scholarly journals Kinetics of substrate hydrolysis and inhibition by mipafox of paraoxon-preinhibited hen brain esterase activity

1986 ◽  
Vol 236 (2) ◽  
pp. 503-507 ◽  
Author(s):  
C D Carrington ◽  
M B Abou-Donia
Keyword(s):  
Kinetic Parameters ◽  
Organophosphorus Compounds ◽  
Model Fit ◽  
Component Model ◽  
Low Concentrations ◽  
Two Component ◽  
High Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Organophosphorus Inhibitor

For the purpose of assessing the neurotoxic potential of organophosphorus compounds, it has been determined that paraoxon-preinhibited hen brain has both neurotoxicant (mipafox)-sensitive (neurotoxic esterase; NTE) and -insensitive esterase components. Several experiments designed to investigate the kinetic parameters governing the reaction of these esterases with two substrates and one organophosphorus inhibitor are presented. First, kinetic parameters for the hydrolysis of phenyl valerate and phenyl phenylacetate were measured. At 37 degrees C, the Km values of NTE for phenyl valerate and phenyl phenylacetate were found to be about 1.4 × 10(-3) and 1.6 × 10(-4) M respectively. At 25 degrees C, the Km of NTE for phenyl valerate was determined to be about 2.4 × 10(-3) M. Secondly, the kinetic constants of NTE for mipafox were measured at both 25 degrees C and 37 degrees C. With either phenyl valerate or phenyl phenylacetate as substrate, the Km at 37 degrees C was determined to be about 1.8 × 10(-4) M, and the phosphorylation constant (k2) was about 1.1 min-1. For phenyl valerate only, the Km at 25 degrees C was found to be about 6 × 10(-4) M, and the k2 was about 0.7 min-1. The data obtained at 25 degrees C were analysed by using a two-component model without formation of Michaelis complex, a two-component model with formation of Michaelis complex on the second component (NTE), or a three-component model without formation of Michaelis complex. The fact that the Michaelis model fit the data significantly better than either of the other two models indicates that the higher apparent Ki values that occur with low concentrations of mipafox are due to formation of Michaelis complex at high concentrations, rather than because of the presence of two NTE isoenzymes, as has been suggested by other investigators.

Uptake of l-valyl-l-Valine and Glycylsarcosine by Hamster Jejunum in Vitro

1982 ◽  
Vol 62 (6) ◽  
pp. 617-626 ◽  
Author(s):  
D. Burston ◽  
R. A. Wapnir ◽  
E. Taylor ◽  
D. M. Matthews
Keyword(s):  
Competitive Inhibition ◽  
Side Chains ◽  
Low Concentrations ◽  
Peptide Component ◽  
High Concentrations ◽  
Kinetics Of Uptake ◽  
Peptide Uptake ◽  
Kinetics Of ◽  
Hydrolysis Of

1. Preliminary observations concerned with the effect of the lipophilic properties of the amino acid side-chains of peptides on their apparent affinity for uptake by rings of everted hamster jejunum showed that of the series glycylglycine, l-alanyl-l-alanine, l-valyl-l-valine and l-leucyl-l-leucine, with increasingly lipophilic side-chains, l-valyl-l-valine, not l-leucyl-l-leucine, was the most powerful inhibitor of uptake of the hydrolysis-resistant dipeptide glycylsarcosine. This apparently anomalous observation indicated a need for further investigation, and this paper reports investigations of the kinetics of uptake of l-valyl-l-valine and of competition for uptake between l-valyl-l-valine and glycylsarcosine. 2. l-Valyl-l-valine was capable of complete competitive inhibition of mediated uptake of glycylsarcosine. Free l-valine did not inhibit mediated uptake of glycylsarcosine. Glycylsarcosine could inhibit mediated uptake of l-valyl-l-valine only partially, but a mixture of glycylsarcosine and l-valine was capable of producing complete inhibition of mediated uptake of l-valyl-l-valine. 3. Investigation of the kinetics of uptake of l-valyl-l-valine indicated two mediated components. Component (a), which disappeared in the presence of free l-leucine, probably represented uptake of free l-valine after hydrolysis of the peptide. Component (b) probably represented peptide uptake. 4. The estimates of Kt obtained for uptake of intact l-valyl-l-valine were many times greater than Ki for inhibition of uptake of glycylsarcosine by l-valyl-l-valine. A possible explanation of the discrepancy is the existence of two pathways for uptake of l-valyl-l-valine and glycylsarcosine, for one of which l-valyl-l-valine has a low Kt (i.e. a high affinity) not readily demonstrable by kinetic analysis. 5. The results suggest that mediated uptake of l-valyl-l-valine is the result of at least two processes, uptake of intact peptide by a mechanism or mechanisms shared with glycylsarcosine and also hydrolysis followed by uptake of free l-valine; estimates of the proportions of intact valine and of free valine taken up by mediated transport suggest that at pH 5 uptake of intact peptide varies from 25% at low concentrations to 55% at high concentrations. They do not explain why l-valyl-l-valine is a stronger inhibitor of uptake of glycylsarcosine than the more lipophilic l-leucyl-l-leucine, but do suggest how such a situation could arise.

Influence of medium on alkaline hydrolysis of succinic acid monomethyl and monopropyl esters

1980 ◽  
Vol 45 (11) ◽  
pp. 2873-2882
Author(s):  
Vladislav Holba ◽  
Ján Benko
Keyword(s):  
Succinic Acid ◽  
Kinetic Parameters ◽  
Alkaline Hydrolysis ◽  
Specific Interactions ◽  
Nonaqueous Media ◽  
The Media ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of alkaline hydrolysis of succinic acid monomethyl and monopropyl esters were studied in mixed aqueous-nonaqueous media at various temperatures and ionic strengths. The results of measurements are discussed in terms of electrostatic and specific interactions between the reactants and other components of the reaction mixture. The kinetic parameters in the media under study are related to the influence of the cosolvent on the solvation sphere of the reactants.

scholarly journals Effect of Pseudomonas moorei KB4 Cells’ Immobilisation on Their Degradation Potential and Tolerance towards Paracetamol

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 820
Author(s):  
Robert Surma ◽  
Danuta Wojcieszyńska ◽  
Jagna Karcz ◽  
Urszula Guzik
Keyword(s):  
Protective Effect ◽  
Kinetic Parameters ◽  
Toxicity Assessment ◽  
Bacterial Cells ◽  
Low Concentrations ◽  
Toxicity Analysis ◽  
Immobilised Cells ◽  
Degradation Activity ◽  
High Concentrations ◽  
The Hill

Pseudomonas moorei KB4 is capable of degrading paracetamol, but high concentrations of this drug may cause an accumulation of toxic metabolites. It is known that immobilisation can have a protective effect on bacterial cells; therefore, the toxicity and degradation rate of paracetamol by the immobilised strain KB4 were assessed. Strain KB4 was immobilised on a plant sponge. A toxicity assessment was performed by measuring the concentration of ATP using the colony-forming unit (CFU) method. The kinetic parameters of paracetamol degradation were estimated using the Hill equation. Toxicity analysis showed a protective effect of the carrier at low concentrations of paracetamol. Moreover, a pronounced phenomenon of hormesis was observed in the immobilised systems. The obtained kinetic parameters and the course of the kinetic curves clearly indicate a decrease in the degradation activity of cells after their immobilisation. There was a delay in degradation in the systems with free cells without glucose and immobilised cells with glucose. However, it was demonstrated that the immobilised systems can degrade at least ten succeeding cycles of 20 mg/L paracetamol degradation. The obtained results indicate that the immobilised strain may become a useful tool in the process of paracetamol degradation.

KINETIC STUDIES ON THE HYDROLYSIS OF BENZOYLCHOLINE BY HUMAN SERUM CHOLINESTERASE

1956 ◽  
Vol 34 (1) ◽  
pp. 637-653 ◽  
Author(s):  
W. Kalow ◽  
K. Genest ◽  
N. Staron
Keyword(s):  
Kinetic Studies ◽  
Reaction Rates ◽  
Serum Cholinesterase ◽  
Initial Reaction ◽  
Ultraviolet Spectrophotometry ◽  
First Order ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

Benzoylcholine stands out from other known substrates of serum cholinesterase because of its high apparent affinity for this enzyme combined with a rapid rate of destruction. The reaction kinetics of the hydrolysis of benzoylcholine can be studied by ultraviolet spectrophotometry, since the absorbance decreases in proportion to the concentration of substrate. Kinetic data obtained by measuring initial reaction rates, and by analyzing continuous hydrolysis curves, are the same within the range of experimental error. The enzymatic data are compatible with the assumption that in the presence of high substrate concentrations a complex consisting of esterase and two substrate molecules is formed. This complex is hydrolyzed more slowly than the complex containing one molecule of substrate which is formed at low concentrations of benzoylcholine. Alkaline hydrolysis of benzoylcholine follows the kinetics of a first order reaction.

scholarly journals Glutathione-Dependent Conversion ofN-Ethylmaleimide to the Maleamic Acid by Escherichia coli: an Intracellular Detoxification Process

2000 ◽  
Vol 66 (4) ◽  
pp. 1393-1399 ◽  
Author(s):  
D. McLaggan ◽  
H. Rufino ◽  
M. Jaspars ◽  
I. R. Booth
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
E Coli ◽  
Transient Activation ◽  
Low Concentrations ◽  
Maleamic Acid ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Strong Activator ◽  
Detoxification Process

ABSTRACT The electrophile N-ethylmaleimide (NEM) elicits rapid K+ efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K+ efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM andN-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-Ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD+-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K+ efflux systems.

scholarly journals Relationship of Exo-β-d-Galactofuranosidase Kinetic Parameters to the Number of Phosphodiesters in Penicillium fellutanum Peptidophosphogalactomannan: Enzyme Purification and Kinetics of Glycopeptide and Galactofuran Chain Hydrolysis

2001 ◽  
Vol 67 (10) ◽  
pp. 4648-4656 ◽  
Author(s):  
Brigitte A. Tuekam ◽  
Yong-Il Park ◽  
Clifford J. Unkefer ◽  
John E. Gander
Keyword(s):  
Kinetic Parameters ◽  
Kinetic Data ◽  
Inorganic Phosphate ◽  
Enzyme Purification ◽  
Degree Of Polymerization ◽  
Apparent Homogeneity ◽  
Relationship Of ◽  
Kinetics Of ◽  
Hydrolysis Of

ABSTRACT Extracellular Penicillium fellutanumexo-β-d-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP2GMii and pP25GMii(containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-β-d-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP2GMii or pP25GMii. Thek cat/Km value for pP25GMii is 1.7 × 103M−1 s−1, that for 1-O-methyl-β-d-galactofuranoside is 1.1 × 104 M−1 s−1, that for pP2GMii is 1.7 × 10 4M−1 s−1, and those for 5-O-β-d-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 105 and 4.1 × 105 M−1s−1, respectively. Variability in thek cat/Km values is due primarily to differences in Km values; thek −1/k 1 ratio likely provides the most influence on Km. k cat increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pPxGMii added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml−1h−1. No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pPxGMii and of related polymers is proportional to thek cat/Km value of each polymer. The kinetic data show that thek cat/Km value increases as the number of phosphodiesters of pPxGMiidecreases, also resulting in an increase in the activity of exo-β-d-galactofuranosidase.

scholarly journals Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate

1969 ◽  
Vol 115 (2) ◽  
pp. 147-162 ◽  
Author(s):  
W. N. Aldridge ◽  
Elsa Reiner
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Organophosphorus Compounds ◽  
Active Centre ◽  
Phosphorus Containing ◽  
High Concentrations ◽  
Influence Of Substrate ◽  
A Site ◽  
Kinetics Of ◽  
Related Compounds

1. The kinetics of the reaction of di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate (haloxon) and related compounds with acetylcholinesterase were studied and found to be unusual. 2. By a progressive reaction haloxon produces a di-(2-chloroethyl)phosphorylated enzyme. The influence of substrate on this reaction leading to a phosphorylated active centre was studied. From competition experiments between inhibitor and substrate values of Km for acetylcholine and acetylthiocholine of 0·79mm and 0·23mm respectively were derived. 3. Haloxon also combines with acetylcholinesterase by a non-progressive reaction, producing a complex that is reversible by dilution and by high concentrations of acetylcholine and acetylthiocholine. From this non-progressive reaction the competition between haloxon and substrate was studied, and it was shown that haloxon combines with a site involved in inhibition by substrate. From competition experiments the following dissociation constants were derived: for combination of haloxon and this site Ki is 4·9μm and for the combination of substrates with this site K88 values are 12mm and 3·3mm for acetylcholine and acetylthiocholine respectively. 4. The non-phosphorus-containing compound 3-chloro-7-hydroxy-4-methylcoumarin was shown to be a good reagent for the site involved in inhibition by substrate; its dissociation constant for the combination with this site is 30μm. 5. In order to interpret the experimental results, theoretical equations were derived for an enzyme with two binding sites to both of which substrate and inhibitor can combine. The equations correlate the activity of the enzyme with the concentration of substrate and inhibitor, for both progressive and non-progressive inhibition. These equations are applicable to reactions of acetylcholinesterase with organophosphorus compounds, carbamates etc. and may be applicable to other enzymes possessing two binding sites.

scholarly journals Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1972 ◽  
Vol 127 (1) ◽  
pp. 271-283 ◽  
Author(s):  
R. G. Hansford
Keyword(s):  
Oxygen Uptake ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Slight Reduction ◽  
Glycerol Phosphate ◽  
Pyruvate Oxidation ◽  
Low Concentrations ◽  
High Concentrations ◽  
Kinetics Of ◽  
Very High

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl2 during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H+ ions, and inhibited by ATP, NADH and Ca2+. 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a `classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.

scholarly journals Kinetics of Alkaline Phosphatase from Pig Kidney. Influence of complexing agents on stability and activity

1976 ◽  
Vol 153 (2) ◽  
pp. 151-157 ◽  
Author(s):  
B P Ackermann ◽  
J Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ion ◽  
Complexing Agents ◽  
Pig Kidney ◽  
Low Concentrations ◽  
Sequential Addition ◽  
Quantitative Examination ◽  
High Concentrations ◽  
Kinetics Of ◽  
Active Centres

Metal ion-complexing agents, like KCN, EDTA etc., inactivate alkaline phosphatase of pig kidney. This inactivation is reversible at low concentrations of the complexing agents and irreversible at high concentrations. The reversible inhibition is probably due to removal of Zn2+ ions from the active site, where they are necessary for catalytic action, whereas the irreversible inhibition results from the removal of Zn2+ ions necessary for preservation of the structure. The inactivation is pseudo-first order. It depends on the concentration, size and charge of the complexing agents. β-Glycerophosphate and Mg2+ ions protect the enzyme from inactivation by complexing agents. Quantitative examination of the effect of substrate leads to a model that is similar to the “sequential model” proposed by D.E. Koshland, G. Nemethy & D. Filmer (1966) (Biochemistry 5, 365-385) to explain allosteric behavior of enzymes. It describes the sequential addition of two substrate molecules at two active centres of the dimer enzyme. The binding of the substrate molecules is accompanied by changes in the conformation, which lead to stabilization of the enzyme against attack by complexing agents.

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