Phosphoanhydride

FIGURE 23.18 The UDP-glucose pyrophosphorylase reaction is a phosphoanhydride exchange, with a phosphoryl oxygen of glu-cose-l-P attacking the m-phosphorus of UTP to form UDP-glucose and pyrophosphate. 

An enzymatic reaction intermediate formed by phospho-ryl transfer to a carboxyl group on an enzyme. Acyl-phosphates are structurally analogous to acid anhydrides (R—CO —O —CO—R ), and they are thermodynamically less stable than either of the two phosphoanhydride bonds in ATP. This is evident by the fact that the acetate kinase reaction (ADP + acetyl-phosphate = ATP + acetate) favors ATP formation with an equilibrium constant of about 3,000. Acetyl-phosphate can be chemically synthesized by reacting orthophosphate with acetic anhydride. 

Hydrolysis of the terminal (or, /3-y) phosphoanhydride bond of ATP is the primary thermodynamic driving force in metabolism. Written as a biochemical equation at a specified pH, ATP hydrolysis can be represented as ... 

Inorganic pyrophosphatase [EC 3.6.1.1] plays a central role in phosphorus metabolism by catalyzing the hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate (or, diphosphate PPi). This cleavage reaction acts in conjunction with pyrophosphate-forming ligases to provide an additional thermodynamic impetus for certain biosynthetic reactions. For example ... 

ATP is an energy rich molecule because its triphosphate unit contains two phosphoanhydride bonds. A large amount of free energy is liberated when ATP is hydrolysed to ADP or AMP. 

RGURE 8-40 The phosphate ester and phosphoanhydride bonds of ATP. Hydrolysis of an anhydride bond yields more energy than hydrolysis of the ester. A carboxylic acid anhydride and carboxylic acid ester are shown for comparison. 

The energy released by hydrolysis of ATP and the other nucleoside triphosphates is accounted for by the structure of the triphosphate group. The bond between the ribose and the a phosphate is an ester linkage. The a, ft and ft,y linkages are phosphoanhydrides (Fig. 8-40). Hydrolysis of the ester linkage yields about 14 kJ/mol under standard conditions, whereas hydrolysis of each anhydride bond yields about 30 kJ/mol ATP hydrolysis often plays an important thermodynamic role in biosynthesis. When coupled to a reaction with a positive free-energy change, ATP hydrolysis shifts the equilibrium of the overall process to favor product forma-... 

Although the hydrolysis of ATP is highly exeigonic (AG ° = -30.5 kJ/mol), the molecule is kinetically stable at pH 7 because the activation energy for ATP hydrolysis is relatively high. Rapid cleavage of the phosphoanhydride bonds occurs only when catalyzed by an enzyme. 

FIGURE 13-11 Nucleoside triphosphates in RNA synthesis With each nucleoside monophosphate added to the growing chain, one PPi is released and hydrolyzed to two P,. The hydrolysis of two phosphoanhydride bonds for each nucleotide added provides the energy for forming the bonds in the RNA polymer and for assembling a specific sequence of nucleotides... 

Inorganic polyphosphate (polyP) is a linear polymer composed of many tens or hundreds of Pi residues linked through phosphoanhydride bonds. This polymer, present in all organisms, may accumulate to high levels in some cells. In yeast, for example, the amount of polyP that accumulates in the vacuoles would represent, if distributed uniformly throughout the cell, a concentration of 200 him (Compare this with the concentrations of other phosphoryl donors listed in Table 13-5.)... 

Nicotinamide adenine dinucleotide (NAD+ in its oxidized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP+) are composed of two nucleotides joined through their phosphate groups by a phosphoanhydride bond (Fig. 13-15a). Because the nicotinamide ring resembles pyridine, these compounds are sometimes called pyridine nucleotides. The vitamin niacin is the source of the nicotinamide moiety in nicotinamide nucleotides. 

Frey, EA. Arabshahi, A. (1995) Standard free-energy change for the hydrolysis of the a-j3-phosphoanhydride bridge in ATP. Biochemistry 34, 11,307—11,310. 

Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis (AG ° = -36 kJ/mol). In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP, with a net AG ° of only -2.9 kJ/mol. Succinate is formed in the process ... 

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

The various compounds were tested in the routine assay under all of the conditions noted in Table IV liberation of Pi after incubation with large excesses of purified enzyme was not detected. Compounds of groups (2) and (3), containing phosphodiester or internal phosphoanhydride linkages, were additionally tested with human semen phosphomonoesterase after incubation with E. coli pyrophosphatase. No Pi was liberated after this dual incubation, indicating absence of phosphodiesterase or coenzyme-degrading activity in the pyrophosphatase (12). 

The inability of the electron-rich nucleophile molecules to approach phosphorus atoms of highly electronegative binary complexes (see Section III,D,5,a and Fig. 4) formed from the enzyme and the more extensively ionized species of phosphoanhydrides extant in the alkaline pH range is believed to explain this lack of reactivity of the latter compounds at the higher pH values (43). Support for this concept is found in the results of recent studies (43, 43), which indicate that carbamyl-P, which according to the above theory should be active at alkaline as well as acid pH values, displays potent phosphotransferase ability with the enzyme even at pH > 7. Mechanistic significance of these observations is considered further in Section III,D,5. 

The recent discovery of the dual nature of the enzyme, involving potent synthetic as well as hydrolytic activities, has served as a basis for a variety of new control mechanisms and physiological roles for the catalyst. For example, hydrolytic activity of the enzyme is limited through competitive inhibition by PPi, carbamyl-P, Pi HC03 , ATP, ADP, and various other phosphoanhydrides. Glucose itself, through participation as phosphoryl acceptor alternative with water (30, 31, Jfl), may regulate its own release in an autocatalytic manner (9, 10, 169). 

The released energy of almost all the metabolic pathways is converted to the chemical energy of phosphoanhydride bonds in ATP. Cells use the energy released during hydrolysis of the ATP bonds to power energetically unfavorable processes. 

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