Hydrolysis of PEP

The hydrolysis of PEP is sufficiently spontaneous to produce ATP from ADP when coupled to this reaction. The sum of the two reactions is equivalent to a spontaneous reaction ... 

PEP). The free energy of hydrolysis of PEP to form the enol form of pyruvate and Pi is on the order of —4 kcal/mol. In aqueous solution, however, the enol form of pyruvate is very unstable. Thus, the hydrolysis of PEP to form pyruvate is a very exergonic reaction. The AGq for this reaction is —14.7 kcal/mol, which corresponds to an equilibrium constant of 6.4 x 10 . PEP is thus an excellent phosphoryl donor and the formation of pyruvate is coupled to ATP synthesis. Since two molecules of pyruvate are formed per glucose catabolized, two ATP are formed. Thus the net yield of ATP is two per glucose oxidized to pyruvate. 

FIGURE 3.14 Hydrolysis and the subsequent tautomerization account for the very large AC of PEP. 

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

This enzyme [EC 3.2.1.17], also called muramidase, catalyzes the hydrolysis of the l,4-/3-linkages between N-acetyl-D-glucosamine and A-acetylmuramic acid in pep-tidoglycan heteropolymers of prokaryotic cell walls. Some chitinases [EC 3.2.1.14] also exhibit this activity. See Kinetic Isotope Effect... 

The reaction is reversible under intracellular conditions the formation of one high-energy phosphate compound (PEP) is balanced by the hydrolysis of another (GTP). 

Fig. 2.18 Simplified scheme of the cycle/aUocation of magnesium in a green plant. Mg is involved - inter aha - in making peptide bonds, in tricarboxylate cycle, hydrolysis of molecules and binding of CO (ribulose-bisphosphatecarboxylase/oxidase or PEP carboxylase in plants which also employs Mg or Mtf+ in some plants (Kai et al. 2003)). Reaction steps in which Mg takes part as biocatalyst are marked by broken lines/arrows. Citrate and other intermediates of the tricarboxylate cycle, particularly malate, are employed by higher plants for extraction of essential metals, including Mg, Fe and Mn (thus the closed loop) from soil via and by means of the roots. This closed loop depicts a manner of autocatalysis. Amino acids which are required for protein biosynthesis are produced by reductive amination from tricarboxylate cycle intermediates and other 2-oxoadds which likewise eventually...

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. 

The problem of undesired hydrolysis of peptide bonds occurring during deprotection can be avoided completely by choosing enzymes devoid of any protease activity. For example by using and 2-bromoethylt l esters as carboxy protecting groups for pep-... 

C4 plants possess two types of photosynthesizing cells in their leaves mesophyll cells and bundle sheath cells. (In C3 plants, photosynthesis occurs in mesophyll cells.) Most mesophyll cells in both plant types are positioned so that they are in direct contact with air when the leaf s stomata are open. In C4 plants, C02 is captured in specialized mesophyll cells that incorporate it into oxaloacetate (Figure 13B). Phosphoenolpymvate carboxylase (PEP carboxylase) catalyzes this reaction. Oxaloacetate is then reduced to malate. Once formed, malate diffuses into bundle sheath cells. (As their name implies, bundle sheath cells form a layer around vascular bundles, which contain phloem and xylem vessels.) Within bundle sheath cells, malate is decarboxylated to pyruvate in a reaction that reduces NADP+ to NADPH. The pyruvate product of this latter reaction diffuses back to a mesophyll cell, where it can be reconverted to PEP. Although this reaction is driven by the hydrolysis of one molecule of ATP, there is a net cost of two ATP molecules. An additional ATP molecule is required to convert the AMP product to ADP so that it can be rephos-phorylated during photosynthesis. This circuitous process delivers CO, and NADPH to the chloroplasts of bundle sheath cells, where ribulose-1,5-bisphosphate carboxylase and the other enzymes of the Calvin cycle use them to synthesize triose phosphates. 

In practice, for most synthetic applications, either acetyl phosphate/acetate kinase or phosphoenolpyruvate/pyruvate kinase are used to regenerate ATP. Because of the ease of preparing AcP, AcP/AcK is the most economical method for large-scale work. Its application is, however, limited to fast phosphorylation reactions where the hydrolysis of AcP is not important. The PEP/pyruvate kinase system is used in instances where the requirement for a strong, stable phosphorylating reagent outweighs the relative inconvenience of preparation of PEP. 

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

Notes - This reaction is a simple dehydration (or ot/ elimination) of 2PG to form phosphoenolpyruvate (PEP), but it has the effect of increasing the energy of hydrolysis of the phosphate bond almost four fold (from -15.6 kJ/mol in 2PG to -61.9 kJ/mol in PEP). This high free energy of hydrolysis is necessary for the next step in glycolysis, which is another substrate level phosphorylation of ADP to form ATP. +1.7 kJ/mol... 

One reaction used by many organisms as a source of ATP is the coupling of ATP production from ADP 4- P with the hydrolysis of phosphoenol pyruvate (PEP). The two reactions are... 

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