Transient Kinetic Analysis Research Articles

Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR.

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Leishmania major (Lm), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg) and Cryptosporidium hominis (Ch), contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the two sequential catalytic activities contained on a single polypeptide chain. It has been suggested that the bifunctional nature of the two catalytic activities may enable substrate channeling. The 3D structures for each of these enzymes reveals distinct features for each species. While three of the four species (Pf, Tg and Ch) contain a junctional region linking the two domains, this is lacking in Lm. The Lm and Pf contain N-terminal amino acid extensions. A multidisciplinary approach using structural studies and transient kinetic analyses combined with mutational analysis has investigated the roles of these unique structural features for each enzyme. Additionally, the possibility of substrate channeling behavior was explored. These studies have identified unique, functional regions in both the TS and DHFR domains that govern efficient catalysis for each species. Surprisingly, even though there are structural similarities among the species, each is regulated in a distinct manner. This structural and mechanistic information was also used to exploit species-specific inhibitor design.

Apparatus for efficient utilization of isotopically-labeled gases in pulse transient studies of heterogeneously catalyzed gas phase reactions

Transient techniques, such as steady state isotopic transient kinetic analysis (SSITKA), are powerful methods for determining various mechanistic and kinetic insights into heterogeneously catalyzed gas-phase reactions.

Understanding FTS selectivity: the crucial role of surface hydrogen.

Monomeric forms of carbon play a central role in the synthesis of long chain hydrocarbons via the Fischer-Tropsch synthesis (FTS). We explored the chemistry of C1Hxad species on the close-packed surface of cobalt. Our findings on this simple model catalyst highlight the important role of surface hydrogen and vacant sites for product selectivity. We furthermore find that COad affects hydrogen in multiple ways. It limits the adsorption capacity for Had, lowers its adsorption energy and inhibits dissociative H2 adsorption. We discuss how these findings, extrapolated to pressures and temperatures used in applied FTS, can provide insights into the correlation between partial pressure of reactants and product selectivity. By combining the C1Hx stability differences found in the present work with literature reports of the reactivity of C1Hx species measured by steady state isotope transient kinetic analysis, we aim to shed light on the nature of the atomic carbon reservoir found in these studies.

Elucidation of deactivation phenomena in cobalt catalyst for Fischer-Tropsch synthesis using SSITKA

Catalyst deactivation is a major problem in Fischer-Tropsch synthesis. It leads to a decrease in hydrocarbon productivity and loss of active sites in the expensive cobalt catalyst, and thus, undermines the overall efficiency of the technology. In the present paper, the effect of deactivation of silica-supported cobalt catalysts and their reductive rejuvenation on the number of active sites and their intrinsic activity (turnover frequency) in Fischer-Tropsch synthesis was studied using a combination of Steady State Isotopic Transient Kinetic Analysis (SSITKA) and catalyst characterization techniques. Catalyst characterization revealed that carbon deposition and agglomeration of cobalt nanoparticles during reaction were responsible for the deactivation. SSITKA experiments showed that the initial rate constant of 2.33μmolg−1s−1 had a loss of 67% of activity after 150h on stream with a reduction in the amount of sites due to deposited carbon by 33.4μmolg−1. The carbon deposition leads to a decrease in the number of carbon chemisorbed intermediates which yield methane through their hydrogenation and desorption. The number of sites for reversible adsorption of CO is less affected by carbon deposition. The surface hydrogenation sites and surface sites favoring stronger reversible adsorption of carbon monoxide deactivate first during the first hours of Fischer-Tropsch synthesis. Catalyst rejuvenation in hydrogen lessens the amounts of deposited carbon species and partially releases the most active sites of carbon monoxide dissociative adsorption and stronger sites of carbon monoxide reversible adsorption. The transient isotopic methods provide an attractive tool to obtain precise information about the mechanisms of deactivation of cobalt catalysts in Fischer-Tropsch synthesis.

Transient Kinetic Analysis of ATP Hydrolysis by the CCT/TRiC Chaperonin

The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis. The phases were assigned by (i) determining their dependence on ATP and K+ concentrations and (ii) by measuring their sensitivity to the mutation Gly345→Asp in subunit CCT4, which decreases cooperativity in ATP binding. The values of the observed rate constants corresponding to the burst phases are found to decrease with increasing ATP and K+ concentrations, thereby indicating that the apo state of CCT/TRiC is in equilibrium between several conformations and that “conformational selection” by ATP takes place before hydrolysis. The amplitude of the lag phase, which follows, decreases with increasing ATP concentrations, thus indicating that it reflects a transition between states with low affinity for ATP and a state with high affinity for ATP that is predominant under steady-state conditions. A kinetic model based on the data is suggested, in which CCT/TRiC is in equilibrium between a relatively large number of states that are distinguished kinetically, in agreement with its proposed sequential allosteric mechanism.

Carbon Number Dependence of Reaction Mechanism and Kinetics in CO Hydrogenation on a Co-Based Catalyst

A detailed mapping of the kinetic parameters involved in the reaction network of CO hydrogenation on a Co-Re/CNT catalyst has been performed. Multicomponent steady-state isotopic transient kinetic analysis (SSITKA) has been used to deconvolute the rates of chain growth and termination to olefins and paraffins with various carbon numbers into the concentration of the surface intermediates and their reactivity, expressed as their rate constants kg, ko, and kp, respectively, at a molecular level. The site coverage of the different products (θCn) measured by the multicomponent analysis of their isotopic distribution allows the study of their effect on chain growth, as well as on olefin and paraffin formation. The insights into the reaction mechanism were gained from the kinetic dependence of different reaction steps on the hydrogen pressure. The results revealed a significant carbon number dependence of the mechanism for the chain growth and termination to paraffin and olefin reactions: an enol-like intermedi...

Modelling a reactor cell for operando IR studies: From qualitative to fully quantitative kinetic investigations

Operando infrared spectroscopy is often used qualitatively to elucidate reaction mechanisms thanks to the observation of the catalyst under actual operating conditions. Besides, in principle such technique can also provide quantitative information that can be used to describe detailed chemical kinetics (spectrokinetic approach). However, the collected kinetic information are relevant only if the gas fluid dynamics inside the reactor cell is well known and can be precisely described. In fact, to perform a reliable kinetic analysis, it is essential to correctly model the flow in the reactor cell and to couple this model with the mass balances. To demonstrate the importance of this aspect, in this work the SSITKA (Steady State Isotopic Transient Kinetic Analysis) approach was exploited within an operando FTIR reactor to study the methanol adsorption onto ceria, herein selected as model probe reaction. A conventional round sample holder with round catalyst wafer and a novel square sample holder with square catalyst pellet have been used. Although the conventional round sample holder shows a satisfactory behaviour, the modified sample holder leads to a virtually perfect plug-flow reactor, which will simplify data computing, provide a better fit of the experimental data and more accurate estimates of the kinetic parameters.

Complex reaction network generation for Steady State Isotopic Transient Kinetic Analysis: Fischer-Tropsch Synthesis

A versatile modeling strategy for Steady State Isotopic Transient Kinetic Analysis (SSITKA) data, acquired in a plug flow reactor, is elaborated with particular attention to complex reaction networks and Fischer-Tropsch Synthesis as example. A spatial discretization scheme optimizing accuracy and CPU time is developed. For low switch time constants, the van Leer and van Albada flux limiter functions used in conjunction with the DASPK solver yields the lowest CPU time for the integration of the resulting ordinary differential equations. For larger switch time constants, conventional central differencing can be applied. A network generation methodology is implemented accounting for the isotopic labeling. It reduces the exponential dependence of the number of considered species on the carbon number to a quadratic dependence. For a reaction network allowing a maximum chain length of 5 carbon atoms a gain in CPU time up to a factor of 10 can be achieved.

A novel transient phase kinetic analysis of the fractional modification of monocyclic enzyme cascades

In this paper we analyse the transient phase, i.e., the time course, of the fractional modification of monocyclic enzyme cascades with a more complete realistic definition for fractional modification as the fraction of the original enzyme converted into the two forms of modified protein, and not only into the free modified protein. This same definition was recently used by our group in an analysis of only its steady state. Hence this contribution extends the above-mentioned one as it includes the transient phase, which provides much more information than a study into only the steady state. The fractional modification definition used herein is more complete than the definitions employed in previous works by other authors and ourselves, which only considered the fraction of molecules of the original enzyme converted into the free modified enzyme. By assuming a minimal set of linearising assumptions, we obtained a general transient phase equation for fractional modification. We also obtained the transient phase equation by assuming rapid equilibrium in the reversible steps involved in the scheme or the reaction. Both equations are valid for the four cyclic enzyme cascades studied herein. Their applicability and goodness were tested by the simulated time progress curves obtained by numerical integration. Finally, a kinetic data analysis is suggested.

On the superior activity and selectivity of PtCo/Nb2O5 Fischer Tropsch catalysts

In this study Co/Nb2O5 catalysts and the effect of Pt-promotion thereon are investigated in comparison with γ-Al2O3- and α-Al2O3-supported catalysts for the Fischer Tropsch (FT) synthesis. Upon Pt-promotion of Co/Nb2O5 the cobalt-weight normalized FT activity was found to increase by a factor of 2.4, while the high C5+ selectivity of 85wt% was maintained. Based on environmental TEM results no indications were found that Pt affected the cobalt particle size in Co/Nb2O5 catalysts. A kinetic study indicates an increased number of active sites upon Pt-promotion whereas Steady-State Isotopic Transient Kinetic Analysis experiments show that a combination of an increased number of active sites and an increased turnover frequency is at the origin of the enhanced activity in Co/Nb2O5 catalysts upon Pt-promotion. Pt was tentatively proposed to bring about more efficient promotion of Co by NbOx being present as smaller clusters.

Dry Reforming of Methane on Rh-Doped Pyrochlore Catalysts: A Steady-State Isotopic Transient Kinetic Study

Increases in worldwide methane production from biological and fossil sources have led to an increased level of interest in the dry reforming of methane (DRM) to produce syngas. Experimental efforts have shown that select pyrochlore materials, such as the Rh-substituted lanthanum zirconate pyrochlore (LRhZ), are catalytically active for DRM, exhibit long-term thermal stability, and resist deactivation. This work seeks to allow further catalyst improvements by elucidating surface reaction kinetics via steady-state isotopic transient kinetic analysis (SSITKA) of dry reforming on the LRhZ pyrochlore. Isotopically labeled CH4 and CO2 were used in multiple SSITKA experiments to elucidate the migration of carbon atoms to product species. Short surface residence times at 650 and 800 °C (<0.6 s) were observed for DRM intermediates involved in reversible reactions, and the participation of all surface metal atoms as active sites for DRM, not only Rh, is suggested. Isotopic responses and kinetic isotope effects are ...

Active Site Dependent Reaction Mechanism over Ru/CeO2 Catalyst toward CO2 Methanation.

Oxygen vacancy on the surface of metal oxides is one of the most important defects which acts as the reactive site in a variety of catalytic reactions. In this work, operando spectroscopy methodology was employed to study the CO2 methanation reaction catalyzed by Ru/CeO2 (with oxygen vacancy in CeO2) and Ru/α-Al2O3 (without oxygen vacancy), respectively, so as to give a thorough understanding on active site dependent reaction mechanism. In Ru/CeO2 catalyst, operando XANES, IR, and Raman were used to reveal the generation process of Ce(3+), surface hydroxyl, and oxygen vacancy as well as their structural evolvements under practical reaction conditions. The steady-state isotope transient kinetic analysis (SSITKA)-type in situ DRIFT infrared spectroscopy undoubtedly substantiates that CO2 methanation undergoes formate route over Ru/CeO2 catalyst, and the formate dissociation to methanol catalyzed by oxygen vacancy is the rate-determining step. In contrast, CO2 methanation undergoes CO route over Ru surface in Ru/α-Al2O3 with the absence of oxygen vacancy, demonstrating active site dependent catalytic mechanism toward CO2 methanation. In addition, the catalytic activity evaluation and the oscillating reaction over Ru/CeO2 catalyst further prove that the oxygen vacancy catalyzes the rate-determining step with a much lower activation temperature compared with Ru surface in Ru/α-Al2O3 (125 vs 250 °C).

Size and Promoter Effects in Supported Iron Fischer–Tropsch Catalysts: Insights from Experiment and Theory

The fundamentals of structure sensitivity and promoter effects in the Fischer–Tropsch synthesis of lower olefins have been studied. Steady state isotopic transient kinetic analysis, switching 12CO to 13CO and H2 to D2, was used to provide coverages and residence times for reactive species on supported iron carbide particles of 2–7 nm with and without promoters (Na + S). CO coverages appeared to be too low to be measured, suggesting dissociative adsorption of CO. Fitting of CH4 response curves revealed the presence of parallel side-pools of reacting carbon. CHx coverages decreased with increasing particle size, and this is rationalized by smaller particles having a higher number of highly active low coordination sites. It was also established that the turnover frequency increased with CHx coverage. To calculate H coverages, new equations were derived to fit HD response curves, again leading to a parallel side-pool model. The H coverages appeared to be lower for bigger particles. The H coverage was suppress...

Dry reforming of methane over 5 wt% Ni/Ce1-xPrxO2-δ catalysts: Performance and characterisation of active and inactive carbon by transient isotopic techniques

The effects of Ce1-xPrxO2-δ support composition (x=0.0–0.8) and reaction temperature (550–750°C) on the catalytic activity and selectivity and important features of the mechanism of the dry reforming of methane (DRM) over supported Ni (5wt%) were investigated. Of particular interest were the effects on the concentration of active and inactive carbon formed, the relative contribution of CH4 and CO2 activation routes towards carbon formation and the structure and morphology of the inactive carbon which was formed. For these carbon characterization studies, steady-state isotopic transient kinetic analysis (13CO2-SSITKA), temperature-programmed oxidation (TPO) following 13CO2/12CH4/He dry reforming, thermal gravimetric analysis coupled with TPO (TGA-TPO), scanning electron miscroscopy (SEM-EDX) and transmission electron microscopy with atomic resolution (HRTEM) and powder X-ray diffraction (XRD) were employed. The relative amount of inactive carbon formed via the CH4 and CO2 activation routes was found to strongly depend on reaction temperature and Pr-dopant support composition. At 550°C, the contribution of the CO2 activation route to the inactive carbon formation (besides that of the CH4 activation route) was 65.7 and 60.1%, respectively, for Ni supported on CeO2 and Ce0.2Pr0.8O2 carriers, whereas at 750°C the respective values were 54.0 and 50.9%. Filamentous carbon and thin layers of graphitic carbon were identified as the main morphologies of inactive carbon. The surface coverage of active carbon that truly participates in the formation of CO was found to depend on support composition and reaction temperature (θC=0.03–0.15at 550°C and 0.07–3.4at 750°C). A pool of inactive reversibly adsorbed CO2 was measured (θ=1.5–4.1) for the first time, which was dependent on support composition and reaction T. The introduction of 20 atom-% Pr in the ceria lattice caused a significant reduction in the rate of inactive carbon formation with marginal decrease in catalyst’s activity and stability after 25h on stream. Further introduction of Pr-dopant (80 atom-%) caused drastic reduction in the deposited carbon after 25h on the reaction stream at 750°C (0.07wt%C) but with appreciable decrease in CH4- and CO2-conversions and H2/CO gas-product ratio (a drop by a factor of 1.85, 1.45 and 1.47, respectively). The latter decrease in catalyst’s performance is correlated with the increase in the pool of inactive adsorbed CO2 in the form of carbonate-like species.

β-MYHC Mutations Linked to Early-Onset HCM and DCM Show Differences in Pre-Steady and Steady State Kinetic Parameters

Hypertrophic Cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young athletes and has an incidence of 1 in 500 people. Dilated cardiomyopathy (DCM) has a prevalence of 1 in 2000 people. Both diseases can be inherited, with HCM being autosomal dominant, and DCM having different genotypic profiles. There are over 400 mutations in cardiac myosin (MYH7) that have been linked to both diseases. Also noteworthy, early-onset HCM and DCM patients typically have a worse prognosis than adult-onset patients. The only intervention for these pediatric patients is a heart transplant. Here we describe the kinetic analysis of several cardiac myosin mutations that have been identified to be unique to early-onset patients. One set of mutations has been identified in pediatric HCM and the other set is DCM. Steady-state characterization of ATP hydrolysis for both mutant sets using an NADH coupled ATPase assay is reported. Additionally, transient kinetic analysis for several mutants was conducted illustrating differences in the cross-bridge kinetics between HCM and DCM mutants, namely ATP-induced dissociation, ADP release, and the binding constants of actin and the different nucleotide states. This data suggests that the motor activity is different among mutants and may provide insight into the mechanistic defects that can lead to both diseases.

Transient kinetic analysis of multipath reactions: An educational module using the IPython software package

A large number of industrial catalytic reactions proceeds via multiple pathways. A transient response method has been successfully utilized for the analysis of kinetics and mechanism of multipath reactions. In this paper, we describe an educational module for teaching kinetics of complex heterogeneously-catalyzed reactions based on the software package for simulation of transient responses in a tubular packed-bed reactor. The module includes the reactor model description, derivation and verification of kinetic equations, analysis of steady-state kinetics, numerical simulation of transient responses, and visualization and analysis of results utilizing the developed IPython notebooks. The use of the module helps students to acquire theoretical knowledge as well as the practical and analytical skills related to the kinetic analysis of multipath reactions.

Transient-State Kinetic Analysis of the RNA Polymerase I Nucleotide Incorporation Mechanism

Eukaryotes express three or more multisubunit nuclear RNA polymerases (Pols) referred to as Pols I, II, and III, each of which synthesizes a specific subset of RNAs. Consistent with the diversity of their target genes, eukaryotic cells have evolved divergent cohorts of transcription factors and enzymatic properties for each RNA polymerase system. Over the years, many trans-acting factors that orchestrate transcription by the individual Pols have been described; however, little effort has been devoted to characterizing the molecular mechanisms of Pol I activity. To begin to address this gap in our understanding of eukaryotic gene expression, here we establish transient-state kinetic approaches to characterize the nucleotide incorporation mechanism of Pol I. We collected time courses for single turnover nucleotide incorporation reactions over a range of substrate ATP concentrations that provide information on both Pol I’s nucleotide addition and nuclease activities. The data were analyzed by model-independent and model-dependent approaches, resulting in, to our knowledge, the first minimal model for the nucleotide addition pathway for Pol I. Using a grid searching approach we provide rigorous bounds on estimated values of the individual elementary rate constants within the proposed model. This work reports the most detailed analysis of Pol I mechanism to date. Furthermore, in addition to their use in transient state kinetic analyses, the computational approaches applied here are broadly applicable to global optimization problems.

Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol.

Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase γ (mtDNA Pol γ) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPol-mediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol γ that may manifest in toxicity.

Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase

The first step in the mitochondrial sulfide oxidation pathway is catalyzed by sulfide quinone oxidoreductase (SQR), which belongs to the family of flavoprotein disulfide oxidoreductases. During the catalytic cycle, the flavin cofactor is intermittently reduced by sulfide and oxidized by ubiquinone, linking H2S oxidation to the electron transfer chain and to energy metabolism. Human SQR can use multiple thiophilic acceptors, including sulfide, sulfite, and glutathione, to form as products, hydrodisulfide, thiosulfate, and glutathione persulfide, respectively. In this study, we have used transient kinetics to examine the mechanism of the flavin reductive half-reaction and have determined the redox potential of the bound flavin to be -123 ± 7 mV. We observe formation of an unusually intense charge-transfer (CT) complex when the enzyme is exposed to sulfide and unexpectedly, when it is exposed to sulfite. In the canonical reaction, sulfide serves as the sulfur donor and sulfite serves as the acceptor, forming thiosulfate. We show that thiosulfate is also formed when sulfide is added to the sulfite-induced CT intermediate, representing a new mechanism for thiosulfate formation. The CT complex is formed at a kinetically competent rate by reaction with sulfide but not with sulfite. Our study indicates that sulfide addition to the active site disulfide is preferred under normal turnover conditions. However, under pathological conditions when sulfite concentrations are high, sulfite could compete with sulfide for addition to the active site disulfide, leading to attenuation of SQR activity and to an alternate route for thiosulfate formation.

Selective hydrogenation of acetylene over Pd/Fiberglass catalysts: Kinetic and isotopic studies

In this study two palladium fiberglass catalysts differing in the location of Pd particles have been synthesized and characterized in detail by means of XPS and HRTEM/STEM. In the first catalyst prepared using H2PdCl4 as a precursor palladium deposits on the external surface of glass fibers, whereas in the second catalyst prepared using ion exchange of [Pd(NH3)4]2+ cations with the glass protons palladium is located in the subsurface layers of glass. It was clearly shown by SSITKA (steady-state isotopic transient kinetic analysis) with the use of doubly labeled acetylene 13C2H2 that gas-phase ethylene is not involved in the selective hydrogenation of acetylene over the second catalyst in contrast to the first one where ethylene is also hydrogenated into ethane. This fact was explained by an ability of glass to absorb predominantly such readily polarizable molecules like acetylene, but not ethylene. As a result, the fiberglass catalyst where Pd clusters are confined in the glass matrix demonstrates high selectivity (ethylene increment ca. 60%) at complete conversion of acetylene. So, Pd fiberglass catalysts were shown to be very promising for the selective hydrogenation processes.