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Role of cis-cis muconic acid in the catalysis of Pseudomonas putida chlorocatechol 1,2-dioxygenase

MELO, Fernando A.; ARAUJO, Ana Paula Ulian de; COSTA FILHO, Antonio José da
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
47.299336%
Chlorocatechol 1,2-dioxygenase (1,2-CCD) is a non-heme iron protein involved in the intradiol cleavage of aromatic compounds that are recalcitrant to biodegradation. In particular, 1,2-CCD catalyzes the conversion of catechol and its halogenated derivatives to cis-cis muconic acid. In this study we describe a series of experiments concerning the interaction of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp1,2-CCD) with cis-cis muconic acid. We used single-injection ITC to show that the reaction product inhibits enzyme kinetics. DSC and EPR measurements probed whether this was accomplished by a direct binding of the product to the enzyme active site. DSC shows that cis-cis muconic acid affects the thermal unfolding of the protein and allowed us to estimate a binding constant. Furthermore, EPR spectra of the Fe(III) center demonstrate that, upon product binding, a significant decrease in resonance intensity is observed, indicating that cis-cis muconic acid binds directly to the active site. Based on the increasing interest for understanding dioxygenases mechanism of action and, moreover, how to control such process, our data indicate that the product of the reaction does play a relevant role in the catalysis and should therefore be taken into account when one thinks about ways of regulating enzyme activity. (C) 2010 Elsevier B.V. All rights reserved.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); CAPES

Estudos da correlação estrutura-função da enzima Clorocatecol 1,2-Dioxigenase de Pseudomonas putida; Studies of the structure-function correlation of the chlorocatechol 1,2-dioxygenase enzyme from Pseudomonas putida

Mesquita, Nathalya Cristina de Moraes Roso
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 13/02/2012 PT
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47.26044%
O intenso uso de compostos orgânicos em conjunto com o grande avanço industrial culminou em um enorme acúmulo de poluentes orgânicos no meio ambiente. Dentre estes poluentes têm-se destacado a presença de hidrocarbonetos aromáticos altamente tóxicos e resistentes à degradação física, química, fotolítica e biológica. Desta maneira, uma nova forma de combater a presença deste tipo de composto no meio ambiente têm sido estudada: o uso de microorganismos, naturais ou geneticamente modificados, capazes de transformá-los em substâncias inertes, como CO2 e água. Tal metodologia é denominada biorremediação. Dentres estes microorganismos destacam-se bactérias dos gêneros Pseudomonas, Aeromonas, Beijerinckia, dentre outros, que têm sido estudadas para esta finalidade. A enzima clorocatecol 1,2-dioxigenase (Pp 1,2-CCD) é uma das proteínas expressas por bactérias do gênero Pseudomonas putida, sendo responsável pela clivagem de hidrocarbonetos aromáticos através da incorporação de ambos os átomos de uma molécula de oxigênio à estrutura do anel aromático, sendo a proteína escolhida para desenvolvermos o presente trabalho. Mais especificamente, nos interessa estudar como o mecanismo de ação da referida enzima é controlado por moléculas extrínsecas...

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Silva, Rafael G.; Pereira, José H.; Canduri, Fernanda; De Azevedo Jr., Walter F.; Basso, Luiz A.; Santos, Diógenes S.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 49-58
ENG
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47.41782%
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Rosado, Leonardo Astolfi; Vasconcelos, Igor Bordin; Palma, Mario Sergio; Frappier, Vincent; Najmanovich, Rafael Josef; Santos, Diógenes Santiago; Basso, Luiz Augusto
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica
ENG
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47.299336%
Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

Effect of heat and thermosonication treatments on peroxidise inactivation kinetics in watercress (nasturtium officinale)

Cruz, Rui M.S.; Vieira, Margarida C.; Silva, Cristina L.M.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2006 ENG
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47.29383%
The effect of heat and the combined heat/ultrasound (thermosonication) treatment on the inactivation kinetics of peroxidase in watercress (Nasturtium officinale) was studied in the temperature range of 40–92.5 C. In the heat blanching processes, the enzyme kinetics showed a first-order biphasic inactivation model. The activation energies and the rates of the reaction at a reference temperature for both the heat-labile and heat-resistant fractions were, respectively, Ea1 = 421 ± 115 kJmol 1 and Ea2 = 352 ± 81 kJmol 1, k184:6 C ¼ 18 14min 1 and k284:6 C ¼ 0:24 0:14min 1. The initial relative specific activity for both isoenzyme fractions were also estimated, being C01 = 0.5 ± 0.08 lmolmin 1mg protein 1 and C02 = 0.5 ± 0.06 lmolmin 1mg protein 1, respectively. The application of thermosonication was studied to enable less severe thermal treatments and, therefore, improving the quality of the blanched product. In this treatment the enzyme kinetics showed a first-order model. The activation energy, the rate of reaction at a reference temperature and the initial relative specific activity were, respectively, Ea3 = 496 ± 65 kJmol 1, k387:5 C ¼ 10 2min 1 and C03 = 1 ± 0.05 lmolmin 1mg protein 1, proving that the enzyme became more heat labile. The present findings will help to design the blanching conditions for the production of a new and healthy frozen product...

A computer program for enzyme kinetics that combines model discrimination, parameter refinement and sequential experimental design.

Franco, R; Gavaldà, M T; Canela, E I
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/09/1986 EN
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A method of model discrimination and parameter estimation in enzyme kinetics is proposed. The experimental design and analysis of the model are carried out simultaneously and the stopping rule for experimentation is deduced by the experimenter when the probabilities a posteriori indicate that one model is clearly superior to the rest. A FORTRAN77 program specifically developed for joint designs is given. The method is very powerful, as indicated by its usefulness in the discrimination between models. For example, it has been successfully applied to three cases of enzyme kinetics (a single-substrate Michaelian reaction with product inhibition, a single-substrate complex reaction and a two-substrate reaction). By using this method the most probable model and the estimates of the parameters can be obtained in one experimental session. The FORTRAN77 program is deposited as Supplementary Publication SUP 50134 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5.

Half-time analysis of the integrated Michaelis equation. Simulation and use of the half-time plot and its direct linear variant in the analysis of some α-chymotrypsin-, papain- and fumarase-catalysed reactions

Wharton, Christopher W.; Szawelski, Ronald J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1982 EN
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47.21384%
Substitution of half-time parameters in the integrated form of the Michaelis–Menten equation for any enzyme-catalysed reaction yields an equation that gives a linear relationship between the half-time of the reaction and the substrate concentration at that point of the reaction. The logarithmic term of the integrated equation becomes a constant as a result of the substitution, which means that the use of the half-time plot of the equation requires calculation only of half-time and substrate-concentration values at various stages of the reaction. The half-time method is both simple and exact, being analogous to an [S0]/vi against [S0] plot. A direct linear form of the half-time plot has been devised that allows very simple estimation of Michaelis parameters and/or initial velocities from progress-curve data. This method involves no approximation and is statistically valid. Simulation studies have shown that linear-regression analysis of half-time plots provides unbiased estimates of the Michaelis parameters. Simulation of the effect of error in estimation of the product concentration at infinite time [P∞] reveals that this is always a cause for concern, such errors being magnified approximately an order of magnitude in the estimate of the Michaelis constant. Both the half-time plot and the direct linear form have been applied to the analysis of a variety of experimental data. The method has been shown to produce excellent results provided certain simple rules are followed regarding criteria of experimental design. A set of rules has been formulated that...

Normalization in the fitting of data by iterative methods. Application to tracer kinetics and enzyme kinetics

Ottaway, J. H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1973 EN
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47.33853%
1. The normalization of biochemical data to weight them appropriately for parameter estimation is considered, with reference particularly to data from tracer kinetics and enzyme kinetics. If the data are in replicate, it is recommended that the sum of squared deviations for each experimental variable at each time or concentration point is divided by the local variance at that point. 2. If there is only one observation for each variable at each sampling point, normalization may still be required if the observations cover more than one order of magnitude, but there is no absolute criterion for judging the effect of the weighting that is produced. The goodness of fit that is produced by minimizing the weighted sum of squares of deviations must be judged subjectively. It is suggested that the goodness of fit may be regarded as satisfactory if the data points are distributed uniformly on either side of the fitted curve. A chi-square test may be used to decide whether the distribution is abnormal. The proportion of the residual variance associated with points on one or other side of the fitted curve may also be taken into account, because this gives an indication of the sensitivity of the residual variance to movement of the curve away from particular data points. These criteria for judging the effect of weighting are only valid if the model equation may reasonably be expected to apply to all the data points. 3. On this basis...

Dynamics of Muscle Glycogenolysis Modeled with pH Time Course Computation and pH-Dependent Reaction Equilibria and Enzyme Kinetics

Vinnakota, Kalyan; Kemp, Melissa L.; Kushmerick, Martin J.
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica
EN
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47.263486%
Cellular metabolites are moieties defined by their specific binding constants to H+, Mg2+, and K+ or anions without ligands. As a consequence, every biochemical reaction in the cytoplasm has an associated proton stoichiometry that is generally noninteger- and pH-dependent. Therefore, with metabolic flux, pH is altered in a medium with finite buffer capacity. Apparent equilibrium constants and maximum enzyme velocities, which are functions of pH, are also altered. We augmented an earlier mathematical model of skeletal muscle glycogenolysis with pH-dependent enzyme kinetics and reaction equilibria to compute the time course of pH changes. Analysis shows that kinetics and final equilibrium states of the closed system are highly constrained by the pH-dependent parameters. This kinetic model of glycogenolysis, coupled to creatine kinase and adenylate kinase, simulated published experiments made with a cell-free enzyme mixture to reconstitute the network and to synthesize PCr and lactate in vitro. Using the enzyme kinetic and thermodynamic data in the literature, the simulations required minimal adjustments of parameters to describe the data. These results show that incorporation of appropriate physical chemistry of the reactions with accurate kinetic modeling gives a reasonable simulation of experimental data and is necessary for a physically correct representation of the metabolic network. The approach is general for modeling metabolic networks beyond the specific pathway and conditions presented here.

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

Hartwell, Supaporn Kradtap; Grudpan, Kate
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
47.41676%
Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

Determining Enzyme Kinetics for Systems Biology with Nuclear Magnetic Resonance Spectroscopy

Eicher, Johann J.; Snoep, Jacky L.; Rohwer, Johann M.
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 06/11/2012 EN
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47.150854%
Enzyme kinetics for systems biology should ideally yield information about the enzyme’s activity under in vivo conditions, including such reaction features as substrate cooperativity, reversibility and allostery, and be applicable to enzymatic reactions with multiple substrates. A large body of enzyme-kinetic data in the literature is based on the uni-substrate Michaelis-Menten equation, which makes unnatural assumptions about enzymatic reactions (e.g., irreversibility), and its application in systems biology models is therefore limited. To overcome this limitation, we have utilised NMR time-course data in a combined theoretical and experimental approach to parameterize the generic reversible Hill equation, which is capable of describing enzymatic reactions in terms of all the properties mentioned above and has fewer parameters than detailed mechanistic kinetic equations; these parameters are moreover defined operationally. Traditionally, enzyme kinetic data have been obtained from initial-rate studies, often using assays coupled to NAD(P)H-producing or NAD(P)H-consuming reactions. However, these assays are very labour-intensive, especially for detailed characterisation of multi-substrate reactions. We here present a cost-effective and relatively rapid method for obtaining enzyme-kinetic parameters from metabolite time-course data generated using NMR spectroscopy. The method requires fewer runs than traditional initial-rate studies and yields more information per experiment...

ERK as a model for systems biology of enzyme kinetics in cells

Futran, Alan S.; Link, A. James; Seger, Rony; Shvartsman, Stanislav Y.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 04/11/2013 EN
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47.41676%
A key step towards a chemical picture of enzyme catalysis was taken in 1913, when Leonor Michaelis and Maud Menten published their studies of sucrose hydrolysis by invertase. Based on a novel experimental design and a mathematical model, their work offered a quantitative view of biochemical kinetics well before the protein nature of enzymes was established and complexes with substrates could be detected. Michaelis-Menten kinetics provides a solid framework for enzyme kinetics in vitro, but what about kinetics in cells, where enzymes can be highly regulated and participate in a multitude of interactions? We discuss this question using the Extracellular Signal Regulated Kinase (ERK) as a model of an important enzyme for which we have crystal structures, quantitative in vitro assays, and a vast list of binding partners. Despite great progress, we still cannot quantitatively predict how the rates of ERK-dependent reactions respond to genetic and pharmacological perturbations. Achieving this goal, which is important from both fundamental and practical standpoints, requires measuring the rates of enzyme reactions in their native environment and interpreting these measurements using simple but realistic mathematical models, the two elements which served as the cornerstones for the seminal 1913 paper.

A Linear Framework for Time-Scale Separation in Nonlinear Biochemical Systems

Gunawardena, Jeremy H.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
Relevância na Pesquisa
47.140557%
Cellular physiology is implemented by formidably complex biochemical systems with highly nonlinear dynamics, presenting a challenge for both experiment and theory. Time-scale separation has been one of the few theoretical methods for distilling general principles from such complexity. It has provided essential insights in areas such as enzyme kinetics, allosteric enzymes, G-protein coupled receptors, ion channels, gene regulation and post-translational modification. In each case, internal molecular complexity has been eliminated, leading to rational algebraic expressions among the remaining components. This has yielded familiar formulas such as those of Michaelis-Menten in enzyme kinetics, Monod-Wyman-Changeux in allostery and Ackers-Johnson-Shea in gene regulation. Here we show that these calculations are all instances of a single graph-theoretic framework. Despite the biochemical nonlinearity to which it is applied, this framework is entirely linear, yet requires no approximation. We show that elimination of internal complexity is feasible when the relevant graph is strongly connected. The framework provides a new methodology with the potential to subdue combinatorial explosion at the molecular level.

Genotype-phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies

Bunge, S.; Clements, P.; Byers, S.; Kleijer, W.; Brooks, D.; Hopwood, J.
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
Publicado em //1998 EN
Relevância na Pesquisa
47.150854%
Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype...

Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase

Hrmova, M.; Varghese, J.; De Gori, R.; Smith, B.; Driguez, H.; Fincher, G.
Fonte: Cell Press Publicador: Cell Press
Tipo: Artigo de Revista Científica
Publicado em //2001 EN
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47.177964%
Background: Barley β-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from β-D-glucans and β-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. Results: When conduritol B epoxide and 2′, 4′-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Oδ1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4I, 4III, 4V-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the −1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 Å) with the Oε2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C1 conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. Conclusions: X-ray crystallography is used to define the three key intermediates during catalysis by β-D-glucan glucohydrolase. Before a new hydrolytic event begins...

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

Johnson, Paley; Whateley, Tony L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/01/1981 EN
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47.33853%
The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol−1·dm3·s−1 for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol−1·dm3·s−1 for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution...

Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[β-imido]triphosphate of smooth-muscle adenylate cyclase

Krall, J. Frederick; Leshon, Steven C.; Korenman, Stanley G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/1982 EN
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47.140557%
Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased Km for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH0 ∼8kJ·mol−1) and accompanied by an increase in entropy (ΔS0 ∼146J·mol−1·K−1). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the Km at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.

Cooperativity and Specificity in Enzyme Kinetics: A Single-Molecule Time-Based Perspective

Qian, Hong
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
EN
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47.263486%
An alternative theoretical approach to enzyme kinetics that is particularly applicable to single-molecule enzymology is presented. The theory, originated by Van Slyke and Cullen in 1914, develops enzyme kinetics from a “time perspective” rather than the traditional “rate perspective” and emphasizes the nonequilibrium steady-state nature of enzymatic reactions and the significance of small copy numbers of enzyme molecules in living cells. Sigmoidal cooperative substrate binding to slowly fluctuating, monomeric enzymes is shown to arise from association pathways with very small probability but extremely long passage time, which would be disregarded in the traditional rate perspective: A single enzyme stochastically takes alternative pathways in serial order rather than different pathways in parallel. The theory unifies dynamic cooperativity and Hopfield-Ninio's kinetic proofreading mechanism for specificity amplification.

Exact and approximate solutions for a century-old problem: A general treatment of Henri-Michaelis-Menten enzyme kinetics

Berberan-Santos, Mario N.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 06/05/2009
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47.305215%
A different view of Henri-Michaelis-Menten (HMM) enzyme kinetics is presented. In the first part of the paper, a simplified but useful description that stresses the cyclic nature of the catalytic process is introduced. The time-dependence of the substrate concentration after the initial transient phase is derived in a simple way that dispenses the mathematical technique known as quasi-steady-state approximation. In the second part of the paper an exact one-dimensional formulation of HMM kinetics is obtained. The whole problem is condensed in a single one-variable evolution equation that is a second-order non-linear differential equation, and the control parameters are reduced to three dimensionless quantities: enzyme efficiency, substrate reduced initial concentration, and enzyme reduced initial concentration. The exact solution of HMM kinetics is obtained as a set of Maclaurin series. From the same equation, a number of approximate solutions, some known, some new, are derived in a systematic way that allows a precise evaluation of the respective level of approximation and conditions of validity. The evolution equation derived is also shown to be well suited for the numerical computation of the concentrations of all species as a function of time for any given combination of parameters.; Comment: 32 pages 10 Figures

Hofmeister specific-ion effects on enzyme activity and buffer pH: Horseradish peroxidase in citrate buffer

Bauduin, Pierre; Nohmie, Fawaz; Touraud, Didier; Neueder, Roland; Kunz, Werner; Ninham, Barry
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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57.21384%
Salt addition to enzymes in buffer always induce the problem of the respective influences of electrostatic interactions and anion specificity on buffer pH and enzyme kinetics. In the present paper the influence of some sodium salts (Na2SO4, NaCl, NaBr and NaNO3) on the pH of a citrate buffer (c = 0.025 M), and on the catalytic constants of horseradish peroxidase (HRP) is studied. First, the pH changes due to the presence of salts in the buffer are examined; second, catalytic constants, KmABTS, VmaxABTS and V maxABTS/KmABTS, are studied as a function of pH in buffer with and without added salt, at various salt concentrations and different pH values due to the salt additions. With a simple electrostatic model, it is possible to show that the glass electrode yields reasonable pH values even in the presence of fairly high 1 : 1 salt concentrations. For the catalytic efficiency, VmaxABTS/KmABTS, a Hofmeister series is found with opposite deviations from the pure pH effect for salting-in and salting-out ions over a large range of salt concentrations. This usual Hofmeister series is a consequence of three, for the moment inseparable salt concentration and specific ion-induced phenomena: global bulk effects, local active site effects and surface effects.