Electrochemical behavior and theoretical studies of arylazo (1-naphthyl-2-cyanoacetamide) derivatives as new corrosion inhibitors for Inconel 800 in chloride solution.

In this study, synthesize and insight the corrosion inhibition properties of two novel derivatives of 1-naphthyl-2-cyanoacetamide (NCDs) [2-cyano-2-((5,6-dimethyl-1H-benzo[d]imidazol-2-yl) diazenyl)-N (naphthalene-1-yl)acetamide] (NCD1) and [2-Cyano-N-(naphthalene-1-yl)-2-[(4,6-dimethyl-1H-pyrazolo [3, 4-b] pyridine-3-yl) hydrazono] acetamide] (NCD2). The characterization of the synthesized NCDs was confirmed through the utilization of Mass fragmentation analysis, 1H-NMR, and IR. The corrosion inhibition performance of NCDs as a novel and environmentally safe corrosion inhibitor has been investigated by electrochemical techniques, a chemical technique, and theoretical studies for its anti-corrosion behavior of Inconel 800 in chloride medium. In addition, the surface morphology and inhibitor adsorption on the Inconel 800 surface were confirmed utilizing SEM, EDX, FTIR, and AFM. The advantages of NCDs include their low toxicity, environmental friendliness, ease of preparation, low odor, contain (N, O, and π-Bonds), and the inhibition efficiency elevated with decreasing solution temperature as well as inhibitor dose increase, yielding increased efficiencies of 91.8% and 95.7% for NCD1 and NCD2, respectively, at the optimum concentration of 21 × 10-6 mol. L-1 and 298 K temperature. An analysis of Tafel plots reveals that NCDs adhere to a mixed and isothermal Langmuir adsorption mechanism. Density Functional Theory (DFT) and Monte Carlo (MC) simulation manifest the two compounds of NCDs can be adsorbed at the Fe (110) surface in a paralleled way, and can have a smaller energy gap (ΔE) value and exhibit higher efficiency. The experimental and theoretical findings confirm that the synthesized compounds obtained are capable of protecting the Inconel 800 from corrosion by creating an anti-corrosion coating on the surface.

Dissociation Kinetics in Quadrupole Ion Traps: Effective Temperatures under Dipolar DC Collisional Activation Conditions.

Ions stored in an electrodynamic ion trap can be forced from the center of the ion trap to regions of higher radio frequency (RF) electric fields by exposing them to a dipolar DC (DDC) potential applied across opposing electrodes. Such ions absorb power from the trapping RF field, resulting in increased ripple motion at the frequency of the trapping RF. When a bath gas is present, ions undergo energetic collisions that result in "RF-heating" sufficient to induce fragmentation. DDC is therefore a broad-band (i.e., mass-to-charge-independent) means for collisional activation in ion traps with added bath gas. Under appropriate conditions, the internal energy distribution of an ion population undergoing dissociation can be approximated with an effective temperature, Teff. In such cases, it is possible to determine thermal activation parameters, such as Arrhenius activation energies and A-factors, by measuring dissociation kinetics. In this work, the well-studied thermometer ion, protonated leucine enkephalin, was subjected to DDC activation under rapid energy exchange conditions and in two separate bath gases, N2 and Ar, to measure Teff as a function of the ratio of DDC and RF voltages. As a result, an empirically derived calibration was generated to link experimental conditions to Teff. It was also possible to quantitatively evaluate a model described by Tolmachev et al. that can be used to predict Teff. It was found that the model, which was derived under the assumption of an atomic bath gas, accurately predicts Teff when Ar was used as the bath gas but overestimates Teff when N2 was the bath gas. Adjustment of the Tolmachev et al. model for a diatomic gas resulted in an underestimate of Teff. Thus, use of an atomic gas can provide accurate activation parameters, while an empirical correction factor should be used to generate activation parameters using N2.

Assessment of Immune Cell Activation in Pemphigus.

(1) Background: Pemphigus is a blistering autoimmune disease of the skin and/or mucous membranes, characterised by the presence of specific autoantibodies directed against structural proteins of the human skin. Recent reports indicate that new haematological parameters, termed Extended Inflammation Parameters (EIP), can be used to assess the activation of immune cells during active inflammation. These include parameters assessing both neutrophil activation (NEUT-RI, NEUT-GI) and the number of activated lymphocytes (RE-LYMP). The aim of this study was to investigate the relationship between changes in NEUT-RI, NEUT-GI and RE-LYMP and the disease activity in patients with pemphigus. (2) Results: The study involved 32 patients with diagnosed different types of pemphigus. Neutrophil activation parameters (NEUT-RI and NEUT-GI) and lymphocytes (RE-LYMP) were significantly higher in these patients compared to the parameters in healthy participants (respectively p = 0.0127, p = 0.0011 and p = 0.0033). The increased quantity of activated lymphocytes (RE-LYMP) also correlated significantly with the extent of skin and/or mucosal lesions in patients assessed by the PDAI scale (p < 0.02). (3) Conclusions: The NEUT-RI, NEUT-GI and RE-LYMP parameters proved to be appropriate markers of inflammation severity in pemphigus, also in relation to local lesions, which was not possible with the inflammation markers (CRP, ESR) used so far on a routine basis.

Hydrolysis data for bis(4-cyanophenyl) phenyl phosphate including rate constants and activation parameters.

Hydrolysis data for Bis(4-cyanophenyl) phenyl phosphate (CPP), introduced as a reactive diluent for phthalonitrile monomers, under pH 4, 7 and 10 are presented. Conversion/time plots collected by HPLC analysis, typical chromatograms and NMR spectra of the substrate and the reaction products are given. Pseudo-first order rate constants are determined for CPP at 25, 50 and 80 °C. Activation parameters were calculated from Arrhenius equation.

Treatment of soil washing wastewater via adsorption of lead and zinc using graphene oxide.

In the present work, graphene oxide (GO) was synthesized via the modified Hummers method and utilized in treating real soil washing wastewater via adsorptive removal of lead (Pb) and zinc (Zn). Characterization analysis of GO was performed using X-ray diffraction, Brunauer-Emmett-Teller method, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and zeta potential analysis. The Van't Hoff, Eyring, and Arrhenius equations were applied to determine the activation and thermodynamic parameters namely activation energy (Ea), standard Gibbs energy change (ΔG°), standard enthalpy change (ΔH°), standard entropy change (ΔS°), change in activation Gibbs energy (ΔG#), change in activation enthalpy (ΔH#), and change in activation entropy (ΔS#). Based on the high coefficient of determination values (0.8882 ≥ R2 ≥ 0.9094) and low values of SSE (0.0292 ≤ SSE ≤ 0.0511) and ARE (0.8014 ≤ ARE ≤ 0.8822), equilibrium data agreed well with the Freundlich isotherm. The maximum adsorption capacity for Pb(II) and Zn(II) was determined to be 11.57 and 4.65 mg/g, respectively. Kinetic studies revealed that pseudo-second-order equation fitted well with the experimental data, which indicates that chemisorption is the rate-determining step of the adsorption system. Results have shown the possibility of GO as a potential adsorbent material in the treatment of soil washing wastewater.

The oriented processes for extraction and recovery of paracetamol compound across different affinity polymer membranes. Parameters and mechanisms.

Membrane processes represent one of the most promising technologies for separation and extraction in modern industries, because they have several advantages. Today these processes are an important research topic, including affinity polymer membranes that are highly efficient for oriented processes. Three affinity polymer membrane types containing lipophilic compounds, methyl cholate (MC) and cholic acid (CA) as extractive agents were prepared and characterized. They have been used to extract active ingredient paracetamol (acetaminophen), from concentrated solutions (0.08-0.01M). Substrate acetaminophen is an important active ingredient and its recovery as a pure compound, is very useful for the pharmaceutical industry. These affinity polymer membranes were adopted to perform experiments on a facilitated extraction process of this substrate at different medium acidities and temperatures. Macroscopic parameters, permeabilities (P) and initial fluxes (J0) for a facilitated extraction of this substrate through each membrane were determined. The results indicate that values of initial fluxes (J0) of the extracted substrate are related to its initial concentration C0 by a saturation law, which allowed to determine microscopic parameters, apparent diffusion coefficients (D*) and association constants (Kass) of formed entity (substrate - extractive agent) (ST). The results show a clear influence of temperature and acidity factors on the evolution of these parameters and membrane performances in this studied process. Activation parameters (Ea, ΔH≠, and ΔS≠) were determined and the values indicate that high performances of these membrane types are certainly related to the movement nature of the substrate across the organic phase, and the structures of the substrate and the extractive agent.

Determination of the interconversion energy barrier of three novel pentahelicene derivative enantiomers by dynamic high resolution liquid chromatography.

Dynamic high resolution liquid chromatography (DHPLC) was used to determine the kinetic and thermodynamic activation parameters of interconversion of three novel pentahelicene derivatives {3,5-bis(trifluoromethyl)benzo[i]pentahelicene, naphtho[1,2-i]pentahelicene and 4-methoxybenzo[i]pentahelicene}. DHPLC was performed on a chiral isopropyl - carbamate cyclofructan 6 (LARIHC CF6-P) column under normal phase conditions. Variation of the column temperature and flow rate was used to study the interconversion process. A computer assisted deconvolution method was employed to determine the individual peak areas and the retention times required for the calculation of apparent enantiomerization energy barriers, enthalphy and entropy of the interconvertion of above defined pentahelicene derivative enantiomers. An ab initio quantum chemistry method was used to estimate theoretical kinetic and thermodynamic interconversion parameters and to evaluate experimental data of these three novel pentahelicene derivative enantiomers.

Inert gas influence on the laminar burning velocity of methane-air mixtures.

Flame propagation was studied in methane-air-inert (He, Ar, N2 or CO2) mixtures with various initial pressures and compositions using pressure-time records obtained in a spherical vessel with central ignition. The laminar burning velocities of CH4-air and CH4-air-inert mixtures obtained from experimental p(t) records of the early stage of combustion were compared with literature data and with those obtained from numerical modeling of 1D flames. The overall reaction orders of methane oxidation were determined from the baric coefficients of the laminar burning velocities determined from power-law equations. For all mixtures, the adiabatic flames temperatures were computed, assuming that the chemical equilibrium is reached in the flame front. The overall activation energy for the propagation stage of the combustion process was determined from the temperature dependence of the laminar burning velocity.

Enzyme surface rigidity tunes the temperature dependence of catalytic rates.

The structural origin of enzyme adaptation to low temperature, allowing efficient catalysis of chemical reactions even near the freezing point of water, remains a fundamental puzzle in biocatalysis. A remarkable universal fingerprint shared by all cold-active enzymes is a reduction of the activation enthalpy accompanied by a more negative entropy, which alleviates the exponential decrease in chemical reaction rates caused by lowering of the temperature. Herein, we explore the role of protein surface mobility in determining this enthalpy-entropy balance. The effects of modifying surface rigidity in cold- and warm-active trypsins are demonstrated here by calculation of high-precision Arrhenius plots and thermodynamic activation parameters for the peptide hydrolysis reaction, using extensive computer simulations. The protein surface flexibility is systematically varied by applying positional restraints, causing the remarkable effect of turning the cold-active trypsin into a variant with mesophilic characteristics without changing the amino acid sequence. Furthermore, we show that just restraining a key surface loop causes the same effect as a point mutation in that loop between the cold- and warm-active trypsin. Importantly, changes in the activation enthalpy-entropy balance of up to 10 kcal/mol are almost perfectly balanced at room temperature, whereas they yield significantly higher rates at low temperatures for the cold-adapted enzyme.

Interacciones moleculares de las soluciones acuosas diluidas de nitrato de sodio a partir de datos viscosimétricos / Molecular interactions of aqueous sodium nitrate solutions from viscometric data / Interacções moleculares de soluções aquosas diluídas de nitrato de sódio a partir de dados viscosimétricos

Se determinaron experimentalmente los tiempos de flujo de soluciones NaNO3 + H2O en el intervalo de concentración molal 0,0000-0,9996 (mol/kg). Se usó un microviscosímetro automático Anton Paar®, modelo AMVn, a temperaturas desde 283,15 K hasta 318,15 K cada 5 K y presión atmosférica de 0,101 MPa. A partir de los datos obtenidos, se calcularon las viscosidades dinámicas (η), los coeficientes de viscosidad A , B y C de la ecuación de Jones-Dole, dB/dT y los parámetros de activación del flujo viscoso (ΔG∞‡, ΔH∞‡ y ΔS∞‡) a dilución infinita. Los coeficientes A, B y C resultaron positivos al igual que dBIdT. De acuerdo con el análisis del signo de este último, el NaNO3 actúa como un soluto formador de la estructura del agua. Por otro lado, los parámetros de activación del flujo viscoso a dilución infinita (ΔG∞‡, ΔH∞‡ y ΔS∞‡) revelaron que el proceso de flujo viscoso es endotérmico con un claro predominio de las interacciones ión-solvente.

Flow times of aqueous sodium nitrate solutions in the molal concentration interval 0.0000 to 0.9996 (mol kg) were determined by using an automatic microviscosimeter AMVn Anton Paar® at temperatures ranging from 283.15 K to 318.15 K every 5 K and atmospheric pressure of 0.101 MPa. From the data obtained, the dynamic viscosities (η), the viscosity coefficients A, B, and C form the Jones-Dole equation, dBIdT and the activation parameters of viscous flow (ΔG∞‡, ΔH∞‡ and ΔS∞‡) at infinite dilution were calculated. The coefficients A, B, and C were positive as well as dBIdT. On the one hand, according to the sign analysis, sodium nitrate acts as a structure-forming solute of the water. On the other hand, the activation viscous flow parameters at infinite dilution (ΔG∞‡, ΔH∞‡ y ΔS∞‡) revealed that the viscous flow process is endothermic with a clear ion-solvent interactions predominance.

Foram determinados experimentalmente os tempos de fluxo de soluções NaNO3 + H2O na gama de concentração molal 0,0000-0,9996 (mol/kg). Foi utilização um microviscosímetro automático Anton Paar® modelo AMVn, a temperaturas desde 283,15 K até 318,15 K cada 5 K e à pressão atmosférica de 0,101 MPa. A partir dos dados obtidos foram calculadas as viscosidades dinâmicas (η), os coeficientes de viscosidade A, B, e C da equação Jones-Dole, dB/dT e os parâmetros de ativação de fluxo viscoso (ΔG∞‡, ΔH∞‡ e ΔS∞‡) a diluição infinita. Os coeficientes A, B, e C foram positivos, como dB/dT. De acordo com a análise do signo deste último, o NaNO3 age como um soluto formador da estrutura da água. Além disso, os parâmetros de ativação do fluxo viscoso à diluição infinita (ΔG∞‡, ΔH∞‡ y ΔS∞‡) revelaram que o processo de fluxo viscoso é endotérmico com uma clara predominância de interações íon-solvente.

Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer-IgE interactions.

BACKGROUND: The IgE-binding DNA aptamer 17.4 is known to inhibit the interaction of IgE with the high-affinity IgE Fc receptor FcεRI. While this and other aptamers have been widely used and studied, there has been relatively little investigation of the kinetics and energetics of their interactions with their targets, by either single-molecule or ensemble methods. METHODS: The dissociation kinetics of the D17.4/IgE complex and the effects of temperature and ionic strength were studied using fluorescence anisotropy and single-molecule spectroscopy, and activation parameters calculated. RESULTS: The dissociation of D17.4/IgE complex showed a strong dependence on temperature and salt concentration. The koff of D17.4/IgE complex was calculated to be (2.92±0.18)×10(-3) s(-1) at 50 mM NaCl, and (1.44±0.02)×10(-2) s(-1) at 300 mM NaCl, both in 1 mM MgCl2 and 25°C. The dissociation activation energy for the D17.4/IgE complex, Ea, was 16.0±1.9 kcal mol(-1) at 50 mM NaCl and 1 mM MgCl2. Interestingly, we found that the C19A mutant of D17.4 with stabilized stem structure showed slower dissociation kinetics compared to D17.4. Single-molecule observations of surface-immobilized D17.4/IgE showed much faster dissociation kinetics, and heterogeneity not observable by ensemble techniques. CONCLUSIONS: The increasing koff value with increasing salt concentration is attributed to the electrostatic interactions between D17.4/IgE. We found that both the changes in activation enthalpy and activation entropy are insignificant with increasing NaCl concentration. The slower dissociation of the mutant C19A/IgE complex is likely due to the enhanced stability of the aptamer. GENERAL SIGNIFICANCE: The activation parameters obtained by applying transition state analysis to kinetic data can provide details on mechanisms of molecular recognition and have applications in drug design. Single-molecule dissociation kinetics showed greater kinetic complexity than was observed in the ensemble in-solution systems, potentially reflecting conformational heterogeneity of the aptamer. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Propiedades viscosimétricas de la DL-alanina en soluciones acuosas de trifluorometanosulfonato de 1-butil-3-metil imidazolio a diferentes temperaturas / Viscometric properties of DL-alanine in aqueous solutions of 1-Butyl-3-methyl imidazolium trifluoromethanesulfonic acid at different temperatures / Propriedades viscosimétricas da DL-alanina em soluções aquosas de trifluorometanossulfonato de 1-butil-3-metil imidazólio a diferentes temperaturas

Se determinaron los tiempos de flujo de la DL-alanina en soluciones acuosas del líquido iónico trifluorometanosulfonato de 1-Butil-3-metil imidazolio (0,1000-1,0000 mol/Kg) usando un viscosímetro Anton Paar® modelo AMVn a temperaturas de 283,15; 288,15; 293,15; 298,15; 303,15; 308,1; 313,15 y 318,15 K y 0,10 MPa. A partir de los datos obtenidos se calcularon las viscosidades absolutas, los coeficientes B de viscosidad, la pendiente y los parámetros de activación del flujo viscoso del estado de dilución infinita. Los valores obtenidos para estos parámetros fueron discutidos en términos de las interacciones presentes en solución.

Flow times of DL-alanine in aqueous solutions of ionic liquid 1-butyl-3-methyl imidazolium trifluoromethanesulfonate (0.1000-1.0000 mol/Kg) were determined, using a viscometer model Anton Paar® AMVn at 283.15, 288.15, 293.15, 298.15, 303.15, 308.1, 313.15 and 318.15 K and 0.10 MPa. From the data obtained the absolute viscosities, viscosity coefficients B, the slope and the activation parameters of viscous flow at state of infinite dilution were calculated. The values obtained for these parameters were discussed in terms of the interactions present in solution.

Foram determinados os tempos de fluxo da DL-alanina em soluções aquosas do líquido iónico trifluorometanossulfonato de 1-butil-3-metil imidazólio (0,100-1,000 mol/Kg) utilizando um viscosímetro Anton Paar® de modelo AMVn a temperaturas de 283,15; 288,15; 293,15; 298,15; 303,15; 308,1, 313,15 e 318,15 K e 0,10 MPa. A partir dos dados obtidos foram calculadas as viscosidades absolutas, os coeficientes de viscosidade B, o gradiente e os parâmetros de ativação de fluxo viscoso no estado de diluição infinita. Os valores obtidos para estes parâmetros são discutidos em termos das interações presentes na solução.

Qgui: A high-throughput interface for automated setup and analysis of free energy calculations and empirical valence bond simulations in biological systems.

Structural information and activity data has increased rapidly for many protein targets during the last decades. In this paper, we present a high-throughput interface (Qgui) for automated free energy and empirical valence bond (EVB) calculations that use molecular dynamics (MD) simulations for conformational sampling. Applications to ligand binding using both the linear interaction energy (LIE) method and the free energy perturbation (FEP) technique are given using the estrogen receptor (ERα) as a model system. Examples of free energy profiles obtained using the EVB method for the rate-limiting step of the enzymatic reaction catalyzed by trypsin are also shown. In addition, we present calculation of high-precision Arrhenius plots to obtain the thermodynamic activation enthalpy and entropy with Qgui from running a large number of EVB simulations.

Dividing a complex reaction involving a hypervalent iodine reagent into three limiting mechanisms by ab initio molecular dynamics.

The electrophilic N-trifluoromethylation of MeCN with a hypervalent iodine reagent to form a nitrilium ion, that is rapidly trapped by an azole nucleophile, is thought to occur via reductive elimination (RE). A recent study showed that, depending on the solvent representation, the S(N)2 is favoured to a different extent over the RE. However, there is a discriminative solvent effect present, which calls for a statistical mechanics approach to fully account for the entropic contributions. In this study, we perform metadynamic simulations for two trifluoromethylation reactions (with N- and S-nucleophiles), showing that the RE mechanism is always favoured in MeCN solution. These computations also indicate that a radical mechanism (single electron transfer) may play an important role. The computational protocol based on accelerated molecular dynamics for the exploration of the free energy surface is transferable and will be applied to similar reactions to investigate other electrophiles on the reagent. Based on the activation parameters determined, this approach also gives insight into the mechanistic details of the trifluoromethylation and shows that these commonly known mechanisms mark the limits within which the reaction proceeds.

Evaluating the thermal vinylcyclopropane rearrangement (VCPR) as a practical method for the synthesis of difluorinated cyclopentenes: experimental and computational studies of rearrangement stereospecificity.

Vinyl cyclopropane rearrangement (VCPR) has been utilised to synthesise a difluorinated cyclopentene stereospecifically and under mild thermal conditions. Difluorocyclopropanation chemistry afforded ethyl 3-(1'(2'2'-difluoro-3'-phenyl)cyclopropyl) propenoate as all four stereoisomers (18a, 18b, 22a, 22b) (all racemic). The trans-E isomer (18a), prepared in 70 % yield over three steps, underwent near quantitative VCPR to difluorocyclopentene 23 (99 %). Rearrangements were monitored by (19) F NMR (100-180 °C). While cis/trans cyclopropane stereoisomerisation was facile, favouring trans-isomers by a modest margin, no E/Z alkene isomerisation was observed even at higher temperatures. Neither cis nor trans Z-alkenoates underwent VCPR, even up to much higher temperatures (180 °C). The cis-cyclopropanes underwent [3,3]-rearrangement to afford benzocycloheptadiene species. The reaction stereospecificity was explored by using electronic structure calculations, and UB3LYP/6-31G* methodology allowed the energy barriers for cyclopropane stereoisomerisation, diastereoisomeric VCPR and [3,3]-rearrangement to be ranked in agreement with experiment.

On the reaction mechanism of the complete intermolecular O2 transfer between mononuclear nickel and manganese complexes with macrocyclic ligands.

The recently described intermolecular O2 transfer between the side-on Ni-O2 complex [(12-TMC)Ni-O2](+) and the manganese complex [(14-TMC)Mn](2+), where 12-TMC and 14-TMC are 12- and 14-membered macrocyclic ligands, 12-TMC=1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane and 14-TMC=1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, is studied by means of DFT methods. B3LYP calculations including long-range corrections and solvent effects are performed to elucidate the mechanism. The potential energy surfaces (PESs) compatible with different electronic states of the reactants have been analyzed. The calculations confirm a two-step reaction, with a first rate-determining bimolecular step and predict the exothermic character of the global process. The relative stability of the products and the reverse barrier are in line with the fact that no reverse reaction is experimentally observed. An intermediate with a µ-η(1):η(1)-O2 coordination and two transition states are identified on the triplet PES, slightly below the corresponding stationary points of the quintet PES, suggesting an intersystem crossing before the first transition state. The calculated activation parameters and the relative energies of the two transition sates and the products are in very good agreement with the experimental data. The calculations suggest that a superoxide anion is transferred during the reaction.

Nanostructured biocompatible thermal/electrical stimuli-responsive biopolymer-doped polypyrrole for controlled release of chlorpromazine: kinetics studies.

Biocompatible nanostructured conductive heparin-doped polypyrrole film was fabricated and employed as a high-capacity cation exchanger for programmable release of neuroleptic drug, chlorpromazine (CPZ) with thermally and electrical dual-stimulation. Releasing behavior were studied at different applied potentials and temperatures by in-situ monitoring of UV absorbance measurements. Three mathematical models (Higuchi, Power, and Avrami equation) were employed to investigate kinetics of the release. Based on the obtained results, the Avrami model found to be more comprehensive than two other ones for mathematical description of electro-stimulated release of CPZ. A quantitative relationship between activation energy parameters (Ea, ΔG(≠), ΔH(≠), and ΔS(≠)) and release conditions (applied potential and temperature) has been developed and established to predict release rate constants at various applied conditions.

Temperature dependence of binding and catalysis for human serum arylesterase/paraoxonase.

The influence of temperature upon the hydrolysis of phenyl acetate, catalysed by purified human serum arylesterase/paraoxonase (E. C. 3.1.8.1), was studied in the temperature range 10 °C-40 °C by spectrophotometry in TRIS buffer, pH 8.0, using both initial rate analysis and progress curve analysis. The kinetic parameters (catalytic constant k(cat); Michaelis constant K(m); product inhibition constant K(p)) were determined by nonlinear regression. All parameters increased with temperature, but the ratios k(cat)/K(m) and K(p)/K(m) remained practically constant. Binding of both substrate and reaction product (phenol) was exothermic. A negative entropic term accounted for about 50% of the enthalpy change for both the binding and catalytic steps. Thermodynamic analysis suggested that: (1) the rate-limiting step is the nucleophilic attack of the carbonyl group of the substrate by a water molecule, (2) the active site is preorganized with no induced fit, (3) the enzyme-bound calcium plays an important role in stabilizing both the substrate and the transition state. The practical implications of these results are discussed.

Application of cinchona-sulfonate-based chiral zwitterionic ion exchangers for the separation of proline-containing dipeptide rotamers and determination of on-column isomerization parameters from dynamic elution profiles.

The interconversion of cis and trans isomers of dipeptides containing C-terminal proline was studied by dynamic chromatography on zwitterionic chiral stationary phases at temperatures ranging from -15°C to +45°C The cis-trans isomers could be separated below 0°C and above 0-10°C plateau formation and peak coalescence phenomena occurred, which is characteristic for a dynamic process at the time-scale of partitioning. At and above room temperature, full coalescence was observed, which allowed separations of enantiomers without interference from interconversion effects. Analysis of the dynamic elution profiles of the interconverting peptides allowed the determination of isomerization rate constants and thermodynamic activation parameters (isomerization enthalpy, entropy and activation energy). In accordance with established results, isomerization rates and thermodynamic parameters were found to depend on the nature of the N-terminal amino acid. Isomerization barriers were only slightly lower than values determined with other methods but significant differences in the relative contributions of the activation enthalpy and entropy as well as isomerization rates pointed toward selector-moderated isomerization dynamics.

Mechanism of the dehydrogenase reaction of DmpFG and analysis of inter-subunit channeling efficiency and thermodynamic parameters in the overall reaction.

The bifunctional, microbial enzyme DmpFG is comprised of two subunits: the aldolase, DmpG, and the dehydrogenase, DmpF. DmpFG is of interest due to its ability to channel substrates between the two spatially distinct active sites. While the aldolase is well studied, significantly less is known about the dehydrogenase. Steady-state kinetic measurements of the reverse reaction of DmpF confirmed that the dehydrogenase uses a ping-pong mechanism, with substrate inhibition by acetyl CoA indicating that NAD(+)/NADH and CoA/acetyl CoA bind to the same site in DmpF. The Km of DmpF for exogenous acetaldehyde as a substrate was 23.7 mM, demonstrating the necessity for the channel to deliver acetaldehyde directly from the aldolase to the dehydrogenase active site. A channeling assay on the bifunctional enzyme gave an efficiency of 93% indicating that less than 10% of the toxic acetaldehyde leaks out of the channel into the bulk media, prior to reaching the dehydrogenase active site. The thermodynamic activation parameters of the reactions catalyzed by the aldolase, the dehydrogenase and the DmpFG complex were determined. The Gibb's free energy of activation for the dehydrogenase reaction was lower than that obtained for the full DmpFG reaction, in agreement with the high kcat obtained for the dehydrogenase reaction in isolation. Furthermore, although both the DmpF and DmpG reactions occur with small, favorable entropies of activation, the full DmpFG reaction occurs with a negative entropy of activation. This supports the concept of allosteric structural communication between the two enzymes to coordinate their activities.