Hydrolysis of phosphate esters

The primary esters, such as D-glucose 6-phosphate, are much more resistant to acid hydrolysis, whereas phosphate esters of glyceric acid or 

Compound Normality of acid Temp- erature cc,) k X 10 Refer- ence 

The mechanism of alkaline hydrolysis of phosphate esters (rupture of P 0 bond) is such that inversion during hydrolysis is unlikely (196), Acid hydrolysis on the other hand catalyzes cleavage of the C 0 bond 

---

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. 

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). 

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. 

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

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. 

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. 

The phosphatases catalyze hydrolysis of phosphate esters to produce inorganic phosphate 667... 

Table 7.6 Relative values ofVm.n alkaline phosphatase" for the hydrolysis of phosphate esters by...

Tabic 7.7 Product ratios in the hydrolysis of phosphate esters and phosphoramidates by alkaline phosphatase"... 

Table 7.9 Product ratios in and phosphatase1 the hydrolysis of phosphate esters by prostatic 7 ...

Table 7.8 Hydrolysis of phosphate esters by prostatic acid phosphatase at pll 5 and 37"C" ...

Phosphate competitively inhibits the hydrolysis of phosphate esters. The appropriate reaction is shown in 128). 

Although metal-promoted hydrolysis of phosphate esters is a topic of very current interest (Section 61.4.4), little work has been published dealing with the effects of metal ions on the hydrolysis of sulfate esters. The acid-catalyzed hydrolysis of aryl sulfates has been shown to occur by an A-l mechanism (Scheme 34).434 Nucleophilic catalysis by amines has been observed in the hydrolysis of p-nitrophenyl sulfate435 and intramolecular carboxyl group catalysis occurs with salicyl sulfate436 as with salicyl phosphate. 

The resistance to hydrolysis of phosphate esters of primary alcohols167 was used to advantage by Seegmiller and Horecker,168 who allowed D-glucose to react with polyphosphoric acid and subjected the resulting mixed phosphate to acid hydrolysis to obtain D-glucose 6-phosphate. 

Lehn and Wipff (72) and Gorenstein and co-workers (73-80) have proposed on the basis of molecular orbital calculations that stereoelectronic effects similar to those observed in esters and amides play also an important role in the hydrolysis of phosphate esters. For instance, calculations suggest that the axial P — OR bond in the trigonal bipyramid conformation 120 is weaker than that in the conformation 121 because in the former, the oxygen atom of the equatorial OR group has an electron pair anti peri planar to the axial P — OR bond. Experimental results tend to support this interesting proposal but additional experiments are needed before unambiguous conclusions can be reached (81). 

These results are common with proteases and also for the hydrolysis of phosphate esters catalysed by alkaline phosphatase in the latter case, there is a pitfall in that the constant value for kcat for phosphate esters is due to a rate-limiting conformational change in... 

---