Derived Rate Constants Research Articles

Coarsening transitional kinetics of γ′ precipitates in a nickel-based single crystal superalloy during thermal exposure

The microstructural stability and coarsening kinetics of γ′ precipitates in a nickel-based single crystal superalloy (Ni-SX) were thoroughly investigated under various thermal exposure treatments. The evolutions of characteristic parameters including γ′ size, morphology, area fraction, and γ channel width were documented. Coarsening rate constants and particle size distributions (PSDs) were calculated and recorded based on measured precipitate sizes at temperatures ranging from 800 ℃ to 1100 ℃ and durations ranging from 100 h to 1010 h. The experimentally derived rate constants were compared with the calculated theoretical values from two mainstream models: matrix-controlled diffusion (LSW model) and interface-controlled diffusion (TIDC model). Notably, transitional coarsening kinetics of γ′ precipitates from the LSW model to the TIDC model was observed at high temperatures over time. Through a comprehensive consideration of key coarsening kinetics parameters such as elemental diffusivity, trans-interface diffusion coefficient, coarsening rate constant, γ/γ′ interfacial area, and solute mass transfer, the transformation of the coarsening kinetics from matrix-controlled diffusion to interface-controlled diffusion was quantitatively discussed. The emergence of the TIDC model as the dominant mechanism was attributed to relatively smaller solute mass transfer through the interface. Moreover, the degradation of γ/γ′ interfacial energy and interfacial area, coupled with the increase in solute diffusion coefficient in the matrix, facilitated the trans-interface diffusion to act as a limiting factor throughout the coarsening process.

Parameter estimation and identifiability analysis for a bivalent analyte model of monoclonal antibody-antigen binding

Surface plasmon resonance (SPR) is an extensively used technique to characterize antigen-antibody interactions. Affinity measurements by SPR typically involve testing the binding of antigen in solution to monoclonal antibodies (mAbs) immobilized on a chip and fitting the kinetics data using 1:1 Langmuir binding model to derive rate constants. However, when it is necessary to immobilize antigens instead of the mAbs, a bivalent analyte (1:2) binding model is required for kinetics analysis. This model is lacking in data analysis packages associated with high throughput SPR instruments and the packages containing this model do not explore multiple local minima and parameter identifiability issues that are common in non-linear optimization. Therefore, we developed a method to use a system of ordinary differential equations for analyzing 1:2 binding kinetics data. Salient features of this method include a grid search on parameter initialization and a profile likelihood approach to determine parameter identifiability. Using this method we found a non-identifiable parameter in data set collected under the standard experimental design. A simulation-guided improved experimental design led to reliable estimation of all rate constants. The method and approach developed here for analyzing 1:2 binding kinetics data will be valuable for expeditious therapeutic antibody discovery research.

Investigation of Methylcyclopentadiene Reactivity: Abstraction Reactions and Methylcyclopentadienyl Radical Unimolecular Decomposition.

Understanding the reactivities of methylcyclopentadiene and the methylcyclopentadienyl radical is important in order to improve our comprehension of the chemical kinetics leading to the formation, decomposition, and growth of the first aromatic ring, as it has been shown that five-membered-ring species are important intermediates in the reaction kinetics of aromatic species. In this work, the rate constants of some key H-abstraction reactions from methylcyclopentadiene to produce the methylcyclopentadienyl radical and the formation of fulvene and benzene from the latter are theoretically determined. Rate constants are evaluated using the ab initio transition state theory-based master equation approach, determining structures and Hessians of all stationary points at the ωB97X-D/aug-cc-pVTZ level, energies at the CCSD(T) level extrapolated to the complete basis set limit, RRKM rate constants using conventional and variational transition state theory, and phenomenological rate constants through the solution of the one-dimensional master equation. Variational corrections are determined in both internal and Cartesian coordinates, and it is found that the choice of the coordinate system can impact the accuracy of the calculated rate constants by up to a factor of 4 for H-abstraction reactions and 2 for the unimolecular decomposition of the methylcyclopentadienyl radical. The calculated rate constants are in good agreement with the available literature data. Prompt dissociation of methylcyclopentadienyl radicals accessed following H-abstraction from methylcyclopentadiene was also investigated, and the corresponding rate constants were determined; the results show that prompt dissociation plays a key role under combustion conditions. Finally, lumping of theoretically derived rate constants to account for methylcyclopentadiene ⇄ methylcyclopentadienyl tautomerism allowed the derivation of a simplified set of rate constants suitable to be inserted into kinetic mechanisms.

Mechanistic studies on the oxidation reaction of antitubercular drug isoniazid and its analogy hydrazides by chlorine dioxide over a wide pH range

Oxidations of the antitubercular drug isoniazid (INH) and its analogy hydrazides, nicotinic hydrazide (NH), picolinoyl hydrazide (PH), and benzohydrazide (BH), by ClO2 were investigated with a focus on the kinetics and reaction mechanisms. In buffered solutions of a wide pH range (from 0.51 to 10.35), the oxidation reactions strictly followed the second-order kinetics, and the apparent second-order rate constant kapp versus pH profiles were established at 25.0 °C and 1.0 M ionic strength. Excess ClO2 could rapidly oxidize the hydrazides to their corresponding aryl acids which were identified by mass spectrometry. For each of the hydrazides, the proposed reaction mechanism involves parallel rate-determining reactions between ClO2 and various protolysis species of the hydrazide, rendering two types of hydrazyl free radicals; the free radicals are quickly oxidized by 3 more equivalents of ClO2 to aryl acids. The derived rate constants for the rate-determining reactions revealed that for each hydrazide a huge reactivity difference existed among the protolysis species (>108 times) and that the enolate form (or its resonant structures) was extremely reactive. Two possible modes of electron transfer in the rate-determining reactions are discussed based on the observed reaction characters. In buffered solution of pH 7.0 and 25.0 °C, the measured values of kapp are 6.20×104, 7.8×103, 2.57×103, and 1.83×103 M−1 s−1 for INH, NH, PH, and BH, respectively, imparting an important data set as a reference to the water treatment. This work may open a new door for resolving water pollution caused by INH and its derivatives.

Desorption kinetics of antipsychotic drugs from sandy sediments by diffusive gradients in thin-films technique

Dynamic processes of organic contaminants in sediments can have important toxicological implications in aquatic systems. The current study used diffusive gradients in thin-films (DGT) devices in sandy sediments spiked with nine antipsychotics and in field sandy sediments. Samplers were deployed for 1 to 30 days to determine the flux of these compounds to DGT devices and the exchange rates between the porewater and sediment solid phase. The results showed a continuous removal of antipsychotics to a binding gel and induced a mobile flux from the DGT device to the adjacent sediment solution. A dynamic model, DGT-induced fluxes in soils and sediments, was used to derive rate constants of resupply of antipsychotics from solid phase to aqueous phase (response time, Tc) and distribution coefficients for labile antipsychotics. The largest labile pool was found for lamotrigine and carbamazepine in spiked sediments. Carbamazepine, clozapine, citalopram, and lamotrigine were resupplied rapidly by sediments with Tc (25–30 min). Tc values of bupropion and amitriptyline were the longest (≈5 h), which exhibited slow desorption rates in sediments. In field sediments, high resupply was found for carbamazepine and lamotrigine, which did not show higher labile pool. The Tc values were obviously higher in the filed sediments (52–171 h). Although the adsorption process is dominant for most studied antipsychotics in both spiked sediments and field sediments, the kinetic resupply of antipsychotic compounds may not be accurately estimated by laboratory-controlled incubation experiments. More studies are needed to explore the mechanisms of desorption kinetics by using in situ DGT technique in the field.

In Situ Photochemical Transformation of Hg Species and Associated Isotopic Fractionation in the Water Column of High-Altitude Lakes from the Bolivian Altiplano.

Photochemical reactions are major pathways for the removal of Hg species from aquatic ecosystems, lowering the concentration of monomethylmercury (MMHg) and its bioaccumulation in foodwebs. Here, we investigated the rates and environmental drivers of MMHg photodegradation and inorganic Hg (IHg) photoreduction in waters of two high-altitude lakes from the Bolivian Altiplano representing meso- to eutrophic conditions. We incubated three contrasting waters in situ at two depths after adding Hg-enriched isotopic species to derive rate constants. We found that transformations mostly occurred in subsurface waters exposed to UV radiation and were mainly modulated by the dissolved organic matter (DOM) level. In parallel, we incubated the same waters after the addition of low concentrations of natural MMHg and followed the stable isotope composition of the remaining Hg species by compound-specific isotope analysis allowing the determination of enrichment factors and mass-independent fractionation (MIF) slopes (Δ199Hg/Δ201Hg) during in situ MMHg photodegradation in natural waters. We found that MIF enrichment factors potentially range from -11 to -19‰ and average -14.3 ± 0.6‰ (1 SE). The MIF slope diverged depending on the DOM level, ranging from 1.24 ± 0.03 to 1.34 ± 0.02 for the low and high DOM waters, respectively, and matched the MMHg MIF slope recorded in fish from the same lake. Our in situ results thus reveal (i) a relatively similar extent of Hg isotopic fractionation during MMHg photodegradation among contrasted natural waters and compared to previous laboratory experiments and (ii) that the MMHg MIF recorded in fish is characteristic for the MMHg bonding environment. They will enable a better assessment of the extent and conditions conducive to MMHg photodegradation in aquatic ecosystems.

Modeling study of coffee extraction at different temperature and grind size conditions to better understand the cold and hot brewing process

AbstractIn this study, the extraction kinetics, based on total dissolved solid (TDS), at different temperatures (4, 23, 50, and 93°C) for different grind sizes (VMD = 139, 643, 1,450, 1,747 μm) were investigated. Coffee extraction proceeded in initial fast extraction stage followed by a significantly slower extraction stage, which correspond to the extraction from surfaces of broken cells and the extraction from intact coffee cells, respectively. Diffusion inside the coffee particle is a very slow process, so breaking the cells is a very efficient way to increase the mass transfer rate. In addition, the ultimate extraction yield increased with increasing brewing temperature and decreasing of particle size. The Weibull distribution, pseudo‐first order and pseudo‐second order model were fitted to the kinetics data, with high coefficients of determination (0.687–0.998), and low root mean square error (0.02–0.26%). Meanwhile, exponential equations were created to correlate the derived rate constants (1/α, k1, and k2) with brewing temperature and particle size to achieve the prediction of brewing extent (TDSt/TDSeqm) at different temperature‐particle size combinations.Practical applicationsThis study investigated the kinetics of coffee extraction at different grind size and temperature conditions with the purposes to better understanding the cold and hot coffee brewing process, as well as to predict the coffee extraction. The findings in this study will have practical applications in three folds: Help manufacturers of coffee brew products (ready to drink or concentrate) to better design and control their coffee extraction process. Aid manufacturers of coffee extraction equipment and coffee brewers to improve their products Provide reference information for coffee store, barista, coffee enthusiast, and consumers to brewing a better cup of coffee

Kinetics of heat-induced changes in foods: A workflow proposal

The goal of kinetic modeling is twofold: i) to increase scientific understanding of the process under study, and ii) to predict product properties in product and process design and shelf life. Reviewing food science literature shows that classical two-step kinetic analysis is most common, by first deriving rate constants for an assumed order of reaction (possibly after linearization to make linear regression possible) and then deriving Arrhenius parameters via linear regression, again after log-linearization. This two-step approach is not without problems and this article proposes an alternative general workflow on the untransformed data using nonlinear, global regression. The basic elements consist of: i) a full statistical analysis of the order of the reaction per temperature, ii) a global analysis of all data simultaneously to estimate Arrhenius parameters while characterizing a possibly varying order via multilevel modeling, iii) evaluation of the resulting model and parameters in terms of fitting and, even more importantly, predictive capacity. The proposed workflow is illustrated with a case study on thermal degradation of carnitin (described in literature as a first-order reaction). A Bayesian approach was used to obtain probability distributions of parameters rather than point estimates, but the common standard frequentist approach can also be applied. Kinetic analysis of the carnitin data for each temperature separately showed that the order varied with temperature between 0.9 and 1.6. Multilevel modeling on all data simultaneously was used to better characterize this variation along with the common Arrhenius parameters. Due to the nature of the Arrhenius equation, reparameterization and rescaling is necessary to avoid strong parameter correlation and numerical difficulties during nonlinear regression. Multilevel modeling of all data showed that the variation of the order with temperature was not that strong as suggested from the separate analyses but it did show that the global order was higher than one. The outcome of the suggested workflow was compared to that of the classical two-step kinetic analysis and showed considerable differences in Arrhenius parameters; this appeared to be due to linearization by taking logarithms of concentration data, at least for this case study. Furthermore, it is illustrated that Bayesian regression leads to better insight into behaviour of parameters and models than least-squares regression in terms of density distributions, parameter correlations and joint confidence intervals. Even more importantly, testing of predictive capacity of kinetic models can be done much more rigorously using the Bayesian approach. • Estimation of uncertainty in reaction order from kinetic data gives insight in reactions. • Multilevel modeling allows variation of reaction order per temperature. • Global regression leads to less uncertainty in model predictions. • Bayesian estimation gives more insight in regression parameters than least-squares. • Logarithmic transformation in Arrhenius regression leads to biased estimates.

Kinetic Investigation on Tetrakis(4-Sulfonatophenyl)Porphyrin J-Aggregates Formation Catalyzed by Cationic Metallo-Porphyrins.

Under mild acidic conditions, various metal derivatives of tetrakis(4-N-methylpyridinium)porphyrin (gold(III), AuT4; cobalt(III), CoT4; manganese(III), MnT4 and zinc(II), ZnT4) catalytically promote the supramolecular assembling process of the diacid 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (H2TPPS4) into J-aggregates. The aggregation kinetics have been treated according to a well-established model that involves the initial formation of a critical nucleus containing m porphyrin units, followed by autocatalytic growth, in which the rate evolves as a power of time. An analysis of the extinction time traces allows to obtain the rate constants for the auto-catalyzed pathway, kc, and the number of porphyrins involved in the initial seeding. The aggregation kinetics have been investigated at fixed H2TPPS4 concentration as a function of the added metal derivatives MT4. The derived rate constants, kc, obey a rate law that is first order in [MT4] and depend on the specific nature of the catalyst in the order AuT4 > CoT4 > MnT4 > ZnT4. Both resonance light scattering (RLS) intensity and extinction in the aggregated samples increase on increasing [MT4]. With the exception of AuT4, the final aggregated samples obtained at the highest catalyst concentration exhibit a negative Cotton effect in the J-band region, evidencing the occurrence of spontaneous symmetry breaking. The role of the nature of the metal derivative in terms of overall charge and presence of axial groups will be discussed.

Understanding the Mechanisms of Early Stage Aluminum Corrosion

Aluminum alloys are widely used in many applications due to their inherent corrosion resistance, which is attributed to the formation of a protective oxide layer. However, the strength and durability of many Al-based materials is reduced in humid environments. Consequently, detailed mechanistic understanding of corrosion reactions over time is necessary to predict the cost, safety, and performance. Most of the current knowledge of atmospheric aluminum corrosion is built on response relationships upon extensive corrosion and typically involve a first-order correlation of corrosion data with environmental conditions. However, mechanistic understanding of corrosion processes is still limited, particularly at the early stages of exposure to humid environments. In addition, the effects of electrolytes such as sodium chloride present in the environment are still not fully understood. This presentation will focus on our recent efforts to reveal the mechanistic dependence of electrochemical aluminum corrosion in humid air in the presence of NaCl. The rate of corrosion is explored through complimentary experimental and theoretical analyses relating corrosion rate to electrolyte chemistry, mass uptake and corrosion products. As an experimental platform, we use quartz crystal microbalance (QCM) electrodes coated with aluminum and sodium chloride using sputtering techniques. Structure, composition, and surface morphology of the samples were characterized by grazing-incidence X-ray diffraction, FTIR spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy, scanning transmission electron microscopy, and electron energy loss spectroscopy. The QCM provides operando data with nanogram resolution on the formation of the corrosion product layer, from which we can derive rate constants for aluminum surface reactions. We find that the reaction rate in dry air is negligible, but a dramatic acceleration is observed at 50% RH and above. In addition, this study provides additional insight into: 1) the influence of the sodium chloride film thickness on the rate and reaction products; 2) the role of surface termination and roughness on aluminum oxide/hydroxide formation; and 3) the electrochemical corrosion reaction rate acceleration as a function of temperature. Finally, based on the interplay between theory and experiment we achieved preliminary understanding of the rate-limiting steps, which we will use to develop validated computational models of early-stage (hours to several days) aluminum corrosion. Figure 1

Determination of rate constants for a thermoneutral H-abstraction reaction: Allylic hydrogen abstraction from 1,5-hexadiene by allyl radical

Thermoneutral H-abstraction reactions, which involve the abstraction by resonantly stabilized radicals from olefins to form other resonantly stabilized radicals, have recently been found to be important in high-temperature combustion. They will shift the distribution of resonantly stabilized radicals and affect the molecular weight growth kinetics, thus changing the prediction for aromatic species. The present work provides the first experimental study on a typical thermoneutral H-abstraction reaction: C3H5-A + 1,5-C6H10 = C3H6 + C6H9-A (R1). A rapid compression machine, together with a fast sampling technique, was used to study the high temperature kinetics of 1,5-hexadiene. Experimental conditions that can be used to derive the rate constant of R1 were first identified by sensitivity analysis of a kinetic model for 1,5-hexadiene combustion. The concentration of propene was found to be highly sensitive to the rate constant of R1 over the temperature range of 893–1007 K at 25 bar. By fitting the measured propene profiles, the rate constant of R1 was deduced under selected conditions. Furthermore, the concentration profiles of propene in 1,5-hexadiene pyrolysis from a flow reactor measurement by Wang et al. (Fuel 208, 779–790, 2017) and oxidation from a jet stirred reactor measurement by Vermeire et al. (Proc. Combust. Inst. 37, 1091–1098, 2019) were also used to derive the rate constant of R1 over wider temperature ranges. The rate coefficient of R1 was further calculated by ab initio transition state theory, with energies obtained at the level of DLPNO-CCSD(T)/aug-cc-pVTZ//B2PLYP/cc-pVTZ. The theoretical predictions were found to be in good agreement with the experimentally derived rate constants. The final rate coefficient determined is: kR1=15.058×T3.425exp(−14492.073/RT)cm3mol−1s−1(500−2000K), with an estimated uncertainty of a factor of 1.5.

Photophysical Properties and Electronic Structure of Zinc(II) Porphyrins Bearing 0-4 meso-Phenyl Substituents: Zinc Porphine to Zinc Tetraphenylporphyrin (ZnTPP).

Six zinc(II) porphyrins bearing 0-4 meso-phenyl substituents have been examined spectroscopically and theoretically. Comparisons with previously examined free base analogues afford a deep understanding of the electronic and photophysical effects of systematic addition of phenyl groups in porphyrins containing a central zinc(II) ion versus two hydrogen atoms. Trends in the wavelengths and relative intensities of the absorption bands are generally consistent with predictions from time-dependent density functional theory calculations and simulations from Gouterman's four-orbital model. These trends derive from a preferential effect of the meso-phenyl groups to raise the energy of the highest occupied molecular orbital. The calculations reveal additional insights, such as a progressive increase in oscillator strength in the violet-red (B-Q) absorption manifold with increasing number of phenyls. Progressive addition of 0-4 phenyl substituents to the zinc porphyrins in O2-free toluene engenders a reduction in the measured lifetime of the lowest singlet excited state (2.5-2.1 ns), an increase in the S1 → S0 fluorescence yield (0.022-0.030), a decrease in the yield of S1 → T1 intersystem crossing (0.93-0.88), and an increase in the yield of S1 → S0 internal conversion (0.048-0.090). The derived rate constants for S1 decay reveal significant differences in the photophysical properties of the zinc chelates versus free base forms. The unexpected finding of a larger rate constant for internal conversion for zinc chelates versus free bases is particularly exemplary. Collectively, the findings afford fundamental insights into the photophysical properties and electronic structure of meso-phenylporphyrins, which are widely used as benchmarks for tetrapyrrole-based architectures in solar energy and life sciences research.

Uranium Bioaccumulation Dynamics in the Mayfly Neocloeon triangulifer and Application to Site-Specific Prediction.

Little is known about the underlying mechanisms governing the bioaccumulation of uranium (U) in aquatic insects. We experimentally parameterized conditional rate constants for aqueous U uptake, dietary U uptake, and U elimination for the aquatic baetid mayfly Neocloeon triangulifer. Results showed that this species accumulates U from both the surrounding water and diet, with waterborne uptake prevailing. Elevated dietary U concentrations decreased feeding rates, presumably by altering food palatability or impairing the mayfly's digestive processes, or both. Nearly 90% of the accumulated U was eliminated within 24 h after the waterborne exposure ceased, reflecting the desorption of weakly bound U from the insect's integument. To examine whether the experimentally derived rate constants for N. triangulifer could be generalized to baetid mayflies, mayfly U concentrations were predicted using the water chemistry and U measured in periphyton from springs in Grand Canyon (United States) and were compared to U concentrations in spring-dwelling mayflies. Predicted and observed mayfly U concentrations were in good agreement. Under the modeled site-specific conditions, waterborne U uptake accounted for 52-93% of the bioaccumulated U. U accumulation was limited in these wild populations due to a combination of factors including low concentrations of bioavailable dissolved U species, slow U uptake rates from food, and fast U elimination.

Mobilisation kinetics of Br, Cd, Cr, Hg, Pb and Sb in microplastics exposed to simulated, dietary-adapted digestive conditions of seabirds

Samples of beached plastics and historical and contemporary consumer plastics containing hazardous elements derived from reaction residues or functional additives have been micronised and subject to extraction conditions representative of the digestive environment of seabirds. Mobilisation of Br, Cd, Cr, Hg, Pb and Sb into NaCl solution, an avian physiologically-based extraction test (PBET) and a dietary-adapted PBET (DA-PBET) incorporating fish oil as part of the avian diet was monitored by ICP-MS over a 168-h period. Kinetic data were subsequently fitted using pseudo-first-order and parabolic diffusion models in order to derive rate constants for the release of hazardous elements during avian digestion of microplastics. Rate constants were variable and dependent on the nature and origin of plastic, type of residue or additive, extractant solution employed and model applied. Resulting estimates of bioaccessibility, defined as the equilibrium or maximum concentration of an element mobilised over the time course relative to its total concentration, were variable but considerable in many cases. Specifically, maximum values of about 65% of Cd and 100% of Pb were observed in consumer polycarbonate-acrylonitrile butadiene styrene exposed to the avian PBET and beached polyurethane exposed to the DA-PBET, respectively. The potential health risks of hazardous elements in microplastics are addressed and criteria for classification based on the European Toy Safety Directive migration (mobilisation) limits are proposed.

An Electrochemical and Spectroscopic Study on Re(CO)3(L)Cl in Dimethylformamide (L = 2,2'‐Bipyridine)

Herein, we present an in‐depth study of the redox chemistry of Re(CO)3(L)Cl (1) in dimethylformamide (dmf). Previous reports on 1 focused on acetonitrile (MeCN) and tetrahydrofuran (thf) as solvent, although the complex exhibits also a good activity in dmf in the electrochemical and photochemical CO2 reduction reaction. The combination of scan rate dependent CV data and IR spectroelectrochemical (IR‐SEC) experiments allows to derive a mechanistic scenario for the coupled electrochemical and chemical steps after reduction of the title compound. The relative reaction rates of competing pathways are distinctly different with regard to MeCN. The dimer [Re(CO)3(L)]2, which is the main species after initial reduction of 1 in MeCN, is barely formed in dmf and [Re(CO)3(L)Cl]– is stable. IR‐SEC data allowed to identify crucial intermediates alongside the reduction pathway of 1, and digital simulation of the CV data allowed to derive rate constants for the individual reaction steps.

Mixture rules and falloff are now major uncertainties in experimentally derived rate parameters for H + O2 (+M) ↔ HO2 (+M)

Rate constants for the title reaction for different bath gas species, M, are essential to accurate combustion predictions. Most experimental studies of the title reaction assume the reaction is in the low-pressure limit. Furthermore, experimental studies for M = H2O usually rely on measurements in H2O/A mixtures, separate measurements in a reference bath gas M = A, and an assumed mixture “rule,” which estimates rate constants in the mixture from those of pure bath gases. We present results from master equation calculations to quantify the uncertainties due to these assumptions in experimental interpretations. Our calculations indicate potential errors due to falloff and mixture rule assumptions that would be imperceptible experimentally over typical variations of pressure and composition yet introduce substantial uncertainties (reaching ∼ 50%) in reported rate constants, which often involve extrapolation to zero pressure and unity mole fraction. Going forward, we recommend that experimentally determined pseudo-second-order rate constants for H + O2 ↔ HO2 for the experimental pressure and mixture composition (which are independent of these assumptions) be reported, that experiments used to derive rate constants in the low-pressure limit or for M = H2O be conducted at lower pressures and higher H2O fractions (where these assumptions are more accurate), and that uncertainty analysis consider uncertainties due to falloff and mixture rule assumptions.

Hindered rotor benchmarks for the transition states of free radical additions to unsaturated hydrocarbons.

The hindered internal rotors of 32 transition states (TSs) formed through four free radicals, namely methyl, vinyl, ethyl, methoxy (ĊH3, Ċ2H3, Ċ2H5, CH3) additions to acetylene, ethylene, allene, propyne, and propene (C2H2/C2H4/C3H4-a/C3H4-p/C3H6) are studied. To validate the uncertainties of rate constants that stem from the use of different electronic structure methods to treat hindered rotors, the rotations of the newly formed C-C and/or C-O rotors in the transition states are calculated using commonly used DFT methods (B3LYP, M06-2X, ωB97X-D and B2PLYP-D3 with two Pople basis sets (6-31+G(d,p), 6-311++G(d,p)) and cc-pVTZ). The hindrance potential energies V(χ) calculated using the M06-2X/6-311++G(d,p) method are benchmarked at the CCSD(T), CCSD(T)-F12, DLPNO-CCSD(T) levels of theory with cc-pVTZ-F12 and cc-pVXZ (X = T, Q) basis sets and are extrapolated to the complete basis set (CBS) limit. The DLPNO-CCSD(T)/CBS method is proven to reproduce the CCSD(T)/CBS energies within 0.5 kJ mol-1 and this method is selected as the benchmark for all of the rotors in this study. Rotational constants B(χ) are computed for each method based on the optimized geometries for the hindrance potential via the I(2,3) approximation. Thereafter, the V(χ) and B(χ) values are used to compute hindered internal rotation partition functions, QHR, as a function of temperature. The uncertainties in the V(χ), B(χ) and QHR calculations stem from the use of different DFT methods for the internal rotor treatment are discussed for these newly formed rotors. For rotors formed by + C2 alkenes/alkynes, the V(χ) and QHR values calculated using DFT methods are compared with the DLPNO-CCSD(T)/CBS results and analysed according to reaction types. Based on comparisons of the DFT methods with the benchmarking method, reliable DFT methods are recommended for the treatment of internal rotors for different reaction types considering both accuracy and computational cost. This work, to the authors' knowledge, is the first to systematically benchmark hindrance potentials which can be used to estimate uncertainties in theoretically derived rate constants arising from the choice of different electronic structure methods.

Kinetic Advantages of the Run‐On Oligomer or Filamentation Mechanism of a DNA Cleaving Enzyme

Filament or run-on oligomer (ROO) formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that cleaves two types of sites, primary and secondary, but cleaves secondary only in the presence of primary. Primary site DNA activates SgrAI to form ROO with rapid DNA cleavage activity and that can incorporate SgrAI/DNA complexes containing secondary site DNA, thereby inducing their cleavage. Structural methods including x-ray crystallography and cryo-electron microscopy have been used to characterize the three-dimensional structure of the ROO of SgrAI bound to DNA. These structures, combined with kinetic FRET measurements of the DNA cleavage reaction, have been used in global modeling and data fitting to derive microscopic rate constants and investigate cooperativity, growth mechanisms, product trapping and to compare to conventional, non-run on oligomer mechanisms. Mutations which disrupt the ROO were tested in vivo for anti-phage activity. The three-dimensional structure of SgrAI bound to primary site DNA and in the run-on oligomer form shows a left-handed helix with 4 SgrAI/DNA complexes per turn, and that could in principle extend indefinitely. Global model data fitting and the derived rate constants show that association of SgrAI/DNA complexes into the run-on oligomer is rate limiting and is also the feature of the mechanism responsible for sequestering activated DNA cleavage of primary and secondary sites on the same DNA molecule as the activating sequences (a.k.a. the primary site sequences). This analysis also shows that only very limited product trapping occurs, and that the run-on oligomers are not limited to dissociating and growing from only the ends. DNA cleavage was found to be ten-fold faster than ROO dissociation, providing a commitment to the enzyme reaction pathway. Finally, simulations with the models and derived rate constants show that the SgrAI run-on oligomer mechanism is advantageous over the conventional, non-run on oligomer mechanism in both speed and sequestration of activated DNA cleavage, particularly in cleavage of secondary sites, not protected by methylation on the host genome. Mutations which reduce ROO formation completely abolish the anti-phage activity of SgrAI in vivo, showing the critical role of filament formation to biological function. Support or Funding Information This work was supported by NSF MCB-1410355 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using a Fluorescent Unnatural Amino Acid to Characterize the Role of Conformational Dynamics in High Fidelity DNA Replication

The DNA polymerase of bacteriophage T7 is a model enzyme for understanding high fidelity DNA replication. Crystal structures of the open, binary (E‐DNA) complex and the closed, nucleotide bound ternary (E‐DNA‐dNTP) complex reveal large structural changes as nucleotide binds and the fingers domain closes around the correct base pair. However, the contribution of this structural transition to DNA replication fidelity has been greatly disputed, and its measurement has been further complicated by the difficulty in site specific fluorescent labeling of the T7 DNA polymerase using traditional cysteine – maleimide coupling chemistry. The work presented here determines the contribution of the motions of the fingers domain of the T7 DNA polymerase to its high DNA replication fidelity, through direct measurement using a fluorescent unnatural amino acid incorporated site‐specifically in the fingers domain of the enzyme. Each step in the pathway of correct nucleotide incorporation was measured using stopped flow and quench flow techniques, and data from all experiments were globally fit in Kintek Explorer to derive rate constants for each step in the pathway. Our data support a model where the conformational change preceding chemistry is not rate‐limiting but is still the major determinant of fidelity, and provides new information on post‐chemistry conformational dynamics of the enzyme and their contribution towards DNA replication fidelity.Support or Funding InformationThe Welch Foundation (F‐1684)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament

Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.