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Menten Model (menten + model)
Selected AbstractsCapillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4ELECTROPHORESIS, Issue 8 2006Minoo Afshar Abstract An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N -despropyl-propafenone (NOR-PPF). Using in,vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N -dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R -PPF only, which can be described by the Michaelis,Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in,vitro kinetics of metabolic steps of a drug. [source] On the Mechanism of Biotransformation of the Anthraquinonic Dye Acid Blue 62 by LaccasesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2009Luciana Pereira Abstract We used the recombinant CotA-laccase from the bacterium Bacillus subtilis to investigate the biotransformation of the commercial anthraquinonic dye Acid Blue 62. Kinetics of dye biotransformation at pH,6 follow a Michaelis,Menten model. NMR and several MS techniques allowed the identification of intermediates and final products of the enzymatic biotransformation. The main final product obtained, 1-[(4-amino-9,10-dioxo-3-sulfo-9,10-dihydroanthracen-1-yl)diazenyl]-4-cyclohexylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid, is formed through the creation of an azo link and has been previously identified as an intermediate compound in the biodegradation of Acid Blue 62 by crude fungal preparations. The identification of 1,4-diamino-9,10-dioxo-3-sulfo-9,10-dihydroanthracene-2-sulfonic acid and of cyclohexanone, in reaction mixtures with CotA-laccase and also its presence in reactions performed with the LAC3 laccase from the fungus Trametes sp. C30, suggest the occurrence of coupling reactions between the intermediate products of dye oxidation. Based on these results, we propose a mechanistic pathway for the biotransformation of Acid Blue 62 by laccases. A bioassay based on the inhibitory effects of the dye and its enzymatic products on the growth of Saccharomyces cerevisiae shows the importance of laccases in reducing dye toxicity. [source] Heterogeneity of the coumarin anticoagulant targeted vitamin K epoxide reduction system.JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2006Study of kinetic parameters in susceptible, resistant mice (Mus musculus domesticus) Abstract Vitamin K epoxide reductase (VKOR) activity in liver microsomes from a susceptible and a genetically warfarin-resistant strain of mice (Mus Musculus domesticus) was analyzed to determine the mechanism of resistance to this 4-hydroxycoumarin derivative. Kinetic parameters for VKOR were calculated for each strain by incubating liver microsomes with vitamin K epoxide ± warfarin. In susceptible mice, an Eadie,Hofstee plot of the data was not linear and suggested the involvement of at least two different components. Apparent kinetic parameters were obtained by nonlinear regression using a Michaelis--Menten model, which takes into account two enzymatic components. Component A presents a high Km and a high Vm, and as a consequence only an enzymatic efficiency Vm/Km was obtained (0.0024 mL/min/mg). Estimated warfarin Ki was 0.17 ,M. Component B presented an apparent Km of 12.73 ,M, an apparent Vm of 0.32 nmol/min/mg, and an apparent Ki for warfarin of 6.0 ,M. In resistant mice, the enzymatic efficiency corresponding to component A was highly decreased (0.0003,0.00066 mL/min/mg) while the Ki for warfarin was not modified. The apparent Vm of component B was poorly modified between susceptible and resistant mice. The apparent Km of component B observed in resistant mice was similar to the Km observed in susceptible mice. These modifications of the catalytic properties are associated with a single nucleotide polymorphism (T175G) in the VKOR-C1 gene, which corresponds to a Trp59Gly mutation in the protein. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:221,229, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20144 [source] The turnover of the H3 deuterons from (2- 13C) glutamate and (2- 13C) glutamine reveals subcellular trafficking in the brain of partially deuterated ratsJOURNAL OF NEUROCHEMISTRY, Issue 2009Sebastián Cerdán Abstract We investigated by 13C NMR the turnover of the H3 deuterons of (2- 13C) glutamate and (2- 13C) glutamine in the brain of partially deuterated rats. Adult animals (150,200 g) fed ad libitum received 50%2H2O or tap water 9 days before infusing (1- 13C) glucose or (2- 13C) acetate for 5, 10, 15, 30, 60, or 90 min. The brains were then funnel-frozen and acid extracts were prepared and analyzed by high-resolution 13C NMR. The deuteration of one or the two H3 hydrogens of (2- 13C) glutamate or glutamine resulted in single (,0.07 ppm) or double (,0.14 ppm) isotopic shifts upfield of the corresponding C2 perprotonated resonance, demonstrating two sequential deuteration steps. The faster monodeuteration generated 3R or 3S (2- 13C, 3- 2H) glutamate or glutamine through the alternate activities of cerebral aconitase or isocitrate dehydrogenase, respectively. The slower process produced bideuterated (2- 13C, 3,3,- 2H2) glutamate or glutamine through the consecutive activity of both enzymes. The kinetics of deuteration was fitted to a Michaelis,Menten model including the apparent Km, and Vmax, values for the observed deuterations. Our results revealed different kinetic constants for the alternate and consecutive deuterations, suggesting that these processes were caused by the different cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, respectively. The deuterations of (2- 13C) glutamate or glutamine followed also different kinetics from (1- 13C) glucose or (2- 13C) acetate, revealing distinct deuteration environments in the neuronal or glial compartments. [source] Modelling the effect of superatmospheric oxygen concentrations on in vitro mushroom PPO activity,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2006Perla A Gómez Abstract The kinetics of polyphenol oxidase (PPO, EC 1.14.18.1) with respect to oxygen concentrations from 5 to 100% using chlorogenic acid (CGA) as substrate was examined. In vitro mushroom PPO activity was determined by measuring the consumption of oxygen during the oxidation reaction. A differential Michaelis,Menten model was fitted to the obtained total depletion curves. The product concentration as well as the concentration of oxygen had a clear inhibitory effect on the reaction rate. However, the inhibitory effect of oxygen was more evident at low product concentration. A linear mixed inhibition model that considered both the product (oxidised CGA) and oxygen as inhibitors was developed. A model with the product as a competitive inhibitor and oxygen as an uncompetitive inhibitor was the most appropriate to explain the reaction kinetics. The values of the inhibition constants calculated from the model were 0.0032 mmol L,1 for Km (Michaelis,Menten constant related to oxygen), 0.023 mmol L,1 for Kmc (constant for competitive inhibition due to the product), 1.630 mmol L,1 for Kmu (constant for uncompetitive inhibition due to oxygen) and 1.77 × 10,4 mmol L,1 s,1 for Vmax (maximum reaction rate). The results indicate that superatmospheric oxygen concentrations could be effective in preventing enzymatic browning by PPO. Copyright © 2006 Society of Chemical Industry [source] Statistical Reconstruction of Transcription Factor Activity Using Michaelis,Menten KineticsBIOMETRICS, Issue 3 2007R. Khanin Summary The basic building block of a gene regulatory network consists of a gene encoding a transcription factor (TF) and the gene(s) it regulates. Considerable efforts have been directed recently at devising experiments and algorithms to determine TFs and their corresponding target genes using gene expression and other types of data. The underlying problem is that the expression of a gene coding for the TF provides only limited information about the activity of the TF, which can also be controlled posttranscriptionally. In the absence of a reliable technology to routinely measure the activity of regulators, it is of great importance to understand whether this activity can be inferred from gene expression data. We here develop a statistical framework to reconstruct the activity of a TF from gene expression data of the target genes in its regulatory module. The novelty of our approach is that we embed the deterministic Michaelis,Menten model of gene regulation in this statistical framework. The kinetic parameters of the gene regulation model are inferred together with the profile of the TF regulator. We also obtain a goodness-of-fit test to verify the fit of the model. The model is applied to a time series involving the Streptomyces coelicolor bacterium. We focus on the transcriptional activator cdaR, which is partly responsible for the production of a particular type of antibiotic. The aim is to reconstruct the activity profile of this regulator. Our approach can be extended to include more complex regulatory relationships, such as multiple regulatory factors, competition, and cooperativity. [source] Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kineticsBIOTECHNOLOGY PROGRESS, Issue 1 2010S. Matosevic Abstract In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200-,m ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His6 -tagged enzymes via Ni-NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop-flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK-catalysed synthesis of L -erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis,Menten model. Results show that the TK kinetic parameters in the IEMR (Vmax(app) = 0.1 ± 0.02 mmol min,1, Km(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the kcat for the microreactor of 4.1 × 105 s,1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His6 -immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell-based systems for TK bioprocess characterization. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | |||||||||||||