) with a dielectric constant of four. The best results were obtained at the scalar relativistic ZORA level for the largest model in conjunction with explicit modeling of the protein environment through the QM/MM procedure, which is also considered to be the highest level of theory used in this work. The protein effects beyond the second coordination sphere were found to be quite substantial (up to 30% changes on some properties), and were found to require an explicit treatment of the protein beyond the second coordination sphere. In addition, the embedding water cage was found to have a nonnegligible influence on the calculated spectroscopic data, which is of the same order as the influence of the protein backbone charges. However, while qualitatively satisfactory, the errors in the calculated spectroscopic parameters are still substantial, and can all be traced back to the fact that the linear-response of the presently available functionals is "too stiff" with respect to the external perturbations at least for the model systems studied here. Ligand field-based approaches are used to correct for systematic errors in the DFT procedures. As a consequence, we propose a new breakdown of the copper hyperfine interaction into Fermi-contact, spin-dipolar and spin-orbit contributions.     

Linear free energy relationship and kinetic isotope effects as measures for the transition state variation. A computational study

Linear free energy relationship (LFER) and kinetic isotope effects (KIEs) are frequently used experimental means to study reaction mechanisms, in particular the nature of transition states (TSs). Density functional theory (B3LYP/6-311+G**) calculations were carried out on a model reaction, acid-catalyzed ionization of phenylethyl alcohol, to analyze how experimentally observable properties, such as nonlinearity in the Hammett and Br?nsted relations and variation in KIE, are related to a variation of the transition state structure and the mechanism. Several conclusions and insights were obtained: (1) Linear Hammett plots with a dual parameter treatment may not be evidence for an invariable TS structure for a series of reactions. (2) Variations of KIEs indeed reflect the variations of TS structures. (3) Nonlinear Br?nsted plots cannot always be taken as evidence for a stepwise mechanism. (4) A TS structure in the gas phase may change much more easily than a TS structure in solution.     

Experimental and theoretical multiple kinetic isotope effects for an SN2 reaction. An attempt to determine transition-state structure and the ability of theoretical methods to predict experimental kinetic isotope effects

The secondary alpha-deuterium, the secondary beta-deuterium, the chlorine leaving-group, the nucleophile secondary nitrogen, the nucleophile (12)C/(13)C carbon, and the (11)C/(14)C alpha-carbon kinetic isotope effects (KIEs) and activation parameters have been measured for the S(N)2 reaction between tetrabutylammonium cyanide and ethyl chloride in DMSO at 30 degrees C. Then, thirty-nine readily available different theoretical methods, both including and excluding solvent, were used to calculate the structure of the transition state, the activation energy, and the kinetic isotope effects for the reaction. A comparison of the experimental and theoretical results by using semiempirical, ab initio, and density functional theory methods has shown that the density functional methods are most successful in calculating the experimental isotope effects. With two exceptions, including solvent in the calculation does not improve the fit with the experimental KIEs. Finally, none of the transition states and force constants obtained from the theoretical methods was able to predict all six of the KIEs found by experiment. Moreover, none of the calculated transition structures, which are all early and loose, agree with the late (product-like) transition-state structure suggested by interpreting the experimental KIEs.     

Transition structures, energetics, and secondary kinetic isotope effects for cope rearrangements of cis-1,2-divinylcyclobutane and cis-1,2-divinylcyclopropane: a DFT study

The minimum energy reaction paths and secondary kinetic isotope effects (KIE) for the Cope rearrangements of cis-1,2-divinylcyclobutane and cis-1,2-divinylcyclopropane obtained by (U)B3LYP calculations are reported. Both reactions proceed through endo-boatlike reaction paths, and have aromatic transition states. The predicted activation energies are in agreement with the experimental data. The reaction paths of the rearrangements are intervened by enantiomerization saddle points of the products (and the reactant in the case of divinylcyclobutane). The calculated KIEs are similar in the two systems, and consistent with the geometries of the transition structures. There is computational evidence that the isotope effect associated with the conversion of a pure sp(2) C-H bond into a pure sp(3) one might be the same in all molecules. The predicted KIEs agree with experiment for divinylcyclopropane, but not for divinylcyclobutane.     

A new strategy of outlier detection for QSAR/QSPR

The crucial step of building a high performance QSAR/QSPR model is the detection of outliers in the model. Detecting outliers in a multivariate point cloud is not trivial, especially when several outliers coexist in the model. The classical identification methods do not always identify them, because they are based on the sample mean and covariance matrix influenced by the outliers. Moreover, existing methods only lay stress on some type of outliers but not all the outliers. To avoid these problems and detect all kinds of outliers simultaneously, we provide a new strategy based on Monte‐Carlo cross‐validation, which was termed as the MC method. The MC method inherently provides a feasible way to detect different kinds of outliers by establishment of many cross‐predictive models. With the help of the distribution of predictive residuals such obtained, it seems to be able to reduce the risk caused by the masking effect. In addition, a new display is proposed, in which the absolute values of mean value of predictive residuals are plotted versus standard deviations of predictive residuals. The plot divides the data into normal samples, y direction outliers and X direction outliers. Several examples are used to demonstrate the detection ability of MC method through the comparison of different diagnostic methods. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.     

A new insight into using chlorine leaving group and nucleophile carbon kinetic isotope effects to determine substituent effects on the structure of SN2 transition states

Chlorine leaving group k(35)/k(37), nucleophile carbon k(11)/k(14), and secondary alpha-deuterium [(kH/kD)alpha] kinetic isotope effects (KIEs) have been measured for the SN2 reactions between para-substituted benzyl chlorides and tetrabutylammonium cyanide in tetrahydrofuran at 20 degrees C to determine whether these isotope effects can be used to determine the substituent effect on the structure of the transition state. The secondary alpha-deuterium KIEs indicate that the transition states for these reactions are unsymmetric. The theoretical calculations at the B3LYP/aug-cc-pVDZ level of theory support this conclusion; i.e., they suggest that the transition states for these reactions are unsymmetric with a long NC-C(alpha) and reasonably short C(alpha)-Cl bonds. The chlorine isotope effects suggest that these KIEs can be used to determine the substituent effects on transition state structure with the KIE decreasing when a more electron-withdrawing para-substituent is present. This conclusion is supported by theoretical calculations. The nucleophile carbon k(11)/k(14) KIEs for these reactions, however, do not change significantly with substituent and, therefore, do not appear to be useful for determining how the NC-C(alpha) transition-state bond changes with substituent. The theoretical calculations indicate that the NC-C(alpha) bond also shortens as a more electron-withdrawing substituent is placed on the benzene ring of the substrate but that the changes in the NC-C(alpha) transition-state bond with substituent are very small and may not be measurable. The results also show that using leaving group and nucleophile carbon KIEs to determine the substituent effect on transition-state structure is more complicated than previously thought. The implication of using both chlorine leaving group and nucleophile carbon KIEs to determine the substituent effect on transition-state structure is discussed.