Showing papers on "Michaelis–Menten kinetics published in 1995"
TL;DR: The results suggest that an experimental strategy for the quantitative analysis of dopamine, and other compounds, in living tissue is to fit a family of concentration versus time curves generated with different iontophoretic current strengths and recorded with a microsensor, to the numerical solution of the diffusion-uptake equation.
170 citations
TL;DR: A Michaelis-Menten-like reaction operating under conditions of dimensional restriction is shown to exhibit new types of synergism and noninteger kinetic orders.
163 citations
33 citations
TL;DR: The present method required as low as a few microgram amount of enzyme and nanogram amount of substrate which is far smaller than those required in a conventional affinity HPLC, which is in good agreement with the literature value.
30 citations
TL;DR: On repeated use of enzyme immobilized on chitosan beads, no loss was observed in enzyme activity even after 10 batches, and Durability of the enzyme was also improved on immobilization.
Abstract: Poly(vinyl alcohol) cross-linked with para-formaldehyde (PVA–F) and natural polysaccharide–chitosan in bead form and salicylic acid–resorcinol–formaldehyde polymeric resin (SRF) in powder form were used for immobilization of β-galactosidase through covalent linkages. Various activation processes and conditions were optimized. Immobilized enzyme showed very good storage stability at room temperature. Durability of the enzyme was also improved on immobilization. On repeated use of enzyme immobilized on chitosan beads, no loss was observed in enzyme activity even after 10 batches. Michaelis constant Km and maximum reaction velocity Vm were calculated for free and immobilized enzyme systems. Effect of pH and temperature on enzyme activity was estimated and energy of activation (Ea) and inactivation constant (Ki) for free and immobilized enzyme were calculated. © 1995 John Wiley & Sons, Inc.
25 citations
TL;DR: Kinetic data indicate that, in anaerobic bacteria under physiological conditions, the superoxide dismutase is not saturable with O2-.
Abstract: Kinetic studies were performed on the superoxide dismutases isolated from the anaerobic bacterium Propionibacterium shermanii as active enzymes with either iron or manganese, which were naturally incorporated into the same molecule depending on the metal supply. Both the Fe- and Mn- forms showed decreasing activity with increasing pH. This suggests the protonation of some groups near the metal, possibly a metal-bound water molecule. Thus the kinetic behaviour of this superoxide dismutase is much more dependent on the protein structure than on the metal incorporated into the active site. The secondary structures of both forms were not influenced by variations in pH, whereas the EPR spectra of the Fe-superoxide dismutase changed as a function of pH. The EPR spectra apparently consist of two overlapping species. Steady-state experiments proved that all iron-containing species show catalytic activity, but the species predominating in the alkaline pH range displays a lower reaction rate. The Michaelis constant and maximal turnover number for the Fe-superoxide dismutase were determined polarographically as Km = 0.54 mmol/l and Vmax. = 2000 mol.s-1 at pH 9.5. These data indicate that, in anaerobic bacteria under physiological conditions, the superoxide dismutase is not saturable with O2-. and the catalytic activity is similar to that of metal-specific Fe- or Mn-superoxide dismutases from aerobic organisms.
15 citations
Journal Article•
TL;DR: Hydlysis reactions obeyed Michaelis-Menten kinetics and exhibited a linear relationship of the initial rate with enzyme concentration, which suggests that the reaction rate in reverse micelle is kinetically controlled and not limited by mass-transfer considerations.
Abstract: In a reverse-micellar system of Aerosol-OT [bis-(2-ethylhexyl) sodium sulphosuccinate]/iso-octane, triacylglycerols (TAG) could be hydrolysed up to 60-98%, depending upon the enzyme source. Hydrolysis reactions obeyed Michaelis-Menten kinetics and exhibited a linear relationship of the initial rate with enzyme concentration, which suggests that the reaction rate in reverse micelle is kinetically controlled and not limited by mass-transfer considerations. The kinetic parameters for hydrolytic reactions are related to enzyme source and physicochemical characteristics of substrate. The Michaelis constant (K m ) and maximum reaction rate (V max. ) for hydrolysis of olive (Olea europaea) TAG by Rhizopus javanicus lipase were significantly higher than that by Candida cylindracea lipase. The kinetic parameters for coconut TAG were lower than that of olive TAG. The K m and V max. for TAG hydrolysis increased with increase in reaction temperature and decreased with increase in reaction pH. Buffer components may have considerable effect on enzyme activity and R-activity (R is the molar ratio of water to surfactant) profile of lipases in reverse micelles. When considering the influence of reaction variables on lipase-catalysed reactions for practical purposes, the initial rate as well as reaction rate over extended periods of time should be studied. The effect of reaction variables, surfactant concentration as an example, could be different on initial rate and degree of hydrolysis at 24 h. Lipase activity is rapidly reduced in reverse-micellar media in absence of any substrate. The effect is severe at higher value of R. The stability of enzyme in reverse micelles is also related to enzyme source. Reverse-micellar systems, in conjunction with other bioseparation techniques, could be used for lipase-catalysed bioconversion of TAG and other sparingly-water-soluble compounds for the production of value-added products.
12 citations
TL;DR: A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7.
Abstract: Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.
12 citations
TL;DR: In this article, the authors investigated the effect of the apparent maximum reaction rate Vmapp and the apparent Michaelis constant Kmapp on the substrate concentration in a system in which an enzyme was immobilized on the external surface of a solid support.
11 citations
TL;DR: A mathematical model was derived to estimate the kinetic parameters and to determine the concentrations of enzyme and substrate and it was indicated that the model gave more precise results.
Abstract: The acid phosphatase hydrolysis of phenylphosphoric acid (at pH 5 and at 25 °C) with a new type of liquid-immersion surface acoustic wave enzyme sensor (SAW) is described. A mathematical model was derived to estimate the kinetic parameters and to determine the concentrations of enzyme and substrate. The inhibition of the product to the enzyme activity was involved in the model. The Michaelis constant was estimated to be 1.17 × 10–4 mol l–1. The recovery of the sensor system was 97.4–104.4%(n= 6). The effects of temperature and pH on the enzyme activity were discussed. The proposed model compared favourably with the linear method which indicated that the model gave more precise results.
9 citations
TL;DR: Diffusivities of the substrate and the product and kinetic parameters, i.e., the Michaelis constant and the maximum velocity, could be estimated by fitting experimental data to a proposed mathematical model and immobilized cell concentration was found to be the main factor causing a decrease in maximum velocity.
Abstract: A method for determining the kinetics of whole-cell enzymes entrapped within calcium alginate gel beads is presented. Diffusivities of the substrate and the product and kinetic parameters, i.e., the Michaelis constant and the maximum velocity, could be estimated by fitting experimental data to a proposed mathematical model. Immobilized cell concentration was found to be the main factor causing a decrease in maximum velocity. The influence of cell concentration in the gel beads on the variation in the maximum velocity could be isolated from the effect of cell concentration on diffusional limitation and evaluated separately. The whole-cell enzymes became partially difficult to access by substrate when the cell population increased. The loss of enzyme accessibility resulted in a decrease in the maximum velocity and apparent initial velocity. This phenomenon of the maximum velocity varying with immobilized cell concentration has not been reported in the literature.
TL;DR: The rate-limiting step in the enzymic reduction of 6,8-dimethylpterin is hydride-ion transfer at acidic pHs as well as neutral pHs.
Abstract: The enzyme kinetics of the reduction of the substrate 6,8-dimethylpterin by chicken and recombinant human dihydrofolate reductases (DHFRs) have been examined over the pH range 5.0-8.0 in the presence of NADPH or (4R)-[2H]NADPH (NADPD). The pH profiles of the catalytic constant (Vmax/[E]o or kcat) showed pH independence for chicken DHFR and little pH dependence for human DHFR. For both DHFRs, the pH profiles of the Michaelis constant (Km(substrate)) and the apparent second-order rate constant (Vmax/Km(substrate)[E]o or kcat/Km(substrate)) indicated that two ionizable groups, deduced to be the substrate and the conserved Glu carboxy group in the enzyme active site, should be ionized in their cationic and anionic forms, respectively, for formation of the enzyme-substrate complex and for catalysis. The pKa values of about 5.3 and 6.5 which were obtained from the pH profiles of Km(substrate) and kcat/Km(substrate) were assigned to the ionizations of the substrate and the enzyme carboxy group, respectively. Deuterium isotope effects on DV and d(V/K) were significant for both enzymes, approximately 3 for chicken DHFR and approximately 4 for recombinant human DHFR, and were pH independent. Thus, the rate-limiting step in the enzymic reduction of 6,8-dimethylpterin is hydride-ion transfer at acidic pHs as well as neutral pHs. The results demonstrate that, compared with dihydrofolate, 6,8-dimethylpterin is a superior substrate for mechanistic investigations as it allows direct study of the effects of both enzyme and substrate ionizations involved in the catalysis and also avoids the obscuration of the catalytic rate by product release.
Journal Article•
TL;DR: Evidence pointing to ascorbate oxidase activity of this enzyme has been obtained and a possible reaction mechanism, which is based on the different enzymatic forms of tyrosinase, is presented.
Abstract: The reaction between mushroom tyrosinase and L-ascorbic acid was studied by oxymetric assays and evidence pointing to ascorbate oxidase activity of this enzyme has been obtained. The activity is clearly linear to enzyme concentration and the Michaelis constant for L-ascorbic acid has a value of 2.69 +/- 0.11 mM. Maximum activity is obtained at pH 7.5. A possible reaction mechanism, which is based on the different enzymatic forms of tyrosinase, is also presented.
Journal Article•
TL;DR: The reaction first-order rate constant was found to decrease as the concentration, in the enzyme assay mixture, of the 6-phosphogluconolactone preparation was increased, which may give rise to determinations involving spurious Michaelis constant and enzyme activity results.
Abstract: Hydrolysis of enzymatically generated 6-phosphogluconolactone in hemolysates has been found to be a first-order reaction, in all cases studied. It follows that the Michaelis constant of the reaction is higher than the highest 6-phosphogluconolactone concentration used in this study, viz., it is higher than 0.30 mmol l-1. The reaction first-order rate constant was found to decrease as the concentration, in the enzyme assay mixture, of the 6-phosphogluconolactone preparation was increased. It follows that this paradoxical effect may give rise to determinations involving spurious Michaelis constant and enzyme activity results. The possible causes of this effect are discussed.
TL;DR: An automatic method for the estimation of Michaelis-Menten and inhibitor constants in biochemical systems was developed using an unsegmented open-closed flow manifold, and the Lineweaver-Burk and Dixon plot methods.
TL;DR: Results obtained show that the enzymatic systems of the type studied here present positive kinetic co-operativity for a Michaelian enzyme in its action on the substrate.
TL;DR: In this paper, pepsin was used in a continuous fluidized bed reactor for the study of clotting of skimmed milk, and the effect of temperature on enzyme activity was studied, and thermoinactivation constant and energy of activation for free and immobilized enzymes were also calculated.
Abstract: Immobilization of pepsin on crosslinked resinous materials SRF (salicylic acid-resorcinol-formaldehyde), Amberlite IRA-400, and poly-(vinyl alcohol) is reported. Enzyme concentration, pH of the coupling medium, and nature and concentration of crosslinking agents were optimized for the better retention of activity of immobilized pepsin. The immobilized systems were characterized through pH, thermal, and storage stabilities. Michaelis constant (K m) and maximum reaction velocity (V m) for the free and immobilized enzymes were calculated from Lineweaver-Burk plots. Effect of temperature on enzyme activity was studied, and the thermoinactivation constant (K ti) and energy of activation (E a) for free and immobilized enzymes were also calculated. The immobilized pepsin was used in a continuous fluidized bed reactor for the study of clotting of skimmed milk. Rate of coagulation was considerably high for the treated milk sample at 50°C and pH 6–6.2.
TL;DR: In this paper, an analysis of the Michaelis-Menten kinetics has been performed assuming the product is unstable, and the explicit time-course equation of the immediate product has been derived under conditions of limiting enzyme concentration.
Journal Article•
TL;DR: It is found that non-catalytic monoclonal antibodies specific for human beta-glucuronidase and, to a lesser extent, bovine serum albumin were capable of enhancing the activity of this enzyme.
Abstract: Catalytic antiidiotypic antibodies exhibit enzyme activity because they possess the internal image of the enzyme that serves as the antigen. However, we have found that non-catalytic monoclonal antibodies specific for human beta-glucuronidase and, to a lesser extent, bovine serum albumin were capable of enhancing the activity of this enzyme. The stability of the enzyme was not increased by these proteins. Enzyme kinetic studies revealed an uncompetitive activation mechanism with a proportional increase in both maximal velocity and Michaelis constant of the enzyme with the increase in the protein concentration. These three proteins apparently bound to the enzyme with subsequent alteration of its affinity for the substrate. Activation of the enzyme by its non-catalytic antibody might not be a rare occurrence and it could play a pathogenic role in certain disease processes.
TL;DR: In this paper, a nonlinear regression program is used to fit data for absorbance and rate vs. time to the model and to the Michaelis-Menten equation.
TL;DR: Exact analytical expressions are derived for the optimal design (minimum overall reaction volume) of N CSTRs in series carrying out an enzyme catalysed Michaelis-Menten one-substrate one-product reversible reaction.
Abstract: Exact analytical expressions are derived for the optimal design (minimum overall reaction volume) of N CSTRs in series carrying out an enzyme catalysed Michaelis-Menten one-substrate one-product reversible reaction. These equations enable the direct calculation of the smallest total reactor volume (holding time) needed for a given overall conversion degree, as well as the individual reactor volume and conversion degrees. Results are also compared with the ones obtained for a single tank and for a plug flow reactor.