PHOSPHATASE ALCALINE INTESTINALE: CINÉTIQUE DE L'HYDROLYSE DU PHOSPHATE DE p-NITROPHÉNYLE

1961 ◽  
Vol 39 (6) ◽  
pp. 1298-1308 ◽  
Author(s):  
Michel Lazdunski ◽  
Ludovic Ouellet
Keyword(s):  
Active Center ◽  
Electrostatic Interactions ◽  
Enzyme Molecule ◽  
Kcal Mole ◽  
Intestinal Alkaline Phosphatase ◽  
High Substrate Concentration ◽  
System P ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Michaelis Constants

The Michaelis constants and the rates at high substrate concentration for the system p-nitrophenyl phosphate – intestinal alkaline phosphatase have been measured at 15.3 °C and 24.4 °C from pH 7.5 to pH 10.4.The experimental data can be interpreted as indicating the presence of three acid groups in the active center of the enzyme. The observed pK of ionization of these groups are 9.02, 8.39, and 7.65 at 15.3 °C, and 9.75, 8.42, and 7.62 at 24.4 °C.These data stress the importance of electrostatic interactions between the groups in the active center and the rest of the enzyme molecule. A heat of ionization of 6 to 7 kcal/mole is attributed to each of these groups. At least one active group of the enzyme would be a thiol function and the other ones imidazole or thiol.

ÉTUDE DES EFFETS ÉLECTROSTATIQUES SUR LE MÉCANISME D'ACTION CATALYTIQUE DE LA PHOSPHATASE ALCALINE INTESTINALE DE VEAU

1965 ◽  
Vol 43 (8) ◽  
pp. 2222-2235 ◽  
Author(s):  
Michel Lazdunski ◽  
Jacques Brouillard ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
High Substrate Concentration ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Activated Complex

The influence of dioxane and ethanol on the rate of hydrolysis of p-nitrophenyl phosphate in the presence of an intestinal alcaline phosphatase can be interpreted as a dielectric constant effect, at high substrate concentration. The dielectric constant effect is a function of the pH of the medium and is maximum around pH 9.4 at 25 °C and pH 9.0 at 15 °C. An interpretation suggesting that the change in diameter of the enzyme molecule becoming an activated complex is minimum at a pH of maximum activity is proposed. The same model can take into account the influence of the ionic strength on the same reaction.

Etude cinétique des interactions électrostatiques entre la phosphatase alcaline intestinale de veau et ses substrats

1974 ◽  
Vol 52 (17) ◽  
pp. 3087-3097 ◽  
Author(s):  
Marius Julien ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constants ◽  
Enzyme Molecule ◽  
Ionic Charge ◽  
Substrate Molecule ◽  
Net Charge ◽  
Organic Part ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Ethanolamine Phosphate

The role played by the ionic charge of the enzyme in the kinetics of the reactions catalyzed by alkaline phosphatases has been investigated. Data obtained from the hydrolysis of p-nitrophenyl phosphate (net charge −2) indicate that the repulsion between the negatively charged enzyme molecule and the substrate molecule is sufficient to take into account, quantitatively, the variation of the Michaelis constant with the pH of the system between pH 8.5 and 10.5. However, the results obtained with ethanolamine phosphate (net charge −1) and p- or o-carboxyphenyl phosphate (net charge −3) show that either the organic part of the substrate molecule is not involved in the formation of the enzyme substrate intermediate or the charge of the enzyme does not have the role suggested from the work with p-nitrophenyl phosphate.Further investigation of this reaction in solutions of various ionic strength and dielectric constants suggests that the active center of the enzyme molecule is positively charged, but that hydrophobic effects are important.

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON THE MYOSIN-CATALYZED HYDROLYSIS OF ADENOSINE TRIPHOSPHATE

1961 ◽  
Vol 39 (7) ◽  
pp. 1444-1453 ◽  
Author(s):  
Gérard E. Pelletier ◽  
Ludovic Ouellet
Keyword(s):  
Experimental Data ◽  
Potassium Chloride ◽  
Active Center ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Kcal Mole ◽  
Hydrogen Ion Concentration ◽  
Michaelis Constants ◽  
Hydrolysis Of ◽  
Ionizable Groups

The Michaelis constants for the system myosin–ATP have been measured at 5 °C and 15 °C from pH 7.0 to pH 10.5, in the presence of 0.4 M potassium chloride and 0.005 M calcium chloride. The experimental data point to the presence, in the active center, of two ionizable groups, with experimental acid pK of 7.35 and 8.6 respectively at 15 °C. The apparent heats of ionization of these groups are about 9 kcal/mole for the first one and close to zero for the second. These data are interpreted as indication of the presence in the active center of myosin of a histidyl and a sulphydryl residue. A mechanism is proposed for the hydrolysis of ATP in the presence of myosin.

Steroids and steroidases. IX. Activation parameters and the mechanism of base- and enzyme-catalyzed isomerizations of androst-5-ene-3,17-dione. The nature of the active center of the Δ5 → Δ4-3-ketosteroid isomerase of Pseudomonas testosteroni

1969 ◽  
Vol 47 (23) ◽  
pp. 4459-4466 ◽  
Author(s):  
J. Bryan Jones ◽  
Donald C. Wigfield
Keyword(s):  
Amino Acids ◽  
Active Center ◽  
Activation Parameters ◽  
Circumstantial Evidence ◽  
Kcal Mole ◽  
Acid Base ◽  
Ketosteroid Isomerase ◽  
Enzyme Action ◽  
Enthalpy Of Activation

Determination of the activation parameters for the acid-, base-, and enzyme-catalyzed isomerizations of androst-5-ene-3,17-dione has revealed that the facility of the enzymic process is mainly due to an extremely low enthalpy of activation of 5.0 kcal mole−1. Further circumstantial evidence regarding the nature of the reacting groups at the active center has also been obtained, and a mechanism of enzyme action is proposed employing tyrosine and histidine as the principal amino acids responsible for catalyzing the isomerization.

THE ROLE OF ALKALINE PHOSPHATASE IN INTESTINAL ABSORPTION I. THE KINETICS OF PHOSPHATASE ACTION ON VARIOUS SUBSTRATES

1953 ◽  
Vol 31 (1) ◽  
pp. 1-7
Author(s):  
Neil B. Madsen ◽  
Jules Tuba
Keyword(s):  
Alkaline Phosphatase ◽  
Average Energy ◽  
Inorganic Phosphorus ◽  
Intestinal Alkaline Phosphatase ◽  
Egg Lecithin ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of intestinal alkaline phosphatase action on sodium β-glycerophosphate, glucose 6-phosphate, and egg lecithin have been studied and compared. The Michaelis constants indicate that the enzyme shows considerably less affinity for lecithin than for the other two substrates, and the approximate ratio of activity with lecithin, glucose 6-phosphate, and sodium β-glycerophosphate is 11 : 78.5 : 100. The energies of activation for the hydrolysis of the three substrates do not differ appreciably and the average energy of activation is 14,500 calories per gram-mole. The similarity of the energies of activation together with results from inhibition studies indicate that in all probability the same enzyme is responsible for the release of inorganic phosphorus from each of the three substrates.

Effects of temperature on measurement of alkaline phosphatase activity.

1985 ◽  
Vol 31 (2) ◽  
pp. 185-190 ◽  
Author(s):  
W H Copeland ◽  
D A Nealon ◽  
R Rej
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Kinetic Properties ◽  
Porcine Kidney ◽  
Human Origin ◽  
Arrhenius Behavior ◽  
Nitrophenyl Phosphate ◽  
Activity Measurements ◽  
Michaelis Constants ◽  
Effects Of Temperature

Abstract We examined the effects of temperature on the activity and steady-state kinetic properties of alkaline phosphatase (EC 3.1.3.1). Purified isoenzymes from human liver, intestine, and placenta were used, as was human serum, and the enzyme from porcine kidney. Phosphatase activity was estimated by two different assay techniques. For all isoenzymes, apparent Michaelis constants for the substrate 4-nitrophenyl phosphate decreased with increased temperature; Km at 37 degrees C was typically half that determined at 25 degrees C. All enzymes of human origin exhibited similar linear Arrhenius relationships over the range examined, 20-37 degrees C (Ea of 30-36 kJ X mol-1). The porcine kidney enzyme obeyed an Arrhenius relationship that was slightly, but significantly, different from the isoenzymes of human origin. Temperature relationships based upon Arrhenius behavior and individual activity measurements are presented. For human alkaline phosphatases, they differed by no more than 10%.

Comparative studies of activity and properties of ferredoxin–NADP+ reductase during cold hardening of wheat

1976 ◽  
Vol 54 (16) ◽  
pp. 1896-1902 ◽  
Author(s):  
Joseph Riov ◽  
Gregory N. Brown
Keyword(s):  
Activation Energy ◽  
Spring Wheat ◽  
Comparative Studies ◽  
Low Temperatures ◽  
Specific Activity ◽  
Cold Hardening ◽  
Heat Inactivation ◽  
Enzyme Molecule ◽  
Enzyme Specific Activity ◽  
Michaelis Constants

Activity and properties of chloroplast ferredoxin–NADP− reductase (EC 1.6.7.1) were studied during cold hardening of two varieties of wheat (Triticum aestivnm), hardy Kharkov (winter wheat) and much less hardy Rescue (spring wheat), to determine whether adaptation to low temperatures involves changes in the activity and properties of this enzyme. Specific activity of ferredoxin–NADP− reductase increased during hardening of both varieties, but the increase was much greater in the more hardy variety, Kharkov 22 MC. No changes were found in the Michaelis constants for NADPH and 2,6-dichlorophenol indophenol, activation energy values, inhibition constants for p-chloromercuriphenylsulfonate, and sensitivity toward cold and heat inactivation of the enzyme from control and cold-hardened seedlings of both varieties. The data suggest that there is a preferential synthesis of ferredoxin–NADP− reductase during hardening of wheat, but the enzyme molecule remains unchanged.

scholarly journals Employment of electrostatic interactions for amperometric detection of carbon nanoparticles in a FIA system

The Analyst ◽  
2016 ◽  
Vol 141 (14) ◽  
pp. 4319-4325 ◽  
Author(s):  
D. Ogończyk ◽  
M. Gocyla ◽  
M. Opallo
Keyword(s):  
Carbon Nanoparticles ◽  
Electrostatic Interactions ◽  
Amperometric Detection ◽  
Nanoparticle Detection ◽  
System P

The development of methods for nanoparticle detection is highly desirable due to their increasing presence in the environment.

The Kinetics of Reactions Catalyzed by Alkaline Phosphatase: The Effects of Added Nucleophiles

1972 ◽  
Vol 50 (12) ◽  
pp. 1360-1368 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Alkaline Phosphatase ◽  
Preceding Paper ◽  
The Other ◽  
Intestinal Alkaline Phosphatase ◽  
Michaelis Constant ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Kinetics Of Formation ◽  
Kinetics Of

An experimental study was made of the hydrolyses of phenyl phosphate and p-nitrophenyl phosphate catalyzed by chicken intestinal alkaline phosphatase. The work was done at pH 8.0 and 10.0, 25.0 °C, and an ionic strength of 0.1 M, and particular attention was paid to the kinetics of formation of the products in the presence of Tris and ethanolamine. It was found that the rates of formation of phenol or p-nitrophenol (P1) and of the phosphorylated nucleophile (P3) were dependent on the concentration of added nucleophile; on the other hand the rate of formation of phosphate (P2) and the Michaelis constant were independent of nucleophile concentration. This result cannot be reconciled with any of the mechanisms discussed in the preceding paper with the exception of mechanism VI, which is an elaboration of one proposed by Trentham and Gutfreund; mechanism VI is[Formula: see text]where W is water and N the alternative nucleophile. ES and E*S are two conformers of the enzyme–substrate complex, and E*S′ and ES′ two forms of the phosphorylated enzyme; only the latter can react with water and only the former with nucleophile.

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