Hydrolysis of phosphates

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

A. Phosphoenolpyruvate.—The mechanisms of hydrolysis of phosphate esters of phosphoenol pyruvic acid (33) have been described in detail, and 0 studies confirm an earlier postulate that attack by water on the cyclic acyl phosphate (34) occurs at phosphorus and not at carbon. In the enolase reaction, the reversible interconversion of 2-phosphoglyceric acid(35)... 

The factors involved and mechanistic pathways in the hydrolysis of phosphate esters, particularly those of a cyclic nature, continue to be the source of much speculation. A further study of the simplest cyclic triester, ethylene methyl phosphate, seems only to have served to consolidate already polarized views. The original, experiments of Westheimer s group employed gc and H nmr spectroscopy and demonstrated that ethylene methyl phosphate (26) hydrolyses under alkaline conditions by... 

Many mechanistic aspects of the hydrolysis of phosphate esters in protic media remain uncertain. In spite of predictions that racemization at phosphorus should be the final outcome if indeed the (hypothetical) metaphosphate intermediate is involved in the solvolysis of monoesters, the results of several studies on the methanolysis of appropriately O-isotopically labelled compounds are consistent with reactions proceeding with inversion of configuration, as observed for all enzymic and non-enzymic systems so far examined this has resulted in the suggestion that if metaphosphate is actually formed, then it must be in a masked form. 

Recently, highly branched macromolecular polyamidoamine dendrimers have been prepared with Co11 bound where the metal ions have additional exchangeable coordination sites.450 These macromolecules show a capacity for catalyzing the hydrolysis of phosphate esters, presumably via intermediate bound phosphoester species. 

Just as intramolecular catalysis has been observed in the hydrolysis of carboxylate esters, a variety of neighbouring groups can participate in the hydrolysis of phosphates. The alkaline hydrolysis of dimethyl phosphoacetoin [47] has found to be ca. 2 x 106 times faster in water at 25°C than that of... 

For the hydrolysis of phosphate esters under mild conditions, metal ions and metal complexes are the most efficient nonenzymatic reagents currently available. However, they do not reach the catalytic efficiency of enzymes, and higher reactivities are desirable in view of applications. To mimic enzymatic dinuclear sites is a strategy to generate more efficient artificial phosphoesterases. 

Westheimer, F., The hydrolysis of phosphate esters, Pure Appl. Chem., 49,1059, 1977. 

Sequences 246 and 249 were tested for their ability to catalyze hydrolysis while in solution rather than while attached to a support. The Zr4 complex of sequence 246 was found to catalyze the hydrolysis of phosphate ester 243b five times faster than the complex of peptide 249. Since the control complex 249 does not catalyze hydrolysis it appears that the small amount of catalysis that was observed was due to free zirconium metal (Scheme 29). 

Hydrolysis of phosphate monoester dianions RO-OPOJ [83] Hydrolysis of axial tetrahydropyranyl acetals [79]... 

Many methods have been developed in which a product of the reaction is chemically modified to produce a substance with a particular spectral property. The inorganic phosphate released by the hydrolysis of phosphate esters may be measured by simple chemical methods (Fiske and Subbarow) after the enzyme reaction has been stopped. Such techniques are often convenient but do not lend themselves to the measurement of initial velocity. 

The classic case of assisted hydrolysis of phosphate diesters is neighbouring... 

Zn Alkaline phosphatase hydrolysis of phosphate esters Peptidases hormone control DNA and RNA polymerases nucleic acid replication and polymerization... 

In DNA and the related single-stranded RNA (ribonucleic acid), the phosphate units are monomeric and play a strictly structural role. Phosphates, however, are also essential to the metabolism of living cells, in which formation and subsequent hydrolysis of phosphate-phosphate bonds... 

Metal ions have been shown to catalyze the hydrolysis of phosphate esters, phosphoric and phosphonic acid halides, and various phosphoric acid anhydrides including acyl phosphates, pyrophosphate derivatives, and ATP. 

Converging lines of evidence have led to a general acceptance of the monomeric metaphosphate mechanism for the hydrolysis of phosphate monoester monoanions. The pH rate profile for aryl and alkyl phosphate monoester hydrolyses commonly exhibits a rate maximum near pH 4. where the concentration of the monoanion is at a maximum. The proposed mechanism is based on these principal points of evidence (a) a general observation of P-O bond cleavage (b) the entropies of activation for a series of monoester monoanions are all close to zero, which is consistent with a unimolecular rather than a bi-molecular solvolysis where entropies of activation are usually more negative by 20 eu7 (c) the molar product composition (methyl phosphate inorganic phosphate) arising from the solvolysis of the monoester monoanion in a mixed methanol-water solvent usually approximates the molar ratio of methanol ... 

Other kinetically allowed mechanistic models, i.e. hydroxide ion attack on the monoanion, can be rejected on the grounds that the required rate coefficients far exceed that found for alkaline hydrolysis of phosphate triesters. At pH > 9 two new reactions appear, one yielding a 1,6-a.nhydro sugar by nucleophilic attack through a five-membered transition state of the 1-alkoxide ion upon C-6 with expulsion of phosphate trianion. The second is apparently general-base catalysis by 1-alkoxide of water attack on C-6 or phosphorus through greater than six-membered cyclic transition states. 

The preceding discussion has emphasized catalysis nevertheless, metal ions may also significantly inhibit the rate of hydrolysis of phosphate esters through chelation at phosphorus. A pertinent example is the sixty-fold decrease in the rate of hydrolysis of 2-aminoethylphosphorothioate in the presence of excess Fe(III)148 149. Such a phenomenon underscores the exacting requirements for observation of metal-ion catalysis and implies that charge neutralization per se is not responsible. One should also note the ineffectiveness of Mg(II) or Zn(II) as catalysts in the above systems, the latter required for the activity of alkaline phosphatase ( . co/i.)150. An attractive, but as yet experimentally untested hypothesis, is that such metal ions may catalyze pseudorotation processes which otherwise would violate the preference rules. 

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