Phosphoenolpyruvate hydrolysis

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 phosphoryl group in phenylphosphate is derived from the -phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol K, 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated several fold by another protein, subunit 3 (24kDa). The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications. ... 

In addition to the aforementioned allenic steroids, prostaglandins, amino acids and nucleoside analogs, a number of other functionalized allenes have been employed (albeit with limited success) in enzyme inhibition (Scheme 18.56) [154-159]. Thus, the 7-vinylidenecephalosporin 164 and related allenes did not show the expected activity as inhibitors of human leukocyte elastase, but a weak inhibition of porcine pancreas elastase [156], Similarly disappointing were the immunosuppressive activity of the allenic mycophenolic acid derivative 165 [157] and the inhibition of 12-lipoxygenase by the carboxylic acid 166 [158]. In contrast, the carboxyallenyl phosphate 167 turned out to be a potent inhibitor of phosphoenolpyruvate carboxylase and pyruvate kinase [159]. Hydrolysis of this allenic phosphate probably leads to 2-oxobut-3-enoate, which then undergoes an irreversible Michael addition with suitable nucleophilic side chains of the enzyme. 

This phosphatase [EC 3.1.3.60] catalyzes the hydrolysis of phosphoenolpyruvate to form pyruvate and phos-... 

Phosphoenolpyruvate (Fig. 13-3) contains a phosphate ester bond that undergoes hydrolysis to yield the enol form of pyruvate, and this direct product can immediately tautomerize to the more stable keto form of pyruvate. Because the reactant (phosphoenolpyruvate) has only one form (enol) and the product (pyruvate) has two possible forms, the product is stabilized relative to the reactant. This is the greatest contributing factor to the high standard free energy of hydrolysis of phosphoenolpyruvate AG ° = -61.9 kJ/mol. 

FIGURE 13-3 Hydrolysis of phosphoenolpyruvate (PB3). Catalyzed by pyruvate kinase, this reaction is followed by spontaneous tautomerization of the product, pyruvate. Tautomerization is not possible in PER and thus the products of hydrolysis are stabilized relative to the reactants Fiesonance stabilization of P, also occurs, as shown in Figure 13-1. 

Proton transfer may proceed directly or via a six-membered cyclic transition state involving a molecule of water. A calculation of the intermediate zwitter-ionic concentration for the hydrolysis of methyl phosphate monoanion, based on the pKa values for methanol and methyl phosphate dianion, predicts the first-order rate coefficient for zwitterion decomposition to be ca. 10 sec-1 at 100°C. This value is in good agreement with the observed rate of hydrolysis and, considering the assumptions involved, with the rate of P-O bond fission of the presumed zwitterionic intermediate (2) formed in the Hg(II) catalyzed solvolysis of phosphoenolpyruvic acid, a model reaction for pyruvate kinase10. 

Recently Benkovic and Schrayl28b and Clark and Kirby,26c have investigated the hydrolysis of dibenzylphosphoenolpyruvic acid and mono-benzylphospho-enolpyruvic acid which proceed via stepwise loss of benzyl alcohol (90%) and the concomitant formation of minor amounts (10%) of dibenzylphosphate and monobenzylphosphate, respectively. The pH-rate profiles for release of benzyl alcohol reveal that the hydrolytically reactive species must involve a protonated carboxyl group or its kinetic equivalent. In the presence of hydroxylamine the course of the reaction for the dibenzyl ester is diverted to the formation of dibenzyl phosphate (98%) and pyruvic acid oxime hydroxamate but remains unchanged for the monobenzyl ester except for production of pyruvic acid oxime hydroxamate. The latter presumably arises from phosphoenolpyruvate hydroxamate. These facts were rationalized according to scheme (44) for the dibenzyl ester, viz. 

The final energy payoff in the glycolytic pathway occurs in the hydrolysis of phosphoenolpyruvate to pyruvate and the concomitant phosphorylation of ADP to ATP. Two molecules of ATP are produced for each molecule of hexose phosphate consumed, bringing the net yield of ATP to two molecules for each molecule of glucose (two molecules of ATP are regenerated in the phosphoglycerate kinase step and two in this step, and two are consumed in the hexoki-nase and phosphofructokinase steps). 

Phosphoglycerate and phosphoenolpyruvate differ only by dehydration between C-2 and C-3, yet the difference in the A G° of hydrolysis is about... 

Mesophyll cells use C02 from the air to convert phospho-enolpyruvate to oxaloacetate (fig. 15.28). Oxaloacetate is reduced to malate, which then moves to the bundle sheath cells that surround the vascular structures in the interior of the leaf. Here malate is decarboxylated to pyruvate in an oxidative reaction that reduces NADP+ to NADPH. The pyruvate returns to the mesophyll cells, where it is phos-phorylated to phosphoenolpyruvate. This phosphorylation is driven by splitting of ATP to AMP and pyrophosphate and subsequent hydrolysis of the pyrophosphate to phosphate. 

Cells drive active transport in a variety of ways. The plasma-membrane Na+-K+ pump of animal cells (a) and the plasma-membrane H+ pump of anaerobic bacteria (b) are driven by the hydrolysis of ATP. Eukaryotic cells couple the uptake of neutral amino acids to the inward flow of Na+ (c). Uptake of /3-gal actosidcs by some bacteria is coupled to inward flow of protons (d). Electron-transfer reactions drive proton extrusion from mitochondria and aerobic bacteria (e). In halophilic bacteria, bacteriorhodopsin uses the energy of sunlight to pump protons (/). E. coli and some other bacteria phosphorylate glucose as it moves into the cell and thus couple the transport to hydrolysis of phosphoenolpyruvate (g). 

In the next reaction, an enol is formed by the dehydration of 2-phosphoglycerate. Enolase catalyzes the formation of phosphoenolpyruvate (PEP). This dehydration markedly elevates the transfer potential of the phosphoryl group. An enol phosphate has a high phosphoryl-transfer potential, whereas the phosphate ester, such as 2-phosphoglycerate, of an ordinary alcohol has a low one. The A G° of the hydrolysis of a phosphate ester of an ordinary alcohol is -3 kcal mofi (-13 kJ mol i), whereas that of phosphoenolpyruvate is -14.8 kcal mofi (- 62 kJ mofi). Why does phosphoenolpyruvate have such a high phosphoryl-transfer potential The phosphoryl group traps the molecule in its unstable enol form. 

On formation, phosphoenolpyruvate is metabolized by the enzymes of glycolysis but in the reverse direchon. These reactions are near equilibrium under intracellular condihons so, when condihons favor gluconeogenesis, the reverse reactions will take place until the next irreversible step is reached. This step is the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pj. 

Attack on Oxygen. Cyclic phosphites or phosphonites (38) react with a-keto-acids to give cyclic acyloxyphosphoranes (39), which have previously been implicated as intermediates in the hydrolysis of phosphoenolpyruvate initial attack on keto-oxygen has been suggested, as shown. Acyclic phosphites gave the Michaelis-Arbusov product (40), presumably because they lack the five-membered ring to stabilize the phosphorane (39). 

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