Showing papers on "Michaelis–Menten kinetics published in 1988"
TL;DR: The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties.
Abstract: The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
85 citations
TL;DR: An unoptimized continuous conversion in an enzyme-membrane-reactor demonstrated that the enzyme could be applied for the stereospecific synthesis of d-mandelic acid.
Abstract: Production, purification and characterization of the NAD(H)-dependent d-mandelate dehydrogenase from Lactobacillus curvatus was studied. An enzyme level of about 150 U/1 could be obtained by anaerobic cultivation in liquid broth. The specific enzyme activity in the crude extract was 1—3 U/mg. Purification by liquidliquid extraction and ion exchange chromatography led to a preparation of 2100 U/mg. The molecular weight of the enzyme was determined to be 60000 (gel filtration on Superose S12) containing two subunits of 30000. A variety of aliphatic and aromatic α-keto acids are accepted as substrates by the mandelate dehydrogenase, for the substrate benzoylformate a Michaelis constant of 2·10-4M was measured. Cu2+-ions and mercury compounds such as HgCl2 or p-chloromercuribenzoate are strong inhibitors at concentrations of 0.1 mM. An unoptimized continuous conversion in an enzyme-membrane-reactor demonstrated that the enzyme could be applied for the stereospecific synthesis of d-mandelic acid.
58 citations
TL;DR: An extracellular acid phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using cation exchange chromatography and chromatofocusing steps and showed positive PAS staining and hence is a glycoprotein (28% glycosylation); the sugar composition suggests the presence of N-linked high mannose-oligosaccharides and galactose.
Abstract: An extracellular acid phosphatase, pH optimum 6.0 from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using cation exchange chromatography and chromatofocusing steps. SDS-PAGE of the purified enzyme exhibited two stained bands at approximately 82-KDa and 70-KDa. The mobility of the active enzyme in gel permeation chromatography indicated the molecular mass to be about 85-KDa. In the concentrated form the enzyme appeared to be purple, the visible absorption spectrum shows a lambda max at 580 nm. On the basis of molecular mass of 82-KDa, the molar extinction coefficient of the enzyme at 280 nm and 580 nm was estimated to be 1.2 x 10(5) M-1 cm-1 and 1.3 x 10(3) M-1 cm-1 respectively. Judging by chromatofocusing, the isoelectric point of the enzyme was about 4.9. The purified enzyme was unstable at 70 degrees C. The enzyme was catalytically very active from 55 degrees to 65 degrees C with a maximum activity at 63 degrees C. The Michaelis constant of the enzyme for p-nitrophenylphosphate was 200 microM with a computed Kcat of 260 per sec. Although the enzyme was insensitive to fluoride, tartrate, and N-ethylmaleimide (NEM), it was competitively inhibited by phosphomycin (Ki = 1.00 mM) and inorganic orthophosphate (Ki = 165 microM). While the enzyme was relatively insensitive to Mn++, Cu++ and Zn++ inhibited the activity 540 fold at a concentration of 100 microM. The enzyme showed positive PAS staining and hence is a glycoprotein (28% glycosylation); the sugar composition suggests the presence of N-linked high mannose-oligosaccharides and galactose. A partial N-terminal amino acid sequence up to the thirty-fourth residue was elucidated.
55 citations
Journal Article•
TL;DR: In this paper, the authors studied the kinetics of sulfide oxidation by dissolved ox ygen in three domestic wastewaters containing concentrations between 2 and 8 mg/L sulfide and 5 and 20 mg/l oxygen.
Abstract: The kinetics of sulfide oxidation by dissolved ox ygen in three domestic wastewaters containing concentrations between 02 and 8 mg/L sulfide and 5 and 20 mg/L oxygen were studied The reaction order with respect to sulfide varied with sulfide concentration but was approximately first order for two of the wastewaters and significantly less for the third The reaction order with respect to oxygen could be described by Michaelis Menton kinetics, with K = 3 mg/L Chemical and biological components of the oxidation process were significant over the concentration range studied The effect of temperature on the rate constant was well described by the Arrhenius equation Rate constant data for 14 wastewater samples are presented and the implications of variability in this data are discussed J Water Pollut Control Fed, 60, 1264 (1988)
51 citations
39 citations
TL;DR: Specific autoantibodies to vasoactive intestinal peptide were present in plasma from 29.6% healthy human subjects who habitually performed muscular exercise, compared to 2.3% healthy subjects who did not.
26 citations
TL;DR: A multiphasic modelling approach to systems containing membrane-bound receptors or catalytic sites and a liposomal preparation as a substrate carrier is described and the significance of experimental membrane partition coefficients is discussed.
Abstract: 1. A multiphasic modelling approach to systems containing membrane-bound receptors or catalytic sites and a liposomal preparation as a substrate carrier is described. Kinetic expressions are derived for a single-substrate enzymic reaction operating at constant liposome concentration or at a fixed substrate/liposome concentration ratio. 2. The assumption that accumulation of exchangeable components into the phospholipid bilayers can be described by linear bulk-phase partition leads to simple relationships between the initial reaction rate and (a) two kinetic coefficients (V and K'm), (b) the partition coefficients of the solutes for the lipid compartments of the membrane (Pms) and liposomal preparations (P1s) and (c) the total concentrations of substrate, membrane lipid and liposomal lipid. K'm is called the effective Michaelis constant. 3. For correct estimation of the coefficients V, K', Pms and P1s extrapolation to zero lipid concentration is required. 4. The distinction is introduced between hydrophilic and hydrophobic aqueous-faced sites, lipid-faced sites and mixed sites, i.e. sites overlapping an aqueous and a lipid region. For hydrophilic aqueous-faced sites K'm is equal to the true Km and for the other types of site to Km/Ps. For lipid-faced and for mixed sites Ps corresponds to the membrane partition coefficient Pms. For binding of homologous compounds to a hydrophobic aqueous-faced binding pocket Ps is the incremental site partition coefficient Pbss, which takes into account the energetic contribution to the binding process due to the hydrophobic tail of the ligands. 5. K'm accounts for any effects due to the facedness and nature of the enzymic sites. The dependence of the systems on the size of the lipidic partition compartment(s) is expressed exclusively by a distribution function F.6. When enzyme assays are performed with a series of chemically different substrates containing the same catalytically sensitive group, independence of K'm from partition indicates a hydrophilic aqueous-faced binding site. For the low-molecular-mass members of the homologous series a linear increase in -log (K'm) with the logarithm of the partition coefficient will be observed with any of the other site types considered 7. Equilibrium relationships for binding of a ligand to a membrane-bound receptor are also derived. 8. The significance of experimental membrane partition coefficients is discussed.
24 citations
TL;DR: The cell-associated β-mannosidase of an alkalophilic Bacillus sp.
Abstract: The cell-associated β-mannosidase of an alkalophilic Bacillus sp. (AM-001) was purified to the electrophoretically homogenous state. The relative molecular mass of the purified enzyme was 94, 000 daltons by SDS-PAGE and the pi was 5.5 by isoelectric focusing. The enzyme was most active at pH 6.0 and 50°C. This β-mannosidase was inhibited by some metal ions and chemical reagents as follows: Ag+, Cd2+, Cu2+, Hg2+, Zn2+, PCMB, SDS, DBS, and NBS, but was not inhibited by D-mannose, D-mannitol, or D-mannonic acid-γ-lactone. Using synthetic substrates such as p-nitrophenyl glycopyranosides, this enzyme showed strong activity only with p-nitrophenyl β-D-mannopyranoside, and no activity with p-nitrophenyl α-D-mannopyranoside. The Michaelis constant (Km) of the enzyme for p-nitrophenyl β-D-mannopyranoside was 1.3mM. The enzyme hydrolyzed β-1, 4-mannooligosaccharides larger than mannobiose.
20 citations
TL;DR: A method is proposed based on fitting the equation for noncompetitive inhibition to data obtained by measuring the rate of the reaction over a range of substrate and inhibitor concentrations, thereby facilitating comparison with results obtained with similar inhibitors, for homologous enzymes, or in other laboratories.
20 citations
TL;DR: The results suggest that the enzyme in solution shifts continuously and reversibly between different configurations, confirmed by parallel studies on the proton-NMR spectrum of water aggregates in the enzyme solution and proton exchange rates.
Abstract: An unconventional hypothesis to the molecular basis of enzyme rhythms is that the intrinsic physical instability of the protein molecules which, in an aqueous medium, tend to move continuously from one conformational state to another could lead, in the population of enzyme molecules, to sizeable long-period oscillations in affinity for substrate and sensitivy to ligands and regulatory effects.To investigate this hypothesis, malate dehydrogenase was extracted and purified from leaves of the plant Kalanchoe blossfeldiana. The enzyme solutions were maintained under constant conditions and sampled at regular intervals for up to 40 or 70 h for measurements of activity as a function of substrate concentration. Km for oxaloacetic acid and sensitivity to the action of 2,3-butanedione, a modifier of active site arginyl residues. The results show that continuous slow oscillations in the catalytic capacity of the enzyme occur in all the extracts checked, together with fluctuations in Km. Apparent circadian periodici...
11 citations
TL;DR: Some implications of substrate channelling in interacting enzyme systems are considered with regard to the energetics and evolution of enzyme action.
TL;DR: A saturation-type exponential function is more curvilinear than the hyperbolic function and more closely approximates enzyme kinetics, and can be further improved by introducing a deceleration term (Vdec), to make the assumption of a steady state unnecessary.
Abstract: Enzyme kinetics are usually described by the Michaelis-Menten equation, where the time-dependent decrease of substrate (-dS/dt) is a hyperbolic function of maximal velocity (Vmax), Michaelis constant (Km), and amount of substrate (S). Because the Michaelis-Menten function in its most general meaning requires an assumption of steady-state, it is less curvilinear than true enzyme kinetics. A saturation-type exponential function is more curvilinear than the hyperbolic function and more closely approximates enzyme kinetics: -dS/dt = Vmax [1 - exp(-S/Km)]. The mathematical representation of enzyme kinetics can be further improved by introducing a deceleration term (Vdec), to make the assumption of a steady state unnecessary. For the action of chymotrypsin on N-acetyltyrosylethylester, the Michaelis-Menten equation yields the following: Vmax = 3.74 mumol/min and Km = 833 mumol. According to decelerated enzyme kinetics, the values Vmax = 4.80 mumol/min, Vdec = 0.0118 mumol/min, and the association constant (Ka) = 0.00111/mumol are more nearly accurate for this reaction (where 1/Ka = 901 mumol approximately Km).
TL;DR: Results indicate that the two reactions proceed through a common intermediary complex, and l-vinylglycine acts as a suicide inactivator for the enzyme.
Abstract: Kynurenine aminotransferase from a yeast, Hansenula schneggii, has been found to catalyze the deamination of an olefinic amino acid, l-vinylglycine, to form α-ketobutyrate and ammonia. The maximum rate of deamination was 0.17 μmol/mg/min at 25°C (pH 8.0), which is approximately 1% of the rate of transamination between L-kynurenine and α-ketoglutarate. Concomitantly with the catalysis, the enzyme lost both the deaminase and aminotransferase activities in a time-dependent manner. The inactivation was irreversible and followed pseudo-first-order kinetics at various concentrations of l-vinylglycine. The Michaelis constant for l-vinylglycine in the inactivation reaction was essentially the same as that in the deamination. These results indicate that the two reactions proceed through a common intermediary complex, and l-vinylglycine acts as a suicide inactivator for the enzyme. The apoenzyme neither catalyzed the deamination nor was inactivated by l-vinylglycine. The enzyme also catalyzes the γ-addition reactio...
Journal Article•
Journal Article•
TL;DR: The enzyme activity was strongly inhibited by L-ascorbic acid, sodium bisulfite, EDTA and KCN, and totally inhibited, by at a concentration of 1mM, however the enzyme was activated by approximately 1.7 times at the same concentration.
Abstract: Acetone powder was prepared from raw arrowroots and the polyphenol oxidases of crude enzyme prepared from acetone powder were identified 5 isoenzymes by staining with catechol containing 005% phenylene diamine The crude enzyme was passed through the columns of ion exchangers and gel permeation to fractionate the polyphenol oxidases The main fraction of polyphenol oxidase appeared to be purified by 94-fold, with the activity yield of 454%, and its molecular weight was determined as 38,500 by poly acrylamide gel electrophoresis The optimal pH and temperature for the enzyme activity were pH 75 and , respectively The purified enzyme showed a high affinity for catechol and pyrogallol The Michaelis constant for catechol was calculated to be 1667mM according to the Lineweaver-Burk method The enzyme activity was strongly inhibited by L-ascorbic acid, sodium bisulfite, EDTA and KCN, and totally inhibited, by at a concentration of 1mM However the enzyme was activated by approximately 17 times at the same concentration
TL;DR: Invertase was ionically immobilized on the poly(ethylene‐co‐vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2‐dimethylaminoacetaldehyde dimethyl acetal to be close to the value for the native one.
Abstract: Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 microg/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m)(') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.
TL;DR: The methods of Padé approximants and Euler transformation are used to construct approximate solutions for the time course of the reversible Michaelis-Menten reaction and the solutions are found to describe the concentrations of the various species quite accurately throughout and beyond the transient phase.
Patent•
12 Feb 1988
TL;DR: In this article, a benzoylformic acid reductase was used to produce alpha-hydroxy acid having an optical purity of 100% and R-type absolute configuration from an alpha-ketoacid.
Abstract: PURPOSE:To produce an alpha-hydroxyacid having an optical purity of 100% and R-type absolute configuration from an alpha-ketoacid, by using a benzoylformic acid reductase extracted from cell of bacterium belonging to Streptococcus genus. CONSTITUTION:Streptococcus faecalis (IFO 12964) is cultured in a tomato juice medium, etc., at 30 deg.C for 15-25 hr under shaking and the obtained cells are disintegrated e.g. by ultrasonic treatment to obtain a benzoylformic acid reductase having a specific activity of about >=100 U/mg. Sodium or potassium salt of alpha-ketoacied of formula (R is 2-4C alkyl, chloromethyl, bromomethyl or benzyl) is added together with reduced-type nicotinamide adenine dinucleotide (NADH) to a phosphate buffer solution having a concentration of from several mM to 2-300 mM and pH of 4-8. The concentration of the additive is about 10-100 times the Michaelis constant of said reductase. Furthermore, the mixture is added with a reagent for regeneration reaction of NADH and the above enzyme and the reaction of the components is started at about 30 deg.C to obtain objective compound.
TL;DR: Characterization studies indicated that the enzyme was capable of cleaving an aminoterminal leucine and phenylalanine residues and showed a wide range of specificity against dipeptides and oligopeptides.
TL;DR: The biological function of fructose‐2,6‐bisphosphate 6‐phosphatase with an apparent Michaelis constant between 7 and 79 μm fructose 2,6-bisph phosphate at Ph 6–7 is suggested to participate in the maintenance of a steady‐state level of fructose 2‐bisPhosphate in a concentration range that fits well with the Michaelis constants of the enzyme.
Patent•
26 Apr 1988
TL;DR: In this article, an enzyme having the following properties is described: Substrate specificity, Km value (Michaelis constant) to L-glutamine is 22mM at 37 deg.C and 6pH (in acetic acid buffer solution); optimum pH, 5-8 in the case of using L- glutamine as substrate; stable pH 5-7; salt resistance.
Abstract: NEW MATERIAL:An enzyme having the following properties. Substrate specificity, Km value (Michaelis constant) to L-glutamine is 22mM at 37 deg.C and 6pH (in acetic acid buffer solution); optimum pH, 5-8 in the case of using L- glutamine as substrate; stable pH, 5-7; salt resistance, exhibits relative activity of >=80% at a salt concentration of 10%(w/v) and >=70% at a salt concentration of 20%(w/v) (6pH, 37 deg.C) taking the enzymatic activity in the absence of salt as 100%. USE:Preparation of food containing salt at high concentration, e.g. seasoning, etc. PREPARATION:For example, Candida famata KM-1 (FERM P-8997) belonging to Candida genus is cultured in a liquid medium under aerobic condition at 25-30 deg.C for 16hr. A material containing a cell wall digesting enzyme is added to the cultured liquid to solubilize the salt-resistant glutaminase in the cultured liquid and the enzyme is separated by conventional method.
TL;DR: In this paper, a nonlinear model of drug kinetics is considered at enzymatic elimination and a method of mathematical description of the concentration variations of drug/free, bound and total/in the course of time are proposed.