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Michaelis-Menten Kinetics (michaelis-menten + kinetics)
Selected AbstractsComparison of biodegradation kinetic parameters for naphthalene in batch and sand column systems by pseudomonas putidaENVIRONMENTAL PROGRESS & SUSTAINABLE ENERGY, Issue 2 2001Jeong-Hun Park Kinetic parameters for the degradation of naphthalene by Pseudomonas putida ( ATCC 17484) were estimated in both batch and column assays, in order to evaluate the role of flow and cell attachment on biodegradation rates. Suspended cells and cells attached to Ottawa sand were used under a variety of biomass levels, column flow-rates, and substrate concentrations. In batch systems, degradation followed zero order kinetics across the entire concentration range, while the columns exhibited decreased rates at concentrations less than 100 (,g/L), describable by Michaelis-Menten kinetics. This is reflected in elevated values of the half-saturation constant, Ks, in columns. We offer the explanation that this may have resulted from reactive heterogeneity within the porous media, imposing a distribution of length-scales for transfer of substrate to the cell surfaces. Well-mixed batch systems are expected to have both shorter and more uniform transfer distances. When kinetic parameters obtained in batch system are used for prediction of degradation in columns, at least two factors,exposed reduction of exposed cell surface are a and heterogeneity of cell distribution,will likely reduce overall column degradation rates. [source] Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyaseFEBS JOURNAL, Issue 14 2002Tadayoshi Kanao The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl -ACL), an ,4,4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl -ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl -ACL. His273 of the , subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA. [source] Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite In Vivo, and acute toxicity in miceJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2009Silvia Romão Abstract The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters Km (19.5 ± 4.5 ,M) and Vmax [1.5 ± 0.4 units of fluorescence/(100 ,g of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:394,405, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20303 [source] Purification and Characterization of an ,-L-Rhamnosidase from Aspergillus terreus of Interest in WinemakingJOURNAL OF FOOD SCIENCE, Issue 2 2001M.V. Gallego ABSTRACT: An enzyme with ,-L-rhamnosidase activity was purified to homogeneity from a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 96 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44 °C, respectively. Using p-nitrophenyl-,-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with KM and Vmax values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnose (K1 2.5 mM). Divalent cations such as Ca2+ Mg2+ Zn2+ and Co2+ stimulated the a-L-rhamnosidase activity, whereas this was inhibited by Hg2+ and Cd2+. Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO2. [source] Allosteric kinetics of human carboxylesterase 1: Species differences and interindividual variability,JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2008Shiori Takahashi Abstract Esterified drugs such as imidapril, derapril, and oxybutynin hydrolyzed by carboxylesterase 1 (CES1) are extensively used in clinical practice. The kinetics using the CES1 substrates have not fully clarified, especially concerning species and tissue differences. In the present study, we performed the kinetic analyses in humans and rats in order to clarify these differences. The imidaprilat formation from imidapril exhibited sigmoidal kinetics in human liver microsomes (HLM) and cytosol (HLC) but Michaelis-Menten kinetics in rat liver microsomes and cytosol. The 2-cyclohexyl-2-phenylglycolic acid (CPGA) formation from oxybutynin were not detected in enzyme sources from rats, although HLM showed high activity. The kinetics were clarified to be different among species, tissues, and preparations. In individual HLM and HLC, there was large interindividual variability in imidaprilat (31- and 24-fold) and CPGA formations (15- and 9-fold). Imidaprilat formations exhibited Michaelis-Menten kinetics in HLM and HLC with high activity but sigmoidal kinetics in those with low activity. CPGA formations showed sigmoidal kinetics in high activity HLM but Michaelis-Menten kinetics in HLM with low activity. We revealed that the kinetics were different between individuals. These results could be useful for understanding interindividual variability and for the development of oral prodrugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5434,5445, 2008 [source] Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheatARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Charles Darkoh Abstract Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu2+ sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5,U/mg of total protein for gluten in a SDS gluten assay (1,U of enzyme activity was defined as the decrease in gel height in millimeters in 1,h) and exhibited a high-affinity Km of 65,µM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20° and 35°C. The enzyme was identified to be a prolyl endoprotease. © 2010 Wiley Periodicals, Inc. [source] Substrate-Dependent Modulation of UDP-Glucuronosyltransferase 1A1 (UGT1A1) by Propofol in Recombinant Human UGT1A1 and Human Liver MicrosomesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2007Yuji Mano In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The glucuronidation of 4-MU followed Michaelis-Menten kinetics with a Km value of 101 µM in the absence of propofol. In the presence of 200 µM propofol, a concentration that causes heterotopic activation of 4-MU glucuronidation (4-MUG), the Vmax value increased to 1.5-fold, while the Km value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3,-glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3,-glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC50 values of 59 and 228 µM, respectively. In addition, a known UGT1A1 modulator, 17,-ethynyloestradiol, stimulated oestradiol 3,-glucuronidation slightly at a concentration of 5 µM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5,100 µM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one glucuronidation substrate. [source] An experiment illustrating the effect of saline stress and ions on the malate dehydrogenase activity in vegetal tissues,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2002Sergio González-Moreno Abstract This paper describes a simple, rapid, and economic protocol to evaluate the effect of saline stress and Na+, K+, Ca2+, and Mg2+ ions on malate dehydrogenase activity in crude extracts from the leaves and roots of Vigna radiata. The main objectives of this work are to illustrate the effect of an environmental stress factor, as well as the in vitro effect of some ions on an enzymatic activity, while introducing the students to the use of Michaelis-Menten kinetics and a graphic procedure to calculate kinetics constants. [source] Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein designBIOTECHNOLOGY JOURNAL, Issue 4 2009Clara Salaheddin Abstract D -Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D -galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D -galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of kcat with both D -glucose and D -galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, kcat/KM is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D -galactose. Batch conversion experiments of D -galactose to 2-keto- D -galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis-Menten kinetics. [source] Immobilization and Characterization of Glucose Oxidase on Single-Walled Carbon Nanotubes and Its Application to Sensing GlucoseCHINESE JOURNAL OF CHEMISTRY, Issue 4 2007Shu-Na Liu Abstract The negatively charged (at pH 8.2) glucose oxidase (GOx, pI ca. 4.2) was assembled onto the surface of single-walled carbon nanotubes (SWNT), which was covered (or wrapped) by a layer of positively charged polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA), via the electrostatic interaction forming GOx-PDDA- SWNT nanocomposites. Fourier transform infrared (FTIR), UV-Vis and electrochemical impedance spectroscopy (EIS) were used to characterize the growth processes of the nanocomposites. The results indicated that GOx retained its native secondary conformational structure after it was immobilized on the surface of PDDA-SWNT. A biosensor (Nafion-GOx-PDDA-SWNT/GC) was developed by immobilization of GOx-PDDA-SWNT nanocomposites on the surface of glassy carbon (GC) electrode using Nafion (5%) as a binder. The biosensor showed the electrocatalytic activity toward the oxidation of glucose under the presence of ferrocene monocarboxylic acid (FcM) as an electroactive mediator with a good stability, reproducibility and higher biological affinity. Under an optimal condition, the biosensor could be used to detection of glucose, presenting a typical characteristic of Michaelis-Menten kinetics with the apparent Michaelis-Menten constant of KappMca. 4.5 mmol/L, with a linear range of the concentration of glucose from 0.5 to 5.5 mmol/L (with correlation coefficient of 0.999) and the detection limit of ca. 83 µmol/L (at a signal-to-noise ratio of 3). Thus the biosensor was useful in sensing the glucose concentration in serum since the normal glucose concentration in blood serum was around 4.6 mmol/L. The facile procedure of immobilizing GOx used in present work would promote the developments of electrochemical research for enzymes (proteins), biosensors, biofuel cells and other bioelectrochemical devices. [source] | ||||||||||