Phosphoenolpyruvic acid , hydrolysis

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. 

Phosphoenolpyruvic acid is another compound (11.6) which releases a comparatively large amount of energy on hydrolysis. Glucose-6-phosphate (glucose-6-phosphoric acid) (11.7) and... 

The free energies of hydrolysis of the phosphorylated intermediates are listed in Table I. It should be noted that with the exceptions of 1,3-diphosphoglyceric acid and phosphoenolpyruvic acid, all other sugar intermediates contain phosphate groups of low energy value. Only when these two intermediates are formed does the energy of glycolysis become available for transfer. 

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)... 

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. 

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. 

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). 

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). 

In contrast to other monosaccharides, activated sialic acid donors are biosynthesized from A -acetylmannosamine (ManNAc) or directly from sialic acids (Sia), including A-acetylneuraminic acid (NeuAc), via a more complex pathway (21). ManNAc is phosphorylated at the at the 6-hydroxyl group and condensed with phosphoenolpyruvate to give A -acetylneuraminic acid-9-phosphate (NeuAc-9-P). Phosphate ester hydrolysis is followed by direct condensation with CTP to give CMP-NeuAc (Figure 3). Sialic acids can intercept this pathway directly via enzymatic reaction with CTP. 

Mammals produce sialic acid by aldolic condensation of phosphoenolpyruvate and Ai-acetylmannosamine 6-phosphate (reaction 12.1). A kinase enzyme catalyses the phosphorylation of A -acetylmannosamine and a phosphatase catalyses the hydrolysis of the phosphate of sialic acid. These phosphorylation and dephosphorylation steps are irreversible, such that the synthesis can be total even with low concentrations of the substrate. A variation of reaction (12.1), observed with the bacterium Neisseria meningitidis, uses non-phosphated /-acetylmannosamine. However, these were not the enzymes used in the preparative synthesis, which used instead a microbial aldolase which catalyses equilibrium (12.2). This enzyme probably plays a catabolic role in these organisms, but it functions in the synthetic sense in the presence of an excess of pyruvate. 

However, the acid phosphatase activity of rat iiver lysosomes has recently been resolved into at least two enzymes [531]. Acid phosphatase is used in subcellular fractionation studies as a marker enzyme for lysosomes. Both acid phosphatase [E.C. 3.1.3.2] and alkaline phosphatase [E.C. 3.1.3.1] activities should not be confused with other specific phosphatases with high specificity requirements for substrate, e.g. glucose-6-phos-phatase, fructose-l,6-diphosphatase, phosphatidate phosphatase. Several assay procedures are available, u.v. estimation can be achieved using phosphoenolpyruvate as a substrate and lactate dehydrogenase in an indicator reaction [539]. Colorimetric assays can be based upon the liberation of phenol from phenylphosphate [540], upon the Uberation of phosphate from sodium /3-glycerophosphate [541], upon the hydrolysis of sodium phenolphthalein phosphate [542], or upon the hydrolysis of p-nitrophenyl phosphate [543]. 

The equilibrium between phosphocreatine and ATP shows that the former compound has a AF of hydrolysis at pH 7.7 of 1100 cal. above that of ATP however, the value is very dependent on pH. The AF of hydrolysis of 1,3-diphosphoglyceric acid, phosphoenolpyruvate, and acetyl phosphate are all of somewhat higher energy, about —15,000 cal. Summaries of such values have been published. ... 

The conversion of 1 glucose to 2 lactates in mammalian tissue leads to the formation of 2 molecules of each of two high-energy phosphate compounds 1,3-diphosphoglyceric acid and phosphoenolpyruvate. These compounds can pass phosphate to ADP and form 4 molecules of ATP. However, 2 molecules of ATP are required to make the reaction proceed, so the net gain is 2 ATP. Of the approximately 50,000 cal. available from the reaction, 28,000 cal. are conserved as (56%), assuming AF of hydrolysis of the terminal phosphate of ATP is —14,000 cal. per mole in vivo. 

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