Phosphomonoesters hydrolysis

The results obtained with this approach are quite impressive. Large rate accelerations (kcat/kuncat = 3 X 104) were observed with the best of the catalysts. A comparison between the best catalyst and a catalytic antibody system designed for phosphomonoester hydrolysis is reported. The combinatorial derived system gives an observed rate constant that is five times larger than that reported for the antibody system. In a control experiment, it was determined that polymers with just one type of carboxylic acid attached did not have catalytic activity. It... 

Fig. 2. Phosphomonoester hydrolysis at active center of alkaline phosphatase.

Fig. 10. A possible mechanism for phosphomonoester hydrolysis at the catalytic site of serine/threonine phosphatase-1 active site.

Aldol condensation of pyruvate and L-aspartate - /3-semialdehyde Phosphomonoesters hydrolysis (p-nitrophenyl phosphate, 3.3 x 10 )... 

Inositol-1,4,5-trisphosphate 5-phosphatase [EC 3.1.3.56], also known as inositol trisphosphate phosphomonoester-ase and inositol polyphosphate 5-phosphatase, catalyzes the hydrolysis of D-myo-inositol 1,4,5-trisphosphate to produce D-myo-inositol 1,4-bisphosphate and orthophosphate. The type I enzyme (but not the type II enzyme) will also hydrolyze inositol 1,3,4,5-tetrakisphosphate at the 5-position. However, neither of the two... 

Alkaline phosphatase (AP) is a (Znn)2-containing phosphomonoester-ase that hydrolyzes phosphomonoesters (RO—POf-) at alkaline pH (7). Ser102 under the influence of one of the zinc(II) ions at the active center 1 (Fig. 2) is directly involved in phosphate hydrolysis (8). On the basis of X-ray structure and NMR studies (9), the mechanism now accepted is that the phosphate substrate, initially recognized by cooperative... 

M aqueous NaOH (done quickly before the subsequent hydrolysis could occur to any extent) showed the monodeprotonation with pKa value of 9.1, which was assigned to the 25a = 25b equilibrium. The pK value was higher than that of 7.3 for 24a under the same conditions, which is ascribable to the proximate phosphate anion interaction with zinc(II) (like 25c). The pendent phosphodiester in 25b underwent spontaneous hydrolysis in alkaline buffer to yield a phosphomonoester-pendent zinc(II) complex 26. Plots of the first-order rate constants vs pH (=7.5 -10.5) gave a sigmoidal curve with an inflection point at pH... 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

While there have been a considerable number of structural models for these multinuclear zinc enzymes (49), there have only been a few functional models until now. Czamik et al. have reported phosphate hydrolysis with bis(Coni-cyclen) complexes 39 (50) and 40 (51). The flexible binuclear cobalt(III) complex 39 (1 mM) hydrolyzed bis(4-nitro-phenyl)phosphate (BNP-) (0.05 mM) at pH 7 and 25°C with a rate 3.2 times faster than the parent Coni-cyclen (2 mM). The more rigid complex 40 was designed to accommodate inorganic phosphate in the in-temuclear pocket and to prevent formation of an intramolecular ju.-oxo dinuclear complex. The dinuclear cobalt(III) complex 40 (1 mM) indeed hydrolyzed 4-nitrophenyl phosphate (NP2-) (0.025 mM) 10 times faster than Coni-cyclen (2 mM) at pH 7 and 25°C (see Scheme 10). The final product was postulated to be 41 on the basis of 31P NMR analysis. In 40, one cobalt(III) ion probably provides a nucleophilic water molecule, while the second cobalt(III) binds the phosphoryl group in the form of a four-membered ring (see 42). The reaction of the phosphomonoester NP2- can therefore profit from the special placement of the two metal ions. As expected from the weaker interaction of BNP- with cobalt(in), 40 did not show enhanced reactivity toward BNP-. However, in the absence of more quantitative data, a detailed reaction mechanism cannot be drawn. 

UMP becomes bound to site B which catalyzes the hydrolysis of the phosphomonoester bond. Adenosine and 3 -AMP by binding at site B could interfere with the breakdown of cyclic 2, 3 -UMP. Similarly, binding of bis (p-nitrophenyl) phosphate at site A could interfere with the breakdown of 3 -AMP. Cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate compete for site A while adenosine competes with 3 -AMP for site B. Unemoto et al. (7) have examined the mutual inhibition of substrates and substrate analogs for the enzyme from halophilic V. alginolyticus. They also concluded that 3 -ribonucleotides and ribonucleo-side 2, 3 -cyclic phosphates are hydrolyzed at different sites. However, because of the nature of the mutual inhibition between 3 -AMP and bis(p-nitrophenyl) phosphate, they suggested that part of the site for the latter substrate overlaps with the 3 -nucleotidase site. At this time the precise mechanism of action of the enzyme is not settled, but clearly there are two active sites, one a 3 -nucleotidase site and a cyclic phosphate diesterase site. Anraku (18) has described this protein as a double-headed enzyme. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

During the nonenzymic acid-catalyzed hydrolysis of phosphomonoesters (22), P-0 cleavage also occurs. 

A large variety of metal ions catalyze the cleavage of RNA through the transesterification mechanism, and some of them can also catalyze hydrolysis of cyclic phosphates (e.g. Pb +, Zn +, and Cd +) and nucleoside phosphomonoesters (e.g. Pb +, La +, and Th +). The most efficient cleavage of RNA in aqueous solutions by free metal ions is achieved by the action of rare metal ions (e.g. Eu +, La +, and Tb +), Pb +, and Zn +. Zn + is only about 4% as efficient as Pb2+,327 other catalytically active metal ions (e.g. AP+, Cd +, Mn +, Cu +, Co +, Ni +, or Mg +) are 1-2 orders of magnitude less efficient than Zn +. As a rule, RNA cleavage has a maximum rate at pH values around the pKaS of the first metal-bound H2O (see Table 1), that is, when the hydroxo-metal species still bear positive charge. ... 

Phosphorus oxychloride is a suitable reagent for preparation of the symmetrically substituted phospho-triesters of type (RO)3PO. The preparation is easily achieved by treatment of phosphorus oxychloride with 3 equiv. of alcohols or their metal salts. The reaction is generally promoted by a base or acid. Titanium trichloride is a particularly effective catalyst for the reaction. Conversion of POCI3 to unsymmetri-cally substituted phosphotriesters is achievable with difficulty. Phosphorochloridates and phosphorodichloridates have been used for the preparation of mixed tertiary phosphoric esters of type (ROlmPOfOROn (ffi = 1, n = 2, or m = 2, n = 1) in a very wide variety. Reaction of phosphorus oxychloride and 1 or 2 equiv. of alcohols followed by hydrolysis forms phosphomonoesters or phosphodi-esters, respectively. The hydrolysis may be generally effected by dilute aqueous alkali. Some phosphoFodichlori te intermediates are easily hydrolyzed by water. For example, the phosphorylation of a ribonucleoside (1 equation 4) with phosphorus oxychloride in an aqueous pyridine-acetonitrile mixture furnishes the nucleoside S -monophosphate (2) in excellent yield. ... 

The data and mechanistic conclusions summarized above come from work with aryl phosphomonoesters as predicted by the steep jSlg value, alkyl ester dianions react at very slow rates. A recent study of methyl phosphate found the rate of the dianion hydrolysis to be below the threshold of detectability, with an estimated rate constant of 2 x 10 20 s 1 at 25 °C.3 Since this value is close to the rate predicted from an extrapolation of the Bronsted plot of aryl phosphomonoester dianions, a similar mechanism is likely to be followed for alkyl and aryl esters. 

Fig. 8 Potential mechanisms for hydrolysis of phosphomonoester monoanions. In mechanism (a), proton transfer from the phosphoryl group to the ester oxygen (probably via the intermediacy of a water molecule) yields an anionic zwitterion intermediate. This may react in either concerted fashion (upper pathway) or via a discrete metaphosphate intermediate in a preassociative mechanism (bottom pathway). Mechanism (b) denotes proton transfer concerted with P-O(R) bond fission. As with (a), such a mechanism could either occur with concerted phosphoryl transfer to the nucleophile (upper pathway) or via a discrete metaphosphate intermediate in a preassociative mechanism (bottom pathway).

The reagent is used for the synthesis of phosphomonoesters of alcohols, particularly of nucleotides. It is coupled to the alcoholic component in pyridine using dicyclo hexylcarbodiimide (DCC) as condensing agent, as illustrated for the case of 5 -0-tritylthymidine. Acid hydrolysis eliminates the protective trityl group, and very mild... 

Hydrolysis reactions of great variety commonly occur on microbial cell surfaces (Fig. 2.4). Phosphatases that degrade phosphomonoesters, urease that degrades urea, carbonic anhydrase that catalyzes the interconversion between bicarbonate and carbon dioxide, and proteases that attack amide bonds in proteins, to name a few, are present. Some of these enzymes are found inside as well as on the surfaces of cells. 

Enzymes which catalyze the reaction type (a) include phosphodiesterases, phospholipases (C and D), nucleotidyl transferases, nucleases, and pyrophos-phokinases. The type (b) reaction involves mainly phosphokinases and phos-phomutases. The hydrolysis of phosphomonoesters (reaction type c) is catalyzed by phosphatases, nucleotidases, ATPases, and so on. Most phosphatases also catalyze the phosphoryl transfer reaction, type (b), if an alcohol is used as an acceptor. 

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