Showing papers on "Michaelis–Menten kinetics published in 1986"
TL;DR: The immunoglobulin MOPC167, which binds the transition state analog p-nitrophenylphosphorylcholine with high affinity, catalyzed the hydrolysis of the corresponding carbonate 1 with first order in hydroxide ion concentration between pH 6.0 and 8.0.
Abstract: The immunoglobulin MOPC167, which binds the transition state analog p-nitrophenylphosphorylcholine with high affinity, catalyzed the hydrolysis of the corresponding carbonate 1. MOPC167 catalysis displayed saturation kinetics with catalytic constant (kcat) = 0.4 min-1 and Michaelis constant (Km) = 208 microM, showed substrate specificity, and was inhibited by p-nitrophenylphosphorylcholine. The rate of the reaction was first order in hydroxide ion concentration between pH 6.0 and 8.0. The lower limit for the rate of acceleration of hydrolysis by the antibody above the uncatalyzed reaction was 770. This study begins to define the rules for the generation of catalytic antibodies.
602 citations
TL;DR: Data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix.
152 citations
TL;DR: Cholesterol oxidase from Nocardia erythropolis, Pseudomonas, and Streptomyces species was active in microemulsion in which cholesterol is well solubilized, and the activity was stable in nonionic microemulsions whereas in cationic and anionic micro emulsions the activity decreased with time.
44 citations
TL;DR: The amount and activity of immobilized glucoamylase were much less than of invertase, and thermal stability was found especially to be improved by immobilization.
Abstract: Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N,N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, Km, was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemarorigin in the same way. The amount and activity of immobilized glucoamylase were much less than those of invertase. 16 references.
38 citations
TL;DR: In this article, the effects of cellulose substrate concentration, cellulase enzyme concentration, and product concentration on the kinetic parameters involved in the enzymatic hydrolysis of celluloses have been studied.
Abstract: The effects of cellulose substrate concentration, cellulase enzyme concentration, and product concentration on the kinetic parameters involved in the enzymatic hydrolysis of cellulose have been studied. The Michaelis constant showed a decreasing trend with a decreasing crystallinity of cellulose substrate while the maximum reaction rate showed an increasing trend. These kinetic parameters were found to be significantly larger when the enzyme concentration was increased. The adsorption kinetic parameters showed an increasing trend as the crystallinity is decreased. It was found that the optimal enzyme loading should be determined by the source, concentration, and crystallinity of cellulose substrate and the initial specific rate of cellulose hydrolysis which is, in large part, influenced by the degree of crystallinity of cellulose substrate. The inhibition constant for cellulase-by cellobiose and that for cellobiase were also determined. These kinetic parameters determined experimentally can be ap...
37 citations
TL;DR: A mathematical analysis for the evaluation of the effectiveness factor and concentration profiles for immobilized enzymes into porous spherical particles is presented in this paper, which has been solved for monosubstrate reactions following reversible Michaelis-Menten kinetics.
25 citations
TL;DR: Prostaglandin-E2 9-ketoreductase might be responsible for the control of the PGE2/PGF2 alpha ratio in human decidua vera and might be an important factor in the cascade of events leading to uterine contractions and parturition.
Abstract: Prostaglandin-E2 9-ketoreductase, the enzyme which catalyzes the reaction from prostaglandin F2α(PGF2α to prostaglandin F2α (PGF2α), has been purified 232-fold from human uterine decidua vera.
The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 29 kDa. Sodium dodecyl sulfate disc gel electrophoresis of the denatured enzyme revealed a molecular mass of 31 kDa. These data suggest that the enzyme consists of a single polypeptide chain.
The rate equation of the enzyme reaction for two substrates was used for the determination of five kinetic constants. The equilibrium constant with respect to PGE2 was 83 μM, the Michaelis constant, Km, for PGE2 was 93 μM. For NADPH, the equilibrium constant was 1.0 μM and Km was 1.6 μM. The maximal velocity for the forward reaction was V1= 217 pmol/min. The inhibition constants for the analgesic agents indomethacin and fentiazac were Ki= 850 μM and Ki= 450 μM and for the steroid progesterone Ki= 1.5 mM, respectively.
Prostaglandin-E2 9-ketoreductase might be responsible for the control of the PGE2/PGF2α ratio in human decidua vera. The enzyme, therefore, might be an important factor in the cascade of events leading to uterine contractions and parturition.
20 citations
TL;DR: In this paper, two flow-injection systems were designed for characterizing the enzymatic hydrolysis of pyrophosphate by inorganic pyrophatase (EC 3.6.1).
19 citations
TL;DR: Computer simulations of a perfused liver system, with well-stirred reservoir and hepatic compartments, were performed to assess whether or not binding, as measured by the partition coefficient (Kp) between the liver and the emergent venous blood, affects the tendency to saturate metabolism.
18 citations
TL;DR: In a Michaelis‐Menten type reversible enzyme reaction the rapid equilibrium kinetics in one direction excludes rapid equilibrium in the reverse direction, which determines that a higher equilibrium concentration of product (or substrate) can be reached only with steady‐state kinetics.
17 citations
TL;DR: The results clearly indicate that, for purposes other than communicating the substrate concentration yielding control of the enzymic activity, usage of Km and its variation with ligand concentration should be avoided and interest instead focused on the slope.
Abstract: When information concerning whether or not a ligand interacts with the same enzyme species as do the substrates, the variation of the Michaelis constant Km (for each substrate) with ligand concentration is sometimes used as a diagnostic. It is shown that the Michaelis constant is of no particular value in this respect and may be misleading. Thus, depending on the mechanism, Km may vary with ligand concentration even though the ligand interacts with species far removed in the mechanism from the substrate-binding steps, and it may stay constant in cases where the ligand competes directly for the free enzyme. In contrast, the slope of a double-reciprocal plot of the kinetic data (= Km/Vmax.) (or, equivalently, the ordinate intercept of a Hanes plot A/v versus A, where A is the substrate concentration) independently of the particular mechanism involved uniquely signifies whether or not such interaction occurs. The results clearly indicate that, for purposes other than communicating the substrate concentration yielding control of the enzymic activity, usage of Km and its variation with ligand concentration should be avoided and interest instead focused on the slope, in accordance with the long-established rules of Cleland [Biochim. Biophys. Acta (1963) 67, 188-196], for which the present analysis provides the formal framework.
TL;DR: A nonlinear continuous-time Markov chain describing a two-step process of cytolytic cells binding to target and the subsequent lysis and release of label is shown to have kinetics which resemble standard enzyme-substrate kinetics.
Abstract: A nonlinear continuous-time Markov chain describing a two-step process of cytolytic cells binding to target and the subsequent lysis and release of label is shown to have kinetics which resemble standard enzyme-substrate kinetics. The Michaelis-Menten saturation function is found as a special case resulting when the target population is in excess. A comparison theorem for the pseudo-steady-state distribution Π is constructed to enable examination of that distribution whose expected value E and variance V satisfy − K m E + ( C T − E ) ( T T − E ) + V = 0 , where K m is the Michaelis half-saturation constant and C T and T T are the initial populations of the two cell types. Using Π as an initial condition, the release of label process is examined. The main result is that the fraction of specific release, f, has the approximate form f = E 1 − e − k 2 t f T T + Gaussian with mean zero , when T t is large, so that a nonlinear regression procedure is appropriate for the determination of the parameters.
TL;DR: Simulation analysis demonstrated that deviations in Tcalc could be at least an order of magnitude higher than previously predicted, and large discrepancies from the simple Michaelis–Menten equation could be caused by "skewed" or "clumped" distributions.
Abstract: Orthophosphate (31Pi) uptake rates by natural Lake Michigan microbial assemblages were measured to test a hypothesis that the instantaneous velocity of 31Pi uptake at low added substrate concentrat...
TL;DR: The method is used to re-examine the oxygen diffusion in a spherical cell with the Michaelis-Menten oxygen uptake kinetics.
Abstract: A numerical method is presented for the solution of reaction diffusion systems in biology. The method is used to re-examine the oxygen diffusion in a spherical cell with the Michaelis-Menten oxygen uptake kinetics.
TL;DR: The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater, and the extent of diffusion control was negligible.
Abstract: The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.
TL;DR: A computer simulation of the ideal stirred tank reactor (CSTR), the ideal plug flow reactor (PFR) and the ideal laminar flow reactors (LFR) is presented, which shows that at low initial substrate concentrations both tube reactors require less enzyme than the tank reactor for a given conversion and inhibition effects are negligible.
TL;DR: The enzyme showed catheptic activity against synthetic substrates such as BANA and BAPNA as well as against denatured hemoglobin and could be of physiological importance since the concentration of phospholipids is increased after endocytosis and autophagy.
Abstract: Buffalo liver cathepsin B was isolated by acid extraction, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-Sephadex chromatography and Sephacryl S-300 chromatography. The enzyme preparation was found to be homogeneous by gel filtration and SDS-polyacrylamide gel electrophoresis but could be resolved into two major and four minor protein bands on polyacrylamide gel electrophoresis in the absence of SDS. The enzyme showed catheptic activity against synthetic substrates such as BANA and BAPNA as well as against denatured hemoglobin. Various physico-chemical and enzymatic properties of the enzyme, such as molecular weight, Stokes radius, frictional coefficient, pH optimum, Michaelis constant, and Vmax, were determined. The values of these parameters were 27,500, 2.41 nm, 1.2, 6.5, 2.08 mM, and 42.4 units/mg, respectively. The hydrodynamic properties suggest a compact and globular conformation for this enzyme. Various compounds were tested for their influence on the activity of cathepsin B. Of these compounds, membrane phospholipids were found to increase significantly the activity of this enzyme. This increase in activity could be of physiological importance since the concentration of phospholipids is increased after endocytosis and autophagy.
TL;DR: The theoretical treatment was proved by determining the Michaelis-Menten parameters of D-glyceraldehyde-3-phosphate dehydrogenase and the first-order rate constant of hydrolysis of the unstable substrate, bisphosphoglycerate.
TL;DR: The analysis of the linear sensitivity of the reaction velocity to rate constants reveals that the characteristics of these valid representations in systems analysis change according to the phase of the Reaction.
Abstract: In relation to the input-output characteristics of enzymatic reactions in the cellular metabolism and biochemical reactors, the validity of the quasi-steady-state and transfer-function representations of reaction velocity has been examined for a basic Michaelis-Menten reaction employing computer simulation, that is, numerical integration of the rate equation. The well-known S-v relationship (relationship between substrate concentration and reaction velocity)derived on the quasi-steady-state assumption is found to be in general a good approximation to the actual velocity throughout the temporal progress of the reaction. The validity of the approximation depends on a ratio of the Michaelis constant to the total enzyme concentration in the reaction system rather than on the individual rate constants. A transfer-function representation is derived on assuming an exponential change in the reaction velocity for the indicial response to the substrate influx rate. The representation has a wider valid region with a decrease in influx rate than with an increase in the influx rate. The validity is most dependent on a ratio of total enzyme concentration to the steady-state concentration of the substrate. The analysis of the linear sensitivity of the reaction velocity to rate constants reveals that the characteristics of these valid representations in systems analysis change according to the phase of the reaction.
TL;DR: When pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.
Abstract: The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.
TL;DR: The technique involves a mathematical analysis which has shown that under specific conditions the peak/plateau blood concentrations of metabolite are related to initial parent compound concentration by the Michaelis-Menten relationship.
TL;DR: The chromophoric substrate can be useful to monitor physical changes that occur in liposomes during the hydrolysis reaction catalyzed by lipoprotein lipase, suggesting base catalysis of Hydrolysis.
TL;DR: In this paper, the reaction cellulase (EC 3.4) with different degrees of polymerization was investigated by the use of a modified Michaelis-Menten equation, valid for enzymatic hydrolysis of linear homopolymers.
Abstract: The reaction cellulase (EC 3.2.1.4)—sodium carboxymethylcellulose (Na-CMC) with different degrees of polymerization (n=140, 640 and 900) was investigated by the use of a modifiedMichaelis-Menten equation, valid for enzymatic hydrolysis of linear homopolymers. TheMichaelis-Menten constant [Km′ (M)=6.31·10−2mol/dm3] and the reaction rate constant (k′+2=4.07·10−6s−1), which correspond to the enzymatic hydrolysis of a single bond in the homopolymer substrates are determined. The free energy (Δ\(G^{ = |} \)=101 kJ/mol), which corresponds to the degradation and formation of a single bond in the enzyme—polymer substrate is also estimated. This energy expressed in electronvolt units is Δ\(E^{ = |} \)=1.39 eV. The ratio between the effective cross section of the reactive substrate bond and the active enzyme center is α=1.22.
Journal Article•
TL;DR: The addition of nitrate, EDTA and dithiothreitol to the enzyme extraction buffer resulted in improved stability of the assimilatory nitrate reductase activity from the food yeast Candida utilis, and suggested possible involvement of a second metal ion, perhaps iron, which was hypothesized to participate in the electron transfer scheme catalysed by this enzyme.
Abstract: The addition of nitrate, EDTA and dithiothreitol to the enzyme extraction buffer resulted in improved stability of the assimilatory nitrate reductase activity from the food yeast Candida utilis at both 4 degrees C and -10 degrees C. By incorporating this critical step in the following sequence the yeast NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) was purified approximately 68-fold by protamine sulphate precipitation, calcium gel adsorption, ion exchange chromatography and gel filtration. Both NADPH-nitrate reductase and NADH-nitrate reductase activities remained in constant association and ratio (2:3) during the entire course of purification. The enzyme showed an absolute requirement of NADPH or NADH for its activity. Maximal enzyme activity was obtained with 10-120 micrograms protein in a 10 min assay at 30 degrees C at pH 6.5, with an apparent Michaelis constant of 0.69 mM for nitrate as substrate. The enzyme is a molybdoflavo-protein involving sulphydryl groups, and is highly sensitive to free reducing agents, heavy metal ions and electron-transfer inhibitors. The results also suggested possible involvement of a second metal ion, perhaps iron, which was hypothesized to participate in the electron transfer scheme catalysed by this enzyme.
Journal Article•
TL;DR: A cellulose-degrading enzyme from Ganoderma lucidum was partially purified by ammonium sulfate precipitation and its enzymatic properties were studied and it acted on native celluose as well as CMC.
Abstract: A cellulose-degrading enzyme from Ganoderma lucidum was partially purified by ammonium sulfate precipitation and its enzymatic properties were studied. The enzyme had an optimum pH for activity at 4.0, and its stability range was pH . The optimum temperature was and the enzyme retained 80% original activity after heated at for 60 min. The activation energy of the enzyme for CMC degradation was caculated and found to be 6.2 Kcal/mole. The enzyme was activited by the addition of , but slightly inactivated by . Various enzyme inhibitors and chemical reagents did not affect the enzyme activity. The enzyme acted on native celluose as well as CMC. The Michaelis constant for CMC was calculated to be 2.4 mg glucose ep/ml.
Journal Article•
TL;DR: The method for computation of the Michaelis-Menten constant and the maximum value of effect with the intermediate conversion of the data in the Lineweaver-Burk plot is described.
Abstract: The method for computation of the Michaelis-Menten constant and the maximum value of effect with the intermediate conversion of the data in the Lineweaver-Burk plot is described.