Transient Kinetic Investigations Research Articles

Steady-State and transient kinetic investigations of the oxidation of NO over Pt/SiO2

The reaction mechanism of the oxidation of NO under conditions of high NO concentration over a 2 wt% Pt/SiO2 was studied using both kinetic and transient isotopic tracing experiments. The reaction mechanism was found to involve both NO adsorption and oxygen adsorption, contrary to the situation under low NO concentration where NO reacts from the gas phase. The kinetic rate data could be fitted to the Langmuir-Hinshelwood model permitting the estimation of rate and equilibrium constants of elementary steps. These steps were investigated using transient responses following independent reactant gas isotopic switches for tracing the nitrogen path and the oxygen path during NO oxidation. These transients could be sufficiently described by a microkinetic model based on two pools of NO intermediates, one pool each for the adsorbed molecular oxygen and atomically adsorbed oxygen. Surface atomic oxygen was found to be formed by two different routes: via a direct dissociation of molecularly adsorbed oxygen and also via an assisted pathway that leads to NO2 formation. The latter surface reaction between adsorbed NO and adsorbed molecular oxygen comprised the kinetically relevant step. Insights from the two experimental and model approaches were in good agreement and provided a coherent reaction mechanism for NO oxidation under high NO reactant concentration.

Comparison of the selective oxidation kinetics between acrolein and methacrolein on Mo/V/W-mixed oxides

The partial oxidation of acrolein and methacrolein on Mo/V/W-mixed oxides was studied by transient kinetic investigation with a new experimental technique. Within the so‑called HETKA (Homologue Exchange Transient Kinetic Analysis) experiments, acrolein (ACR) was stepwise replaced by methacrolein (MAC), and vice versa under constant reaction conditions. Conversion and yield decrease significantly when ACR is substituted by MAC. Evidence is given that the lower reaction rate with MAC is caused by the formation of coke deposits on the catalyst surface. Furthermore, a temperature programmed reaction (TP reaction) was carried out in which both aldehydes were fed at the same time. Compared with the TP reactions with only one aldehyde, the conversion and yield curves are shifted to higher temperatures. Regarding acrylic acid, the maximum yield and selectivity are not influenced by the contemporaneous presence of both aldehydes. On the contrary, the selectivity to methacrylic acid is increased significantly.

Caged neurotransmitters and other caged compounds: design and application.

The approaches using caged neurotransmitters described here enable transient kinetic investigations to be made with membrane-bound proteins (receptors) on a cell surface with the same time resolution as was previously possible only with proteins in solution.

Pre-Steady-State Kinetic Analysis of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase from Mycobacterium tuberculosis Reveals Partially Rate-Limiting Product Release by Parallel Pathways

As part of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the conversion of DXP into 2-C-methyl-D-erythritol 4-phosphate (MEP) by consecutive isomerization and NADPH-dependent reduction reactions. Because this pathway is essential to many infectious organisms but is absent in humans, DXR is a target for drug discovery. In an attempt to characterize its kinetic mechanism and identify rate-limiting steps, we present the first complete transient kinetic investigation of DXR. Stopped-flow fluorescence measurements with Mycobacterium tuberculosis DXR (MtDXR) revealed that NADPH and MEP bind to the free enzyme and that the two bind together to generate a nonproductive ternary complex. Unlike the Escherichia coli orthologue, MtDXR exhibited a burst in the oxidation of NADPH during pre-steady-state reactions, indicating a partially rate-limiting step follows chemistry. By monitoring NADPH fluorescence during these experiments, the transient generation of MtDXR·NADPH·MEP was observed. Global kinetic analysis supports a model involving random substrate binding and ordered release of NADP(+) followed by MEP. The partially rate-limiting release of MEP occurs via two pathways--directly from the binary complex and indirectly via the MtDXR·NADPH·MEP complex--the partitioning being dependent on NADPH concentration. Previous mechanistic studies, including kinetic isotope effects and product inhibition, are discussed in light of this kinetic mechanism.

Mechanism of Potentiation of a Dysfunctional Epilepsy-Linked Mutated GABAA Receptor by a Neurosteroid (3α, 21-Dihydroxy-5α-pregnan-20-one): Transient Kinetic Investigations

The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.

Transient Kinetic Investigation of GTP Hydrolysis Catalyzed by Interferon-γ-induced hGBP1 (Human Guanylate Binding Protein 1)

Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of P(i) formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds.

A Protecting Group for Carboxylic Acids That Can Be Photolyzed by Visible Light

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (epsilon(390) = 13651 M(-)(1) cm(-)(1)). Experiments are performed at 400 nm (epsilon(400) = 12232 M(-)(1) cm(-)(1)) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 degrees C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 micros with a quantum yield of 0.11 +/- 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between approximately 10(12) neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators.

Toward the development of new photolabile protecting groups that can rapidly release bioactive compounds upon photolysis with visible light.

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C). The caging group is attached through an ester linkage to the carboxyl functionality of beta-alanine, which activates the inhibitory glycine receptor in the mammalian central nervous system. Such caged compounds play an important role in transient kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged beta-alanine, the caging group is released within 5 micros. Quantum yields of 0.03 and 0.002 were obtained in the UV region (308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments, using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293 cells transiently transfected with cDNA encoding the alpha1 homomeric, wild-type glycine receptor. The experiments demonstrated that neither DANP-caged beta-alanine nor its byproducts inhibit or activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged beta-alanine is water-soluble and stable and can be used for transient kinetic measurements.

Acceleration in catalyst development by fast transient kinetic investigation

This study intends to illustrate the application of transient kinetics in combinatorial research. Thus, it is shown through various case studies how high-throughput transient kinetics may both accelerate the search for new catalytic materials and bring fundamental insights in reaction mechanisms. As a first case study, the cracking reactions of the C6 isomers (n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, and 2,3-dimethylbutane) over a fluidised catalytic cracking (FCC) catalyst at temperatures between 300 and 650 °C have been studied in a temporal-analysis-of-product (TAP) reactor. A mathematical model taking into account the extra- and intracrystalline transport phenomena was used to determine the diffusion, sorption, and kinetic parameters for the hydrocarbons studied. Sorption and diffusion decrease with increasing branching in agreement with literature data. Except product distribution, the activation energies for the overall cracking of the C6 isomers are similar for all isomers. As a second case study, a “paralleled” TAP reactor with a multisample holder is shown to be an efficient tool for acquiring adsorption/desorption parameters for a series of zeolites, tested to evaluate their potential as catalyst supports in the oxidative dehydrogenation of ethane. Finally, the applied combinatorial study was found also suitable for discovering new materials precursors of dense oxygen-conducting membranes. From high-throughput transient TAP and TPD experiments, it was possible to derive a strategy directing the rapid combinatorial evolution within the available parameter space. It was shown that high-quality information is accessible from these data, providing all parameters necessary for a well fitting description with a kinetic model.

Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis.

The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor.

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.

In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.

Kinetics and thermodynamics of the reaction of peroxides with manganese-reconstituted horseradish peroxidase: a stopped-flow transient kinetic investigation

The reaction of manganese-reconstituted horseradish peroxidase (MnHRP) with peroxides of different nature has been investigated by stopped-flow kinetics in order to understand the nature of the active site of the enzyme, the differences in the mechanism of the formation of MnHRP–I with different peroxides and possible resolution of the intermediate(s) involved. The reaction of m-ClC6H4CO3H with MnHRP shows that only the unionised species of the peracid reacts. tert-Butyl hydroperoxide reacted with MnHRP, although it does not with native HRP. The reaction shows saturation kinetics at room temperature for the formation of MnHRP–I at high concentrations of ButO2H. This observation provides direct evidence for a reversible binding step of the enzyme and peroxide before the product formation. The thermodynamic parameters for the formation of MnHRP–I are similar to those for HRP–I formation, indicating that the mode of formation of both peroxide compounds may be similar.

25] Development and application of caged ligands for neurotransmitter receptors in transient kinetic and neuronal circuit mapping studies

Publisher Summary This chapter describes the instrumentation and theory involved in transient kinetic investigations of neurotransmitter receptor-mediated reactions on the surface of nervous system cells and in the membranes of Xenopus laevis oocytes in which many receptors have been expressed. Photolabile, biologically inert precursors of neurotransmitters (caged neurotransmitters) that can be photolyzed to the neurotransmitters in the microsecond time region form an integral part of the transient kinetic techniques. The use of these techniques was illustrated in investigations of the formation of transmembrane channels, receptor-drug interactions, and integration of excitatory and inhibitory signals by single central nervous system neuron that have temporal and spatial information. The use of caged neurotransmitters in identifying cells that contain a specific receptor and cells that secrete the neurotransmitter in a circuit of cells controlling a measurable response has also been illustrated in the chapter.

Kinetic and transient kinetic investigations of the synthesis of oxygenates from carbon monoxide/hydrogen mixtures on supported rhodium catalysts

Abstract A detailed kinetic investigation has been undertaken on the CO/H 2 reaction on promoted rhodium catalysts at high pressures. It has been found that hydrocarbon production is inhibited by increasing the carbon monoxide pressure and enhanced by increasing the hydrogen pressure. Ethanol production is generally negative order with respect to carbon monoxide and positive order with respect to hydrogen. Both acetaldehyde and acetic acid formation are positive order with respect to carbon monoxide and hydrogen pressures. The results are interpreted in terms of a model in which the availability of hydrogen is a critical factor in determining the activity and selectivity of the various catalysts. Promotion of rhodium by reducible oxides causes a change in the orders of reaction, the net effect being that the reactions become much less sensitive to hydrogen pressure. It is concluded that such oxide promoters enhance the availability of hydrogen by increasing the effective surface hydrogen pressure on the catalyst adjacent to active sites. Transient kinetic experiments have been performed in which the reaction mixture has been switched on and off and products analysed at short intervals of time. The results are in agreement with the conclusions of the kinetic experiments concerning the orders of reaction and the sensitivity to hydrogen pressure. No evidence has been found for long delays in the production of ethanol when the catalyst is exposed to the CO/H 2 reaction mixture. Possible reasons for differences observed in this work and in other published work are considered. The overall conclusion is that the formation of ethanol is rapid, but that the detection of ethanol may be delayed by adsorption effects in the catalyst bed.