Metabolite Pools and Metabolic Branching as Factors of in-vivo Isotope Discriminations by Kinetic Isotope Effects

Abstract Inter- and intra-molecular non-statistical isotope distributions do not only require the existence of a kinetic isotope effect on a defined enzyme catalyzed reaction, but also the prerequisite that this reaction is located at a metabolic branching point. Furthermore a metabolic and isotopic balance demand that the extent of the isotopic shift is reciprocal to the products' yields. On this base the (13)C-enrichment of L-ascorbic acid in position C-1 and the depletion of glycerol in C-1 are interpreted. The (13)C-pattern of natural malic acid is discussed as a consequence of isotope effects on the carboxylation of pyruvate and PEP and on the pyruvate dehydrogenase reaction. The patterns of natural products synthezised by transfer of "active acetaldehyde" is proposed to be due to an isotope effect on the thiamine pyrophosphate containing lyase reaction. An isotope effect on the reduction of "active formaldehyde" to "active methyl" and the existence of corresponding pools is responsible for (13)C-enrichments and depletions of natural products in positions bearing these intermediates. Finally a model for the main nitrogen pools and for isotope discriminations between α-amino, ω-amino-N and amide pools in plants is proposed.     

Metabolite Pools and Metabolic Branching as Factors of in-vivo Isotope Discriminations by Kinetic Isotope Effects

Abstract

Inter- and intra-molecular non-statistical isotope distributions do not only require the existence of a kinetic isotope effect on a defined enzyme catalyzed reaction, but also the prerequisite that this reaction is located at a metabolic branching point. Furthermore a metabolic and isotopic balance demand that the extent of the isotopic shift is reciprocal to the products' yields. On this base the ¹³C-enrichment of L-ascorbic acid in position C-1 and the depletion of glycerol in C-1 are interpreted. The ¹³C-pattern of natural malic acid is discussed as a consequence of isotope effects on the carboxylation of pyruvate and PEP and on the pyruvate dehydrogenase reaction. The patterns of natural products synthezised by transfer of “active acetaldehyde” is proposed to be due to an isotope effect on the thiamine pyrophosphate containing lyase reaction. An isotope effect on the reduction of “active formaldehyde” to “active methyl” and the existence of corresponding pools is responsible for ¹³C-enrichments and depletions of natural products in positions bearing these intermediates. Finally a model for the main nitrogen pools and for isotope discriminations between α-amino, ω-amino-N and amide pools in plants is proposed.     

Mercury Isotopes in Earth and Environmental Chemistry

In Earth and environmental chemistry magnetic isotopes are the universal means to identify reaction mechanisms. Mass-independent fractionation of isotopes as a signature of mechanism occurs by two ways: first, via magnetic isotope effect (MIE), which is controlled by magnetic, or hyperfine, coupling between unpaired electrons and magnetic nuclei in paramagnetic species (in radicals, particularly), and, second, via nuclear volume effect (NVE), which is induced by the difference in volumes of isotopic nuclei. MIE is the dependence of the reaction rates on the nuclear magnetic moment of reactants and fractionates magnetic and nonmagnetic isotopes; NVE fractionates isotopes with different nuclear volumes. Both effects, MIE and NVE, are supposed to coexist in condensed phases. Decisive test for their differentiation is illustrated by example of radical pairs with mercury nuclei: if isotope fractionation is controlled by MIE, the ratio Δ²⁰¹Hg/Δ¹⁹⁹Hg is expected to be in the limits 1.05–1.25 for isotopic enrichment and 0.80–0.92 for impoverishment. If isotope fractionation is controlled by NVE, this ratio is estimated to be in the range 0.50–0.62. In contrast to MIE-induced two-directional fractionation, which is controlled by direction of coherent spin conversion of radical pair (triplet–singlet or vice versa), the NVE induces one-directional, universal isotope fractionation, almost independent on the reaction mechanism.     

The reversibility of dissimilatory sulphate reduction and the cell-internal multi-step reduction of sulphite to sulphide: insights from the oxygen isotope composition of sulphate

Dissimilatory sulphate reduction (DSR) leads to an overprint of the oxygen isotope composition of sulphate by the oxygen isotope composition of water. This overprint is assumed to occur via cell-internally formed sulphuroxy intermediates in the sulphate reduction pathway. Unlike sulphate, the sulphuroxy intermediates can readily exchange oxygen isotopes with water. Subsequent to the oxygen isotope exchange, these intermediates, e.g. sulphite, are re-oxidised by reversible enzymatic reactions to sulphate, thereby incorporating the oxygen used for the re-oxidation of the sulphur intermediates. Consequently, the rate and expression of DSR-mediated oxygen isotope exchange between sulphate and water depend not only on the oxygen isotope exchange between sulphuroxy intermediates and water, but also on cell-internal forward and backward reactions. The latter are the very same processes that control the extent of sulphur isotope fractionation expressed by DSR. Recently, the measurement of multiple sulphur isotope fractionation has successfully been applied to obtain information on the reversibility of individual enzymatically catalysed steps in DSR. Similarly, the oxygen isotope signature of sulphate has the potential to reveal complementary information on the reversibility of DSR. The aim of this work is to assess this potential. We derived a mathematical model that links sulphur and oxygen isotope effects by DSR, assuming that oxygen isotope effects observed in the oxygen isotopic composition of ambient sulphate are controlled by the oxygen isotope exchange between sulphite and water and the successive cell-internal oxidation of sulphite back to sulphate. Our model predicts rapid DSR-mediated oxygen isotope exchange for cases where the sulphur isotope fractionation is large and slow exchange for cases where the sulphur isotope fractionation is small. Our model also demonstrates that different DSR-mediated oxygen isotope equilibrium values are observed, depending on the importance of oxygen isotope exchange between sulphite and water relative to the re-oxidation of sulphite. Comparison of model results to experimental data further leads to the conclusion that sulphur isotope fractionation in the reduction of sulphite to sulphide is not a single-step process.     

Fluctuations of fragment observables

This contribution presents a review of our present theoretical as well as experimental knowledge of different fluctuation observables relevant to nuclear multifragmentation. The possible connection between the presence of a fluctuation peak and the occurrence of a phase transition or a critical phenomenon is critically analyzed. Many different phenomena can lead both to the creation and to the suppression of a fluctuation peak. In particular, the role of constraints due to conservation laws and to data sorting is shown to be essential. From the experimental point of view, a comparison of the available fragmentation data reveals that there is a good agreement between different data sets of basic fluctuation observables, if the fragmenting source is of comparable size. This compatibility suggests that the fragmentation process is largely independent of the reaction mechanism (central vs. peripheral collisions, symmetric vs. asymmetric systems, light ions vs. heavy-ion-induced reactions). Configurational energy fluctuations, that may give important information on the heat capacity of the fragmenting system at the freeze-out stage, are not fully compatible among different data sets and require further analysis to properly account for Coulomb effects and secondary decays. Some basic theoretical questions, concerning the interplay between the dynamics of the collision and the fragmentation process, and the cluster definition in dense and hot media, are still open and are addressed at the end of the paper. A comparison with realistic models and/or a quantitative analysis of the fluctuation properties will be needed to clarify in the next future the nature of the transition observed from compound nucleus evaporation to multi-fragment production.     

Kinetic isotope studies of the gas-phase reaction (H,D,Mu)+HBr→(H,D,Mu)H+Br

Reaction rate constants and kinetic isotope effects for the gas-phase reaction A + HBrAH+Br, where A can be H, D, or Mu, have been studied using variational transition state theory with semiclassical tunneling on two semiempirical potential energy surfaces and a newab initio surface. The rate constants and kinetic isotope effects are compared to experimental results. This comparison is used to test the potential energy surfaces, to determine which regions of the potential energy surface control the reaction rates and the kinetic isotope effects, and to learn whether or not any of the potential energy surfaces is accurate for a wide enough range of features to predict both the reaction rate constants and the kinetic isotope effects for this chemical system.     

New trends in baro-enzymology

Abstract

The recent interest on high pressure biotechnology (mainly in food industry) requires fundamental studies on the pressure behavior of biochemical constituants¹ In this laboratory, we use pressure as a thermodynamic parameter, such as temperature, to determine the energetic quantities of enzyme reactions². Two essential requirements for the study of the mechanism of enzyme action are that, first, a simple rate constant has to be measured (determination of a composite rate constant, such as kcat, can lead to ambiguous results) and, second, a maximum number of physico-chemical ways must be used to perturb the system under study. To assess this simple usually very rapid rate constant, cryoenzymology was used. It was then possible to probe, at constant pressure, the thermodynamics of the interconversion of two successive intermediates, thereby obtaining the classical ΔG? ΔS? and ΔH? parameters. Ify however, we can also vary another intensive parameter, namely the pressure, it is possible to determine the activation volume (ΔV?) of the reaction. In addition to pressure and temperature, a third variable has to be considered also : the nature of the cryosolvent which allows experiments to be extended to subzero temperatures in the first place (with the medium being kept fluid) but which can also act as a perturbing agent thereby inducing controlled and reversible changes in equilibrium and me processes. The interdependence of the two variables, namely temperature and pressure, which is predicted by the general equation for the standard variation of free energy ΔG = f(T, P), is presented. This approach will be illustrated using different model reactions where data are analysed according to the classical transition state theory.     

Hydrogen absorption mechanism and location in intermetallic compounds

Conclusions The purpose of this review is to acquaint the reader with the new field of ternary hydrides and to expose him to its relevance to modern energy technology. Studying their fundamental properties is one fascinating aspect in view of their novel and unusual nature. The methods of hyperfine interactions have an important role to play in their microscopic understanding, as we have illustrated through a few examples. The absorption mechanism of hydrogen has been shown to be closely related to the nature of surface overlayers and their catalytic activity. Similar measurements will be of importance also in many of the other catalytic reactions used in industry. The microscopic nature of hyperfine interaction tools also helps in determining the hydrogen site occupancy. In addition, such methods can in principle yield dynamic information which are useful in investigating relevant properties such as diffusion rates of hydrogen.Work supported by the U. S. Department of Energy     

Interaction of therapeutic ultrasound with purified enzymes in vitro

The effects of ultrasound on the rates of the catalytic reactions of four purified enzymes in vitro have been extensively investigated under a wide range of biochemical and physical exposure conditions. In general, it can be concluded that therapeutic intensities of continuous wave 0.88 MHz ultrasound had no detectable direct effects on the rates of the reactions catalysed by creatine kinase, lactate dehydrogenase, hexokinase and pyruvate kinase. Some minor effects were noted. These were: an indirect effect resulting from mixing within the sample chamber caused by quartz wind streaming; an effect on partially-hydrated cross-linked enzyme systems which appears to be the result of increased fluid penetration of the solid matrix in the presence of ultrasound; and an increase in the rate of spontaneous dissociation of a multimeric enzyme system. It is, therefore, concluded that a direct interaction between ultrasound and the catalytic functioning of individual enzyme molecules is unlikely to be the primary step in any acousto-biological interaction, and that this primary interaction appears to be occurring at a higher level of organizational complexity.     

Simulation of dual carbon–bromine stable isotope fractionation during 1,2-dibromoethane degradation*

We performed a model-based investigation to simultaneously predict the evolution of concentration, as well as stable carbon and bromine isotope fractionation during 1,2-dibromoethane (EDB, ethylene dibromide) transformation in a closed system. The modelling approach considers bond-cleavage mechanisms during different reactions and allows evaluating dual carbon–bromine isotopic signals for chemical and biotic reactions, including aerobic and anaerobic biological transformation, dibromoelimination by Zn(0) and alkaline hydrolysis. The proposed model allowed us to accurately simulate the evolution of concentrations and isotope data observed in a previous laboratory study and to successfully identify different reaction pathways. Furthermore, we illustrated the model capabilities in degradation scenarios involving complex reaction systems. Specifically, we examined (i) the case of sequential multistep transformation of EDB and the isotopic evolution of the parent compound, the intermediate and the reaction product and (ii) the case of parallel competing abiotic pathways of EDB transformation in alkaline solution.     

Carbon and secondary deuterium kinetic isotope effects on SN2 methyl transfer reactions

Carbon and secondary deuterium kinetic isotope effect (KIE) for three types of S_N2 methyl transfer reactions have been predicted theoretically at a DFT level in gas phase and in aqueous solution modelled by the PCM continuum solvent model. No correlation between these isotope effects and geometrical features of the corresponding transition states (TSs), force constants of the imaginary frequency or Gibbs free energies of the studied reactions has been found. These findings suggest that comparative analysis of the magnitudes of the studied KIEs should be constrained to interpretation of the TS localization on the reaction coordinate only in a series of very closely related reactions. Copyright © 2007 John Wiley & Sons, Ltd.     

采用量子波包方法和准经典轨线方法研究S(~3P)+HD反应

利用量子波包方法和准经典轨线方法在一个新的3A′′势能面上研究了S(3P)+HD→SD+H和SH+D反应的动力学性质.计算得到了两个反应通道在碰撞能为0.8—2.2 eV范围内的J=0反应几率、积分反应截面、内同位素因子和产物转动取向因子.这些结果揭示了S(3P)+HD反应非常明显的内同位素效应.通过对势能面和反应轨线的分析,我们发现了SD+H反应通道一个新的反应机制.S(3P)+HD反应的内同位素效应可以利用新发现的反应机制和反应的质量组合来进行解释.     

Molecular dynamics study of reaction kinetics in viscous media

Predicted by stochastic models and observed experimentally in a number of isomerization reactions, viscosity-induced solvent effects manifest themselves in a significant departure of the reaction rates from the values expected on the basis of transition state theory. These effects are well understood within the framework of stochastic models; however, the predictive power of such models is limited by the fact that their parameters are not readily available. Experiment and molecular dynamics (MD) simulations can provide such information and can serve as the testing grounds for various stochastic models. In real solvents, a change in viscosity is inevitably associated with variation of at least one of the three factors – temperature, pressure, or solvent identity, resulting in different solvent–solvent and solvent–solute interactions. A model is proposed in which solvent viscosity is manipulated through mass scaling, which allows one to maintain other factors constant for a series of viscosities. This approach was tested on MD simulations of the kinetics of two model isomerization reactions in Lennard–Jones solvents, whose viscosity was varied over three orders of magnitude. The results reproduce the Kramers turnover and a strong negative viscosity dependence of the reaction rates in the high viscosity limit, somewhat weaker than η ^?1.     

超高速分子摄影术——飞秒泵浦-探测方法在分子超快动力学研究中的应用

飞秒泵浦-探测技术是一种可以在原子运动时间尺度上实时观测化学反应的有力手段.利用飞秒泵浦-探测技术,可以实时观测化学反应过渡态,在分子层次上了解基元化学反应的过程和机理,从而深入地了解化学反应的本质和历程.文章介绍了飞秒泵浦-探测技术的基本原理以及在分子超快动力学研究中的应用,并结合作者所在研究小组的工作,展示了基于该技术的几种典型的飞秒光谱方法在分子超快动力学研究领域的主要成果.最后,展望了该技术的发展前景和方向.     

Evaluation of reduced combustion kinetic mechanisms using global sensitivity-based similarity analysis (GSSA)

Reduced combustion kinetic mechanisms, instead of detailed ones, are often used in computational fluid dynamics (CFD) simulations for reduced and frequently even affordable computational cost. The criterion for the evaluation of a reduced mechanism usually focuses on its prediction error for the global properties such as the ignition delay time, while ignoring the detailed features of reaction kinetics such as reaction pathways. In our opinion, good reduced mechanisms should have similar predicting behaviors as the detailed ones, and these behaviors include model predictions for specific targets, prediction error bars, and uncertainty sources for the errors. In this work, a new approach using global sensitivity-based similarity analysis (GSSA) is proposed to compare reduced mechanisms with detailed ones. The similarity coefficient for the reduced mechanism is calculated by similarity method based on Euclidean distance between sensitivity indices of the reduced mechanism and those of the detailed mechanism. The larger the similarity coefficient, the higher the degree of similarity between the reduced and detailed mechanisms. To demonstrate this similarity method, directed relation graph with error propagation (DRGEP) is employed to simplify both the GRI 3.0 mechanism without the NOx chemistry and the JetSurF mechanism consisting of 1459 reactions, resulting in reduced mechanisms with different sizes which can accurately predict the ignition delay times for corresponding fuel mixtures. Similarity analysis is then employed to evaluate these reduced mechanisms. The result shows that the actual reaction kinetic features cannot be replicated by some of the reduced mechanisms. First, the rankings of the important reactions obtained by reduced mechanisms are not consistent with those obtained by the detailed mechanism. Second, by investigating the sensitive reactions, the actual impact of uncertainties in reaction rates on the ignition delay times cannot be presented by reduced mechanisms. The similarity analysis on reduced mechanisms can be used to select a reduced mechanism which shows much better performance to replicate the actual combustion reaction kinetics. GSSA can provide information on the uncertainty sources induced by the reactions parameters of reduced mechanisms for target predictions, which is important for further reduced model optimization and for the sensitivity analysis of CFD simulations.     

功率超声对酶促反应的影响

本文评述了功率超声在水溶液和有机中对酶促反应的影响,对固定化酶的影响,同时探讨了功率超声影响酶促反应的可能机理,并并评价功能超声作为一种工业生物化学反应促进手段的可能性。     

Probing isotope effects in chemical reactions using single ions

Isotope effects in reactions between Mg+ in the 3p{2}P{3/2} excited state and molecular hydrogen at thermal energies are studied through single reaction events. From only approximately 250 reactions with HD, the branching ratio between formation of MgD+ and MgH+ is found to be larger than 5. From an additional 65 reactions with H2 and D2 we find that the overall fragmentation probability of the intermediate MgH2+, MgHD+, or MgD2+ complexes is the same. Our study shows that few single ion reactions can provide quantitative information on ion-neutral reactions. Hence, the method is well suited for reaction studies involving rare species, e.g., rare isotopes or short-lived unstable elements.     

An investigation of lipase catalysed sonochemical synthesis: A review

Ultrasonic irradiation has recently gained attention of researchers for its process intensification in numerous reactions. Earlier ultrasound was known for its application either to deactivate enzyme activity or to disrupt the cell. However, in recent years, practice of ultrasonic irradiation began to emerge as a tool for the activation of the enzymes under mild frequency conditions. The incorporation of ultrasound in any of enzymatic reactions not only increases yield but also accelerates the rate of reaction in the presence of mild conditions with better yield and less side-products. To attain maximum yield, it is crucial to understand the mechanism and effect of sonication on reaction especially for the lipase enzyme. Thus, the influence of ultrasound irradiation on reaction yield for different parameters including temperature, enzyme concentration, mole ratio of substrates, solvents ultrasonic frequency and power was reviewed and discussed. The physical effect of cavitation determined by bubble dynamics and rate of reaction through kinetic modelling also needs to be assessed for complete investigation and scale up of synthesis. Thus, prudish utilisation of ultrasound for enzymatic synthesis can serve better future for sustainable and green chemistry.