Hydrolysis of Pyrophosphate

FIGURE 15.20 The adenylyl cyclase reaction yields 3, 5 -cyclic AMP and pyrophosphate. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase. 

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 ... 

FIGURE 24.7 The acyl-CoA synthetase reaction activates fatty acids for /3-oxidation. The reaction is driven by hydrolysis of ATP to AMP and pyrophosphate and by the 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... 

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... 

Another way in which organisms affect the chemistry of natural waters is by accelerating the rate of a reaction that normally proceeds by nonenzymic processes. Hydrolysis of pyrophosphate and tripolyphosphate to orthophosphate has been shown to proceed at a slow rate in the absence of microorganisms in natural waters. However, in the presence of microorganisms, the rate is greatly accelerated (9, 10, 57). 

The hydrolysis of pyrophosphate to phosphate is catalyzed by yeast inorganic pyrophosphatase, which is a dimeric enzyme having two identical subunits.290 It now appears that three cations aTe required per active site, two bound to the enzyme and the third to the phosphate (equation 4). 

SM2/AM1 and SM3/PM3 models were used to study the hydrolysis of pyrophosphate, which is coupled to virtually all biosynthetic reactions. However, the authors concluded that extreme care must be taken when applying semiempirical methods to compounds containing second-row atoms, since they may produce anomalously high atomic charges [98]. On the other hand, a study on syn and anti conformations of solvated cyclic 3 ,5 -adenosine monophosphate indicated that SM3/PM3 and SM2/AM1 models are inexpensive yet accurate approaches... 

Once citrulline is in the cytosol, argininosuccinic acid is formed by condensation of citrulline with aspartate. This is where the second nitrogen atom enters the cycle. Argininosuccinate synthetase, a homotetramer of a 46-kd polypeptide catalyzes the reversible reaction accompanied by hydrolysis of ATP to AMP and pyrophosphate. The subsequent hydrolysis of pyrophosphate shifts the equilibrium to the right and results in the consumption of two high-energy phosphate bonds. 

A similar conclusion was obtained with aspirin 3 as substrate (27), but the hydrolysis of pyrophosphate was inhibited (ii5). 

The chain-elongation reaction catalyzed by DNA polymerases is a nucleophilic attack by the 3 -hydroxyl group of the primer on the innermost phosphorus atom of the deoxynucleoside triphosphate (Figure 5.22). A phosphodiester bridge forms with the concomitant release of pyrophosphate. The subsequent hydrolysis of pyrophosphate by pyrophosphatase,... 

Second, the mechanism of elongation is similar the 3 -OH group at the terminus of the growing chain makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate. Third, the synthesis is driven forward hy the hydrolysis of pyrophosphate. In contrast with DNA polymerase, however, RNA polymerase does not require a primer. In addition, RNA polymerase lacks the nuclease capability used hy DNA polymerase to excise mismatched nucleotides. 

Hydrolytic driving force. The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic reactions such as the synthesis of DNA. This hydrolytic reaction is catalyzed in Escherichia coli hy a pyrophosphatase that has a mass of 120 kd and consists of six identical subunits. For this enzyme, a unit of activity is defined as the amount of enzyme that hydrolyzes 10 pmol of pyrophosphate in 15 minutes at 37°C under standard assay conditions. The purified enzyme has a of 2800 units per milligram of enzyme. 

A proper inference. What information do the A G° data given in Table 14,1 provide about the relative rates of hydrolysis of pyrophosphate and acetyl phosphate ... 

This reaction is readily reversible. However, pyrophosphate is rapidly hydrolyzed in vivo to orthophosphate by an inorganic pyrophosphatase. The essentially irreversible hydrolysis of pyrophosphate drives the synthesis of UDP-glucose. 

The synthesis of UDP-glucose exemplifies another recurring theme in biochemistry many biosynthetic reactions are driven by the hydrolysis of pyrophosphate. 

At this stage, orotate couples to ribose, in the form of 5-phosphoribosyl-l-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide bases. PRPP is synthesized from ribose-5-phosphate, formed by the pentose phosphate pathway, by the addition of pyrophosphate from ATP. Orotate reacts with PRPP to form orotidylate, a pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The enzyme that catalyzes this addition, pyrimidine phosphoribosyltransferase, is homologous to a number of other phosphoribosyltransferases that add different groups to PRPP to form the other nucleotides. Orotidylate is then decarboxylated to form uridylate (IMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. 

The A G° of this reaction is close to 0, because the free energy of hydrolysis of the ester bond of aminoacyl-tRNA is similar to that for the hydrolysis of ATP to AMP and PPj. As vv e have seen many times, the reaction is driven by the hydrolysis of pyrophosphate. The sum of these three reactions is highly exergonic ... 

We have already encountered an acyl adenylate intermediate in fatty acid activation (Section 22.2.2). The major difference between these reactions is that the acceptor of the acyl group is CoA in fatty acid activation and tRNA in amino acid activation. The energetics of these biosyntheses are very similar both are made irreversible by the hydrolysis of pyrophosphate. 

Long chain fatty acids are are bound to Fatty acid binding protein for transport within the cytosol. They are impermeable to the inner mitochondrial membrane. They are thus esterified in the cytosol by microsomal Fatty acyl CoA synthetase in a reaction identical to the one shown above. Again the reaction is driven by the hydrolysis of pyrophosphate. The enzyme involves an acyl AMP intermediate ... 

Figure 9.8 outlines how matrix vesicles increase and decrease the concentration of pyrophosphate. NTP-PPi hydrolase synthesizes pyrophosphate from stromal fluid nucleotides, mostly ATP (ATP —> AMP + PPi). Many cells secrete ATP into the extracellular fluid and it passes into the blood plasma where it affects a variety of cells independently of its function in intracellular energy metabolism. In mice, a nonfunctional ANK protein or a deletion of NTP-PPi hydrolase decreases the extracellular pyrophosphate concentration and the phenotype exhibits extensive mineralization. Thus, the hydrolysis of pyrophosphate appears to be a major function of alkaline phosphatase (TNAP) after the calcium phosphate precipitate has raptured the matrix vesicles. Rapid mineralization of collagen and the rest of the osteoid matrix ensue without a need to transport any more Ca2+ or Pi to the region. 

The reaction is freely reversible, but it is rendered practically irreversible by hydrolysis of pyrophosphate (PPi) through the action of pyrophosphatase. 

The reaction is driven forward by hydrolysis of pyrophosphate to inorganic phosphate. Argininosuccinate formation is considered as the rate-limiting step for urea synthesis. This reaction incorporates the second nitrogen atom of the urea molecule donated by aspartate. 

As in DNA synthesis, the reaction is driven to the right by hydrolysis of pyrophosphate, so the overall reaction is... 

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