Home About us Contact
|
Home About us Contact | ||
|
|
||
Kinetic Mechanism (kinetic + mechanism)
Selected AbstractsKinetic mechanism for p38 MAP kinase ,FEBS JOURNAL, Issue 18 2005A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 µm, KI = 240 µm, and KI > 2000 µm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 µm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source] Nucleoside transporter and nucleotide vesicular transporter: Two examples of mnemonic regulationDRUG DEVELOPMENT RESEARCH, Issue 1-2 2001Raquel P. Sen Abstract According to their relevant roles in the regulation and availability of extracellular levels of purinergic signals, the nucleoside transporter and the nucleotide vesicular transporter are subject to acute regulation. The plasma membrane nucleoside transporter has been shown to exhibit several regulatory mechanisms, such as regulation by long-term signals, phosphorylation/dephosphorylation processes, and allosteric modulation. The present work reviews studies concerning allosteric modulation of nucleoside and nucleotide vesicular transporters, as the first reported examples of mnemonic behavior in transporter proteins, presenting kinetic and allosteric cooperativity. This fact implies that the protein can exhibit different conformations, each one with specific kinetic parameters. Transport substrates are able to induce slow conformational changes between the different forms of the transporter. This kinetic mechanism can provide several physiological advantages, since it allows strict control of transport capacity by changes in substrate concentrations. This allosteric modulation has been confirmed in several experimental models, the nucleoside transporter in chromaffin and endothelial cells from adrenal medulla and the nucleotide vesicular transporter in the chromaffin cell granules and rat brain synaptic vesicles. Taking into account these considerations, the mnemonic regulation described here could be a widespread mechanism among transporter proteins. Drug Dev. Res. 52:11,21, 2001. © 2001 Wiley-Liss, Inc. [source] Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivativesFEBS JOURNAL, Issue 24 2008Diana Lindberg The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre-steady-state measurements on a series of styrene oxide derivatives. A preference for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans -stilbene oxide was established, with E -values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme formation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by 13C-NMR spectroscopy of 18O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans -stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency. [source] 7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic targetFEBS JOURNAL, Issue 20 2006Characterization, inhibition studies Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S -adenosyl- l -methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5,-phosphate-dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a pKa of 8.0, which was attributed to the active-site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: KmAdoMet = 0.78 ± 0.20 mm, KmKAPA = 3.8 ± 1.0 µm, kcat = 1.0 ± 0.2 min,1, KiKAPA = 14 ± 2 µm. Amiclenomycin and a new analogue, 4-(4c -aminocyclohexa-2,5-dien-1r -yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: Ki = 12 ± 2 µm, kinact = 0.35 ± 0.05 min,1, and Ki = 20 ± 2 µm, kinact = 0.56 ± 0.05 min,1, for amiclenomycin and compound 1, respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound 1, respectively, which make these inactivators particularly efficient. compound 1 (100 µg·mL,1) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound 1 specifically targets DAPA AT in vivo. [source] Kinetics and thermodynamics of nick sealing by T4 DNA ligaseFEBS JOURNAL, Issue 21 2003Alexey V. Cherepanov T4 DNA ligase is an Mg2+ -dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a ,processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a ,nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (< 10 µm) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (, 0.2 min,1) and a subsequent slow ligation (, 2 × 10,3 min,1). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal·mol,1), transadenylation of the nick (0.9 kcal·mol,1), and nick-sealing (16.3,18.8 kcal·mol,1) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase,AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis. [source] Functional analysis of disease-causing mutations in human galactokinaseFEBS JOURNAL, Issue 8 2003David J. Timson Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia. [source] Multiple criticality and staged ignition of methane in the counterflowINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 12 2009Wei Liu The counterflow ignition of methane was investigated, with particular interest in the role of radical versus thermal runaway. Simulation with different kinetic mechanisms showed that the ignition response is qualitatively sensitive to the kinetic mechanism adopted, either exhibiting or not exhibiting two ignition turning points in the S-curve response. For the former situation, ignition could take place in a staged manner with either increasing temperature or decreasing strain rate of the counterflow, characterized by the first ignition event being radical induced with negligible thermal effect and the second ignition event requiring thermal feedback. Sensitivity analysis and a recently developed chemical explosive mode analysis were performed to identify the dominant reactions in the radical runaway stage, revealing the importance of branching pathways involving the HO2 radical. Counterflow experiments were also conducted, yielding useful information on the ignition response. The study identifies items of further research to sharpen the comprehensiveness and accuracy of the methane oxidation mechanism. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 764,776, 2009 [source] Kinetics and mechanism of the oxidation of carbon by NO2 in the presence of water vaporINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 4 2009M. Jeguirim The kinetics and mechanism of the oxidation of carbon by NO2 in absence and presence of water vapor were studied in a fixed bed reactor. The rate of carbon oxidation by NO2 is enhanced in the presence of water vapor in the range of temperature 300,400°C. The benefit effect of water is attributed to the intermediate formation of traces of nitric and nitrous acids, which enhance the rate of the carbon oxidation without modifying the global mechanism reaction. Therefore, water acts as a catalyst for the carbon oxidation by NO2. A kinetic mechanism derived from this parametric study shows a decrease in the activation energy of carbon oxidation by NO2 in the presence of water vapor. This result is in agreement with the experimental observation. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 41: 236,244, 2009 [source] Thermal dimerization kinetics of 3-(p -bromo-phenyl)-pyridazinium benzoyl methylid in solutionsINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 5 2008V. Melnig 3-(p -Bromo-phenyl)-pyridazinium-benzoyl methylid (BPPBM) participates in solution at 3 + 3 dipolar thermal dimerization that can be spectrally monitored by the extinction in its visible intramolecular charge transfer (ICT) band. The attenuation of ICT band intensity shows the decrease of the BPPBM concentration with the increasing of dimer concentration. The complex kinetics of light-assisted dimerization process was studied taking into account that the thermodynamic equilibrium is reached after more than 24 h. On the basis of general order of reaction theory, we found that the dimerization reaction must be described as a multistep mechanism. The rate constants of the dimerization reactions in ethanol (k = 0.00897 s,1) and benzene (k = 0.00774 s,1) solutions were correlated with the BPPBM and dimer structural features established by using the HyperChem 5.02 simulation program package. A kinetic mechanism of 3 + 3 dipolar thermal dimerization for the studied ylid is proposed. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 230,239, 2008 [source] Experimental and modeling study of the autoignition of cyclopenteneINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 1 2008M. Yahyaoui Ignition delay times of cyclopentene,oxygen,argon mixtures were measured behind reflected shock waves. Mixtures contained 0.5% or 1% of hydrocarbons for equivalence ratios ranging from 0.5 to 1.5. Reflected shock wave conditions were as follows: temperatures from 1300 to 1700 K and pressures from 7 to 9 atm. When compared with the previous results obtained under similar conditions, it can be observed that the reactivity of cyclopentene is much lower than that of cyclohexene, but very close to that of cyclopentane. A kinetic mechanism recently proposed for the combustion of cyclopentene in a flame has been used to model these results, and a satisfactory agreement is obtained. The main reaction pathways have been derived from the flow rate, simulated temporal profiles of products, and sensitivity analyses. © 2007 Wiley Periodicals, Inc. Int J Chem Kinet 40: 25,33, 2008 [source] A high-temperature chemical kinetic model for primary reference fuelsINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 7 2007Marcos Chaos A chemical kinetic mechanism has been developed to describe the high-temperature oxidation and pyrolysis of n -heptane, iso -octane, and their mixtures. An approach previously developed by this laboratory was used here to partially reduce the mechanism while maintaining a desired level of detailed reaction information. The relevant mechanism involves 107 species undergoing 723 reactions and has been validated against an extensive set of experimental data gathered from the literature that includes shock tube ignition delay measurements, premixed laminar-burning velocities, variable pressure flow reactor, and jet-stirred reactor species profiles. The modeled experiments treat dynamic systems with pressures up to 15 atm, temperatures above 950 K, and equivalence ratios less than approximately 2.5. Given the stringent and comprehensive set of experimental conditions against which the model is tested, remarkably good agreement is obtained between experimental and model results. © 2007 Wiley Periodicals, Inc. Int J Chem Kinet 39: 399,414, 2007 [source] Role of phenoxy radicals in PCDD/F formationINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 9 2002Sukh Sidhu In this work, the role of phenoxy radicals in polychlorinated dibenzo- p -dioxins and polychlorinated dibenzofurans (PCDD/F) formation was investigated by studying the slow oxidation of 2-chlorophenol (2-CP) and 2-chloroanisole (2-CA) at a gas-phase concentration of 4 ppm (,2.1 × 104 ,g/m3) over a temperature range of 400,800°C. Residence times were maintained at 2.0 ± 0.10 s. PCDD/F reaction products were dibenzofuran, dibenzo- p -dioxin, 4-chlorodibenzofuran, 1-chlorodibenzo- p -dioxin, 4,6-dichlorodibenzofuran, and 1,6-dichlorodibenzo- p -dioxin (1,6-DCDD). Major products observed in these experiments were 2,6-dichlorophenol, 3-phenyl-2-propenal, 1-indanone, 1,3-isobenzofurandione, and 3-phenyl-2-propenoyl chloride. The 2-CP and 2-CA experiments, along with the variable concentration 2-CA experiments, showed that the concentration of radicals present in the oxidation system has a significant effect on the PCDD/F product distribution and ultimately the PCDD/PCDF ratio. Also, the observation of dichlorinated phenoxy phenol and dichlorinated dihydroxybiphenyl, the proposed intermediate species in the radical,radical mechanism, suggests that radical,radical mechanism dominates gas-phase PCDD/F formation. This information will be helpful in constructing a detailed kinetic mechanism of PCDD/F formation/destruction in combustor postcombustion zone. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 531,541, 2002 [source] The gas-phase oxidation of n -hexadecaneINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 10 2001R. Fournet Since n -hexadecane or cetane is a reference fuel for the estimation of cetane numbers in diesel engines, a detailed chemical model of its gas-phase oxidation and combustion will help to enhance diesel performance and reduce the emission of pollutants at their outlet. However, until recently the gas-phase reactions of n -hexadecane had not been experimentally studied, prohibiting a validation of oxidation models which could be written. This paper presents a modeling study of the oxidation of n -hexadecane based on experiments performed in a jet-stirred reactor, at temperatures ranging from 1000 to 1250 K, 1-atm pressure, a constant mean residence time of 0.07 s, and high degree of nitrogen dilution (0.03 mol% of fuel) for equivalence ratios equal to 0.5, 1, and 1.5. A detailed kinetic mechanism was automatically generated by using the computer package (EXGAS) developed in Nancy. The long linear chain of this alkane necessitates the use of a detailed secondary mechanism for the consumption of the alkenes formed as a result of primary parent fuel decomposition. This high-temperature mechanism includes 1787 reactions and 265 species, featuring satisfactory agreement for both the consumption of reactants and the formation of products. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 33: 574,586, 2001 [source] A Mu-class glutathione S -transferase from gills of the marine shrimp Litopenaeus vannamei: Purification and characterizationJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2007Carmen A. Contreras-Vergara Abstract Glutathione S -transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S -transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione,agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of ,25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 ,mol/(min mg), and the Km values for CDNB and GSH are very similar (390 and 335 ,M, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver,Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:62,67, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20162 [source] Rapid estimation of chemical kinetics by implicit calibration.JOURNAL OF CHEMOMETRICS, Issue 2 2003Abstract This study continues the development of a method, implicit calibration, for estimating kinetic parameters from on-line measurements of batch reactions. The basic idea of implicit calibration is to combine non-linear parameter estimation with the calibration of measured spectra with concentrations calculated by an assumed kinetic model. A new example is studied, an esterification reaction with a rather complicated kinetic mechanism, where activities, instead of concentrations, and NIR spectra are used as measurements. The emphasis in the study is on estimating the uncertainty of the kinetic parameters. Two approaches, linearization and bootstrap, are applied. In the case studied, the two approaches give closely similar estimates of the uncertainty. As well, a new way is introduced to control the rigidity of the implicit calibration, based on minimizing the lack of fit of the model. It is also shown that ,mixed implicit calibration', i.e. implicit calibration combined with a few off-line calibrated concentrations, greatly enhances the identifiability of the kinetic model. Copyright © 2003 John Wiley & Sons, Ltd. [source] Population balance modeling of particle size distribution in monomer-starved semibatch emulsion polymerizationAICHE JOURNAL, Issue 12 2009Shahriar Sajjadi Abstract The evolution of particle size distribution (PSD) in the monomer-starved semibatch emulsion polymerization of styrene with a neat monomer feed is investigated using a population balance model. The system under study ranges from conventional batch emulsion to semicontinuous (micro)emulsion polymerization depending on the rate of monomer addition. It is shown that, contrary to what is often believed, the broadness of PSD is not necessarily associated with the length of nucleation period. The PSDs at the end of nucleation are found to be independent of surfactant concentration. Simulation results indicate that at the completion of nucleation the particle size is reduced and the PSD narrows with decreasing rate of monomer addition despite nucleation time increasing. The broad distribution of particles frequently encountered in semibatch emulsion polymerizations is therefore attributed to stochastic broadening during the growth stage. The zero-one-two-three model developed in this article allows perceiving that the dominant kinetic mechanism may be different for particles with different sizes. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Aqueous polymerization of ethyl acrylate initiated by ceric ion-reducing agent systems in nitric acid mediumPOLYMER INTERNATIONAL, Issue 3 2001V Ramana Reddy Abstract Kinetic study of aqueous polymerization of ethyl acrylate (EA) was carried out at 30,°C in dilute nitric acid medium by employing ammonium ceric nitrate (ACN),methyl cellosolve (MC) and ACN,ethyl cellosolve (EC) as redox initiator systems. The ceric ion consumption was found to be first order with respect to ceric ion concentration with both initiator systems. The formation of complexes between Ce(IV) and reducing agents (RA) was observed. The order with respect to Ce(IV), reducing agents and monomer was evaluated for aqueous polymerization of EA by Ce(IV),MC and Ce(IV),EC redox initiator systems. The overall activation energy, Eoverall, for aqueous polymerization of EA was evaluated in the temperature region of 27,40,°C with both initiator systems. A kinetic mechanism for aqueous polymerization of EA initiated by redox initiator systems is presented. © 2001 Society of Chemical Industry [source] Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2,-IVaPROTEIN SCIENCE, Issue 8 2010Marta Toth Abstract Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2,-IVa (APH(2,)-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2,)-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (kcat/Km) of 1.5 × 103 to 1.2 × 106 (M,1 s,1). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2,)-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes. [source] Probing nonnucleoside inhibitor-induced active-site distortion in HIV-1 reverse transcriptase by transient kinetic analysesPROTEIN SCIENCE, Issue 8 2007Qing Xia Abstract Nonnucleoside reverse transcriptase inhibitors (NNRTI) are a group of structurally diverse compounds that bind to a single site in HIV-1 reverse transcriptase (RT), termed the NNRTI-binding pocket (NNRTI-BP). NNRTI binding to RT induces conformational changes in the enzyme that affect key elements of the polymerase active site and also the association between the two protein subunits. To determine which conformational changes contribute to the mechanism of inhibition of HIV-1 reverse transcription, we used transient kinetic analyses to probe the catalytic events that occur directly at the enzyme's polymerase active site when the NNRTI-BP was occupied by nevirapine, efavirenz, or delavirdine. Our results demonstrate that all NNRTI,RT,template/primer (NNRTI,RT,T/P) complexes displayed a metal-dependent increase in dNTP binding affinity (Kd) and a metal-independent decrease in the maximum rate of dNTP incorporation (kpol). The magnitude of the decrease in kpol was dependent on the NNRTI used in the assay: Efavirenz caused the largest decrease followed by delavirdine and then nevirapine. Analyses that were designed to probe direct effects on phosphodiester bond formation suggested that the NNRTI mediate their effects on the chemistry step of the DNA polymerization reaction via an indirect manner. Because each of the NNRTI analyzed in this study exerted largely similar phenotypic effects on single nucleotide addition reactions, whereas each of them are known to exert differential effects on RT dimerization, we conclude that the NNRTI effects on subunit association do not directly contribute to the kinetic mechanism of inhibition of DNA polymerization. [source] 3-Fluoro-2,4-dioxa-3-phosphadecalins as Inhibitors of Acetylcholinesterase.CHEMISTRY & BIODIVERSITY, Issue 3 2009A Reappraisal of Kinetic Mechanisms, Diagnostic Methods Abstract A systematic survey of the acetylcholine-mimetic 2,4-dioxa-3-phosphadecalins as irreversible inhibitors of acetylcholinesterase revealed hitherto overlooked properties as far as the kinetic mechanisms of interaction are concerned. As a support to past and future work in this field, we describe the kinetics of eight reaction schemes that may be found in irreversible enzyme modification and compare them with two mechanism of reversible, slow-binding inhibition. The relevant kinetic equations and their associated graphical representations are given for all mechanisms, and concrete examples illustrate their practical use. Since irreversible inhibition is a time-dependent phenomenon, kinetic analysis is greatly facilitated by fitting the appropriate integrated rate equations to reaction-progress curves by nonlinear regression. This primary scrutiny provides kinetic parameters that are indispensable tools for diagnosing the kinetic mechanism and for calculating inhibition constants. Numerical integration of sets of differential equations is an additional useful investigation tool in critical situations, e.g., when inhibitors are unstable and/or act as irreversible modifiers only temporarily. [source] Kinetic mechanism for p38 MAP kinase ,FEBS JOURNAL, Issue 18 2005A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 µm, KI = 240 µm, and KI > 2000 µm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 µm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source] Multiple criticality and staged ignition of methane in the counterflowINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 12 2009Wei Liu The counterflow ignition of methane was investigated, with particular interest in the role of radical versus thermal runaway. Simulation with different kinetic mechanisms showed that the ignition response is qualitatively sensitive to the kinetic mechanism adopted, either exhibiting or not exhibiting two ignition turning points in the S-curve response. For the former situation, ignition could take place in a staged manner with either increasing temperature or decreasing strain rate of the counterflow, characterized by the first ignition event being radical induced with negligible thermal effect and the second ignition event requiring thermal feedback. Sensitivity analysis and a recently developed chemical explosive mode analysis were performed to identify the dominant reactions in the radical runaway stage, revealing the importance of branching pathways involving the HO2 radical. Counterflow experiments were also conducted, yielding useful information on the ignition response. The study identifies items of further research to sharpen the comprehensiveness and accuracy of the methane oxidation mechanism. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 764,776, 2009 [source] A shock tube study of cyclopentane and cyclohexane ignition at elevated pressuresINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 10 2008Shane M. Daley Ignition delay times for cyclopentane/air and cyclohexane/air mixtures were measured in a shock tube at temperatures of 847,1379 K, pressures of 11,61 atm, and equivalence ratios of , = 1.0, 0.5, and 0.25. Ignition times were determined using electronically excited OH emission monitored through the shock tube endwall and piezoelectric pressure measurements made in the shock tube sidewall. The dependence of ignition time on pressure, temperature, and equivalence ratio is quantified and correlations for ignition time formulated. Measured ignition times are compared to kinetic modeling predictions from four recently published mechanisms. The data presented provide a database for the validation of cycloalkane kinetic mechanisms at the elevated pressures found in practical combustion engines. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 624,634, 2008 [source] Capturing pressure-dependence in automated mechanism generation: Reactions through cycloalkyl intermediatesINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 3 2003David M. Matheu Chemical kinetic mechanisms for gas-phase processes (including combustion, pyrolysis, partial oxidation, or the atmospheric oxidation of organics) will often contain hundreds of species and thousands of reactions. The size and complexity of such models, and the need to ensure that important pathways are not left out, have inspired the use of computer tools to generate such large chemical mechanisms automatically. But the models produced by existing computerized mechanism generation codes, as well as a great many large mechanisms generated by hand, do not include pressure-dependence in a general way. This is due to the difficulty of computing the large number of k(T, P) estimates required. Here we present a fast, automated method for computing k(T, P) on-the-fly during automated mechanism generation. It uses as its principal inputs the same high-pressure-limit rate estimation rules and group-additivity thermochemistry estimates employed by existing computerized mechanism-generation codes, and automatically identifies the important chemically activated intermediates and pathways. We demonstrate the usefulness of this approach on a series of pressure-dependent reactions through cycloalkyl radical intermediates, including systems with over 90 isomers and 200 accessible product channels. We test the accuracy of these computer-generated k(T, P) estimates against experimental data on the systems H + cyclobutene, H + cyclopentene, H + cyclohexene, C2H3 + C2H4, and C3H5 + C2H4, and make predictions for temperatures and pressures where no experimental data are available. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 35: 95,119, 2003 [source] KINETICS of QUALITY CHANGE DURING COOKING and FRYING of POTATOES: PART I. TEXTUREJOURNAL OF FOOD PROCESS ENGINEERING, Issue 4 2003F. NOURIAN ABSTRACT Kinetics of texture change during cooking and frying of potatoes were evaluated in this study. Potatoes were cut into cylinders (diameter × height: 20 mm × 20 mm for cooking, and 10 mm × 20 mm for frying) and cooked in a temperature controlled water bath at 80,100C or fried in a commercial fryer at 160,190C for selected times. the cooked samples were water cooled while the fried samples were air cooled immediately after the treatment. Test samples were then subjected to a single cycle compression test in a computer interfaced Universal Testing Machine and three textural properties (hardness, stiffness and firmness) were derived from the resulting force-deformation curves. Texture parameters of cooked potatoes decreased with progress of cooking time and the rate of texture changes associated with each temperature was found to be consistent with two pseudo first-order kinetic mechanisms, one more rapid than the other. Textural values of fried potatoes were found to increase with frying time and also followed a first order kinetic model. Temperature sensitivity of rate constants was adequately described by Arrhenius and z-value models. [source] Design of flexible reduced kinetic mechanismsAICHE JOURNAL, Issue 11 2001Avinash R. Sirdeshpande Reduced mechanisms are often used in place of detailed chemistry because the computational burden of including all the species continuity equations in the reactor model is unreasonably high. Contemporary reduction techniques produce mechanisms that depend strongly on the nominal set of problem parameters for which the reduction is carried out. Effects of variability in these parameters on the reduced mechanism are the focus of this work. The range of validity of a reduced mechanism is determined for variations in initial conditions. Both sampling approaches and quantitative measures of feasibility, such as the flexibility index and the convex hull formulation, are employed. The inverse problem of designing a reduced mechanism that covers the desired range of initial conditions is addressed using a multiperiod approach. The effect of the value of a user-defined tolerance parameter, which determines whether the predictions made by the reduced mechanism are acceptable, is also assessed. The analytical techniques are illustrated with examples from the literature. [source] Prediction of Polymer Properties in LDPE ReactorsMACROMOLECULAR MATERIALS & ENGINEERING, Issue 4 2005Gary J. Wells Abstract Summary: A new analysis tool is presented that uses the governing kinetic scheme to predict properties of low-density polyethylene (LDPE) such as the detailed shape of the molecular weight distribution (MWD). A model that captures mixing details of autoclave reactor operation is used to provide a new criterion for the onset of MWD shouldering. Kinetic effects are shown to govern the existence of MWD shoulders in LDPE reactors, even when operation is far from perfectly-mixed. MWD shoulders occur when the mean reaction environment has a relatively high radical concentration and has a high polymer content, and is at a low temperature. Such conditions maximize long chain formation by polymer transfer and combination-termination, while limiting chain scission. For imperfectly-mixed reactors, the blending of polymer-distributions produced in different spatial locations has a small effect on the composite MWD. However, for adiabatic LDPE autoclaves, imperfect mixing broadens the stable range of mean reactor conditions, and thereby increases the possibility for MWD shouldering. Polymer MWD produced in an LDPE autoclave reactor by various kinetic mechanisms. [source] Challenges of introducing quantitative elementary reactions in multiscale models of thin film depositionPHYSICA STATUS SOLIDI (B) BASIC SOLID STATE PHYSICS, Issue 9 2010Alessandro Barbato Abstract The implementation of detailed surface kinetic mechanisms describing the thin film growth dynamics into models of chemical vapor deposition (CVD) reactors has been a challenge for many years. In this article we review the literature concerning the study of the dynamics of the Si(100)2,×,1 surface and introduce a multiscale model that captures the main features of its reactivity. The model combines the results of ab initio calculations with an atomistic description of the Si surface, obtained using a 3D-kinetic Monte Carlo (KMC) model that explicitly accounts for the 2,×,1 surface reconstruction and the formation and diffusion of Si dimers on a hydrogenated surface. At the atomistic scale, we determined pre-exponential factors and activation energies of hydrogen desorption reactions proceeding through the 2H, 3H, and 4H mechanisms. The calculated kinetic constants were embedded in the KMC model and used to simulate literature TPD experimental data. The simulations were used to fit the activation energies of hydrogen desorption reactions, which showed that DFT calculations performed with B3LYP functionals are likely to overestimate hydrogen desorption energies by up to 9,kcal,mol,1, which was confirmed by successive ab initio calculations. Two examples of the solution of the KMC model in conjunction with a reactor scale model are provided, in which the coupling was performed adopting both a hierarchic and a two-way coupling strategy. We found that in the plasma deposition of nanocrystalline silicon performed at low substrate temperatures the growth proceeds through a layer-by-layer mechanism on a surface almost completely covered by hydrogen. The application of the same model to the simulation of the thermal CVD of Si showed that at intermediate growth temperatures, when the hydrogen surface concentration is high, a new hydrogen desorption mechanism, in which Si adatoms play an important role, is active. Length scales encountered in multiscale modeling of thin films deposition. [source] The Oxidation Kinetics of Mercury in Hg/O/H/Cl SystemASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 3-4 2005Y. Qiao The potential for regulation of mercury emission from coal-fired boilers is a concern for the electric utility industry. Field data show a wide variation in the fraction of mercury that is emitted as a vapor versus that retained in the solid products. The reason for this variation is not well understood. Near the end of the flue gas path, mercury exists as a combination of elemental vapor and HgCl2 vapor. The data show that HCl2 is more likely to be removed form the flue gas. The need to describe accurately mercury reaction products and their concentration-time correlation prompted investigation of mercury chemical kinetic mechanisms and their application to real combustion systems. This paper uses chemical equilibrium analysis to study the speciation of mercury in flue gases during coal combustion and gasification. The paper presents a simple kinetic model of mercury oxidation in the Hg/O/H/Cl system. The results porn the model calculation are in reasonable agreement with the Mamani-Paco and Heleble [1] experimental data [source] 3-Fluoro-2,4-dioxa-3-phosphadecalins as Inhibitors of Acetylcholinesterase.CHEMISTRY & BIODIVERSITY, Issue 3 2009A Reappraisal of Kinetic Mechanisms, Diagnostic Methods Abstract A systematic survey of the acetylcholine-mimetic 2,4-dioxa-3-phosphadecalins as irreversible inhibitors of acetylcholinesterase revealed hitherto overlooked properties as far as the kinetic mechanisms of interaction are concerned. As a support to past and future work in this field, we describe the kinetics of eight reaction schemes that may be found in irreversible enzyme modification and compare them with two mechanism of reversible, slow-binding inhibition. The relevant kinetic equations and their associated graphical representations are given for all mechanisms, and concrete examples illustrate their practical use. Since irreversible inhibition is a time-dependent phenomenon, kinetic analysis is greatly facilitated by fitting the appropriate integrated rate equations to reaction-progress curves by nonlinear regression. This primary scrutiny provides kinetic parameters that are indispensable tools for diagnosing the kinetic mechanism and for calculating inhibition constants. Numerical integration of sets of differential equations is an additional useful investigation tool in critical situations, e.g., when inhibitors are unstable and/or act as irreversible modifiers only temporarily. [source] | ||||||||||||||||