Phosphate Pyrophosphate Hydrolysis

The same strategy has been applied to the recognition of pyrophosphate ions P20) (PPi). A-9 (Figure 10.32) binds these ions over 2000 times more tightly than phosphate ions, permitting the real-time monitoring of pyrophosphate hydrolysis. 

This enzyme [EC 3.1.3.9] catalyzes the hydrolysis of o-glucose 6-phosphate to yield o-glucose and orthophosphate. Some glucose phosphatases also catalyze transphosphorylation reactions from carbamoyl phosphate, hexose phosphates, pyrophosphate, phosphoenolpyru-vate and nucleoside di- and triphosphates, using D-glu-cose, D-mannose, 3-methyl-D-glucose, or 2-deoxy-D-glu-cose as phosphoryl acceptors. See Isotope Exchange (Reactions Away from Equilibrium)... 

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. 

Recently, Czamik et al. have reported the use of the acyclic protonated amine host 9 as a chemosensor of pyrophosphate. Typical fluorescence sensing methods rely on the ability of a complexed anion to quench the fluorophore. The fluorescence intensity of host 9, however, is actually increased upon complexation of anions and its 2200-fold selectivity of pyrophosphate over phosphate allows for real-time assay of pyrophosphate hydrolysis by inorganic pyrophosphatase.18... 

The Czarnik group then prepared compound 2, which has two polyamine chains at the 1- and 8-positions of anthracene, as a fluorescence chemosensor toward pyrophosphate (PPi) [14]. To effectively bind both phosphates of PPi simultaneously, the polyamine chains were geometrically preorganized. The dissociation constant (K ) for the 2-PPi complex was found to be 2.9 xM, whereas that for 2-phosphate was 6.3 mM. In particular, chemosensor 2 displayed a 2200-fold pyrophosphate/phosphate discrimination at pH 7 (Scheme 2). Such high ion selectivity allows a real-time assay of pyrophosphate hydrolysis catalyzed by inorganic phosphatase. 

Let us take the case of hexokinase which transfers a phosphate residue from ATP to glucose with the formation of ADP and glucose-6-phosphate. The hydrolysis of the pyrophosphate linkage of ATP gives 12,000 cdories and that of glucose-6-phosphate only yields 2,000-4,000 cal. During the... 

In E. coli GTP cyclohydrolase catalyzes the conversion of GTP (33) into 7,8-dihydroneoptetin triphosphate (34) via a three-step sequence. Hydrolysis of the triphosphate group of (34) is achieved by a nonspecific pyrophosphatase to afford dihydroneopterin (35) (65). The free alcohol (36) is obtained by the removal of residual phosphate by an unknown phosphomonoesterase. The dihydroneoptetin undergoes a retro-aldol reaction with the elimination of a hydroxy acetaldehyde moiety. Addition of a pyrophosphate group affords hydroxymethyl-7,8-dihydroptetin pyrophosphate (37). Dihydropteroate synthase catalyzes the condensation of hydroxymethyl-7,8-dihydropteroate pyrophosphate with PABA to furnish 7,8-dihydropteroate (38). Finally, L-glutamic acid is condensed with 7,8-dihydropteroate in the presence of dihydrofolate synthetase. 

The concepts of destabilization of reactants and stabilization of products described for pyrophosphate also apply for ATP and other phosphoric anhydrides (Figure 3.11). ATP and ADP are destabilized relative to the hydrolysis products by electrostatic repulsion, competing resonance, and entropy. AMP, on the other hand, is a phosphate ester (not an anhydride) possessing only a single phosphoryl group and is not markedly different from the product inorganic phosphate in terms of electrostatic repulsion and resonance stabilization. Thus, the AG° for hydrolysis of AMP is much smaller than the corresponding values for ATP and ADP. 

The reaction proceeds via attack by a phosphate oxygen of glucose-l-phosphate on the a-phosphorus of UTP, with departure of the pyrophosphate anion. The reaction is a reversible one, but—as is the case for many biosynthetic reactions —it is driven forward by subsequent hydrolysis of pyrophosphate ... 

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthase I (reaction 1, Figure 29-9). A cytosolic form of this enzyme, carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Chapter 34). Carbamoyl phosphate synthase I, the rate-hmiting enzyme of the urea cycle, is active only in the presence of its allosteric activator JV-acetylglutamate, which enhances the affinity of the synthase for ATP. Formation of carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphate donor. Conversion of the second ATP to AMP and pyrophosphate, coupled to the hydrolysis of pyrophosphate to orthophosphate, provides the driving... 

Of the three organic phosphorus insecticides—hexaethyl tetraphosphate, tetraethyl pyrophosphate, and parathion—the first two have been shown to be mixtures (36) that contain tetraethyl pyrophosphate as the principal active ingredient. Several methods have been proposed for the determination of this compound in the commercial products (25, 35). All are based on the separation of the tetraethyl pyrophosphate from the related ethyl phosphates, followed by its hydrolysis to diethyl orthophosphoric acid and titration with standard alkali. Both hexaethyl tetraphosphate and tetraethyl pyrophosphate are soluble in water and are rapidly hydrolyzed to monoethyl and diethyl orthophosphoric acid. This rapid hydrolysis to nontoxic products greatly limits the duration of the in- secticidal effectiveness of tetraethyl pyrophosphate, but it also eliminates the danger of toxic residues on the crops treated. 

A mechanism proposed 87) for the alkaline hydrolysis of tetraethyl pyrophosphate, which is markedly accelerated by HPO e ions, has been substantiated by isotopic labeling 88). The nucleophilic attack by HPOJp on the symmetrical pyrophosphate 131 is considered to lead initially to the unsymmetrical P P1-diethyl pyrophosphate dianion 132 which decomposes spontaneously under the conditions of reaction to give the diethyl phosphate anion and POf 102. The latter reacts with water to form inorganic phosphate and with alcohols suclj as methanol and ethylene glycol to produce alkyl phosphates. 

Phosphate in combination with NaCl has a beneficial effect on the waterbinding capacity of processed meat products for a detailed description, see Schmidt.227 The effect of phosphates is suggested to be alterations in pH or ionic strength, sequestration of metal ions, dissociation of actomyosin and depolymerisation of myosin.103,104,228,229 However, before action, added phosphates must be hydrolysed by muscle phosphatases or non-enzymatically. Belton et al.230 studied the hydrolysis of pyrophosphate and tripolyphosphate in comminuted chicken meat using 31P NMR spectroscopy, and found that the rate of hydrolysis was dependent on the length of ageing period of the muscle as well as the presence of NaCl. Li et al.231 studied the hydrolysis of various types of phosphates in intact chicken muscle with a similar approach by 31P NMR spectroscopy and thereby demonstrated differences in rate of hydrolysis of various phosphates. The findings of these studies... 

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