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.

Influence of pH on the Kinetics of Reactions Catalyzed by Alkaline Phosphatase

1973 ◽  
Vol 51 (7) ◽  
pp. 1096-1103 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Alkaline Phosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Coordinated Water ◽  
Ph Range ◽  
Barbital Buffer ◽  
Influence Of Ph ◽  
Kinetics Of ◽  
Hydrolysis Of

An experimental study has been made of the kinetics of the hydrolysis of p-nitrophenyl phosphate catalyzed by chicken-intestinal alkaline phosphatase. The work was done in barbital buffer (carbonate above pH 9.6), and covered the pH range from 7.0 to 10.0. A sufficiently wide range of substrate concentration was used to allow reliable values of [Formula: see text] and [Formula: see text] to be determined. The results lead to pK values of 8.1 and 8.6 for the free enzyme, and it is concluded that the Michaelis complex and the phosphoryl intermediate ionize only on the acid side, the former also having a pK of 8.1. It is suggested that the group of pK 8.1 is probably an α-amino group and that the group of pK 8.6 probably corresponds to the ionization of a Zn(II)-coordinated water molecule.

scholarly journals Chicken Intestinal Alkaline Phosphatase

1960 ◽  
Vol 43 (6) ◽  
pp. 1149-1169 ◽  
Author(s):  
M. Kunitz
Keyword(s):  
Alkaline Phosphatase ◽  
Zinc Ions ◽  
Magnesium Ions ◽  
Intestinal Alkaline Phosphatase ◽  
Reversible Inactivation ◽  
Higher Temperature ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Zn Ions ◽  
Denaturation Of Proteins

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.

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.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

scholarly journals The effect of pH on the kinetics of arylsulphatases A and B

1983 ◽  
Vol 213 (3) ◽  
pp. 603-607 ◽  
Author(s):  
C O'Fagain ◽  
B M Butler ◽  
T J Mantle
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Substrate Binding ◽  
Acid Base ◽  
Effect Of Ph ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

L-Phenylalanine inhibition of human alkaline phosphatases with p-nitrophenyl phosphate as substrate.

1982 ◽  
Vol 28 (12) ◽  
pp. 2426-2428 ◽  
Author(s):  
T Komoda ◽  
S Hokari ◽  
M Sonoda ◽  
Y Sakagishi ◽  
T Tamura
Keyword(s):  
Alkaline Phosphatase ◽  
Human Placenta ◽  
Placental Alkaline Phosphatase ◽  
Specific Inhibition ◽  
Human Intestine ◽  
Alkaline Phosphatases ◽  
Substrate Complex ◽  
Nitrophenyl Phosphate ◽  
Organ Specific ◽  
Alkaline Phosphatase Isoenzyme

Abstract With p-nitrophenyl phosphate as the substrate, there reportedly is no organ-specific inhibition of alkaline phosphatase (EC 3.1.3.1) activity by L-phenylalanine. However, we found that at pH 10.0, with p-nitrophenyl phosphate as the substrate, L-phenylalanine obviously inhibits the alkaline phosphatase isoenzyme from human placenta, whereas there is little if any inhibition of the isoenzyme from human intestine. Because of the differing effects of substrates (p-nitrophenyl phosphate and phenyl phosphate) and their enzymic products (p-nitrophenol and phenol) for L-phenylalanine action on the placental alkaline phosphatase isoenzyme, we suggest that the isoenzyme--inhibitor--substrate complex and the effect of released phosphate on L-phenylalanine inhibition of the isoenzyme activity differ from each other.

A comparison of four serological tests for the detection of banana bunchy top virus in banana

1996 ◽  
Vol 47 (3) ◽  
pp. 403 ◽  
Author(s):  
ADW Geering ◽  
JE Thomas
Keyword(s):  
Alkaline Phosphatase ◽  
Completion Time ◽  
Polyclonal Antibodies ◽  
Sandwich Elisa ◽  
Nitrocellulose Membrane ◽  
Serological Tests ◽  
Banana Bunchy Top Virus ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Enzyme Amplification

Four different serological tests for detection of banana bunchy top virus (BBTV) in banana sap are compared: (i) a triple-antibody sandwich ELISA for BBTV, utilising anti-BBTV polyclonal antibodies for virus capture, and anti-BBTV monoclonal antibodies, alkaline phosphatase-labelled sheep anti-mouse antibodies, and p-nitrophenyl phosphate for detection (ELISA-NPP); (ii) an alternative enzyme-substrate system for ELISA involving an amplification step (AmpakTM enzyme amplification kit) (ELISA-A); (iii) a colorimetric dot immunobinding assay (DIBA-C), in which the enzyme-substrate system was alkaline phosphatase and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; (iv) an enhanced chemiluminescent form (DIBA-ECL), in which the enzyme-substrate system was horseradish peroxidase and luminol. For both DIBA-C and DIBA-ECL, maximum sensitivity was obtained by pre-coating the nitrocellulose membrane with anti-BBTV polyclonal antibodies, by using 0.05 M sodium carbonate (pH 9.6) as the coating buffer, and by clarifying the sap by centrifugation and extraction with chloroform or dichloroniethane. Treatment of the sap before centrifugation by snap-freezing at -70�C, or heating at either 30 or 50�C for 10 min, had no effect on sensitivity; heating at 70�C for 10 min eliminated antigenicity. ELISA-NPP, ELISA-A, and DIBA-ECL had equivalent sensitivity, but DIBA-C was up to 8-fold less sensitive than the former 3 assays. ELISA-NPP was adjudged to be the best compromise between sensitivity, cost and completion time.

On the molecular specificity of steroid-enzyme combinations. The kinetics of β -hydroxysteroid dehydrogenase

1955 ◽  
Vol 144 (914) ◽  
pp. 116-132 ◽  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Hydroxyl Group ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Molecular Specificity ◽  
Planar Molecules ◽  
High Concentrations ◽  
Kinetics Of ◽  
Androstane Derivatives ◽  
Marked Tendency

β -Hydroxysteroid dehydrogenase is a purified enzymic protein of bacterial origin which catalyzes oxidations of 3 β - and 17 β -hydroxysteroids to their respective ketones with diphosphopyridine nucleotide as a hydrogen acceptor. The reaction kinetics of this enzyme with a variety of steroids are not in accordance with the predictions of the theory of Michaelis & Menten (1913), since the velocity of oxidation shows a marked tendency to decline at high concentrations of substrate. The behaviour of these compounds may be fully analyzed on the assumption of the formation of an enzyme-substrate complex involving two substrate molecules. The theory for bimolecular complex formation and its implications are examined. Affinity constants have been calculated for various steroids and conclusions drawn as to the structural requirements favouring attachment to the enzyme surface. Phenolic compounds of the oestra-1:3:5(10)-triene-3-ol family are most firmly bound. Planar molecules of the androst-4-ene, androst-5-ene or 5 α -androstane series show intermediate affinity, while testane (5 β -androstane) derivatives which deviate considerably from planarity are most poorly bound to the enzyme surface. The presence of oxygenated functions at positions 3 and 17 promotes high affinity, whereas an additional 11 α - or 11 β ?-hydroxyl group opposes this effect. Conclusions have been drawn as to the manner of attachment of substrates to the enzyme surface. Certain correlations between the molecular requirements for efficient binding of steroids to the enzyme surface and their physiological activities are demonstrated.

scholarly journals Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

Biosensors ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 145
Author(s):  
Antonio Guerrieri ◽  
Rosanna Ciriello ◽  
Giuliana Bianco ◽  
Francesca De Gennaro ◽  
Silvio Frascaro
Keyword(s):  
Trichoderma Viride ◽  
Kinetic Studies ◽  
Pt Electrode ◽  
Allosteric Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Influence ◽  
Ph Dependent ◽  
Kinetics Of ◽  
Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

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