Phosphoenolpyruvate from oxaloacetate

Decarboxylation drives the condensation of malonyl ACP and acetyl ACP. In contrast, the condensation of two molecules of acetyl ACP is energetically unfavorable. In gluconeogenesis, decarboxylation drives the formation of phosphoenolpyruvate from oxaloacetate. 

The carboxylation reaction produces an activated carboxyl group in the form of a high-energy carboxybiotin intermediate. The cleavage of this bond and release of CO2 in the phosphoenolpyruvate carboxykinase reaction or the transfer of the CO2 to acceptors in other reactions in which biotin participates allows endergonic reactions to proceed. Thus, the formation of phosphoenolpyruvate from oxaloacetate is driven by the release of CO2 (AG° = -4.7 kcal/mol) and the hydrolysis of GTP (AG° = -7.3 kcal/mol). 

The Jirst indirect route in glucose synthesis involves the formation of phosphoenolpyruvate from pyruvate without the intervention of pyruvate kinase. This route is catalyzed by two enzymes. At first, pyruvate is converted into oxaloacetate. This reaction occurs in the mitochondria as the pyruvate molecules enter them, and is catalyzed by pyruvate carboxylase according to the scheme... 

As noted in the discussion of anaplerotic reactions (Table 16-2), phosphoenolpyruvate can be synthesized from oxaloacetate in the reversible reaction catalyzed by PEP carboxykinase ... 

Phosphoenolpyruvate is produced from oxaloacetate in animals and plants by reaction (33), which is catalyzed by phosphoenolpyruvate carboxykinase. 

Finally, oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase in the cytosol. The CO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. Recall that, in glycolysis, the presence of a phosphoryl group traps the unstable enol isomer of pyruvate as phosphoenolpyruvate (Section 16.1.7). In gluconeogenesis, the formation of the unstable enol is driven by decarboxylation—the oxidation of the carboxylic acid to CO2—and trapped by the addition of a phosphate to carbon 2 from GTP. The two-step pathway for the formation of phosphoenolpyruvate from pyruvate has a AG° of + 0.2 kcal mol ( + 0.13 kj moP ) in contrast with +7.5 kcal mol ( + 31 kj mol ) for the reaction catalyzed by pyruvate kinase. The much more favorable AG° for the two-step pathway results from the use of a molecule of ATP to add a molecule of CO2 in the carboxylation step that can be removed to power the formation of phosphoenolpyruvate in the decarboxylation step. Decarboxylations often drive reactions otherwise highly endergonic. This metabolic motif is used in the citric acid cycle (Section IS.x.x), the pentose phosphate pathway (Section 17.x.x), and fatty acid synthesis (Section 22.x.x). 

Fig 8. Production of blood glucose from glycogen (by glycogenolysis) and from alanine, lactate, and glycerol (by gluconeogenesis). PEP = phosphoenolpyruvate OAA = oxaloacetate. 

The anabolic reactions of gluconeogenesis take place in the cytosol. Oxaloacetate is not transported across the mitochondrial membrane. Two mechanisms exist for the transfer of molecules needed for gluconeogenesis from mitochondria to the cytosol. One mechanism takes advantage of the fact that phosphoenolpyruvate can be formed from oxaloacetate in the mitochondrial matrix (this reaction is the next step in gluconeogenesis) phosphoenolpyruvate is then transferred to the cytosol, where the remaining reactions take place (Figure 19.12). The other mechanism relies on the fact that malate, which is another intermediate of the citric acid cycle, can be transferred to the cytosol. There is a malate dehydrogenase enzyme in the cytosol as well as in mitochondria, and malate can be converted to oxaloacetate in the cytosol. 

C4 plants green plants in which the primary product of CO2 fixation is not 3-phosphoglycerate (cf. C3 plants) but a C4 acid such as oxaloacetate, malate or aspartate. These plants possess two types of photi>-synthesizing cells. In mesophyll cells near the leaf surface, CO2 is fixed into C4-compounds. This prefixation of CO2 is due to the action of the cytosolic enzyme, phosphoeno/pyruvate carboxylase (EC 4.1.1.31), which carboxylates phosphoenolpyruvate to oxaloacetic acid (see Hatch-Slack-Kortschak cycle). The Calvin cycle (see) operates in the the vascular bundle cells of C4 plants, and CO2 for the Calvin cycle is derived from the decarboxylation C4 compounds rather than directly from the atmosphere. This Kranz anatomy , i.e. photosynthetically active bundle sheath cells with a photosynthetically active layer... 

Figure 17.14 Anaerobic metabolic pathways involved in SA production in engineered Mannheimia succiniciproducens PALFK strain. PALFK was engineered to metabolize sucrose and glycerol simultaneously by deleting the fructose phosphotransferase gene ifruA) from the genomic DNA of M. succiniciproducens PALK. X indicates knocked-out gene. PEP, phosphoenolpyruvate OAA, oxaloacetate ...

The steps of the citrate cycle involving dicarboxylic acids— from succinate to oxaloacetate—are even richer in correlations with other metabolic pathways. Fumarate is a fragment of the breakdown of tyrosine it is also formed from aspartate in the course of the formation of urea (see below). Oxaloacetate can be converted by reversible transamination into aspartate, one of the nonessential amino acids. Another pathway leads from oxaloacetate to phosphoenolpyruvate and hence to the synthesis of carbohydrates. 

CO2 fixation by enzymes such as phosphoenolpyruvate carboxykinase is essential to produce succinic acid from glucose through a reversed path in a partial TCA cycle. This is depicted in Fig. 9.4, where an extra carbon is connected in the process to convert phosphoenolpyruvate to oxaloacetate. The desired C4 pathway, with succinate as an end product, is therefore evidently distinguished from the C3 pathway that generates a variety of undesired by-products. Apart from CO2 (gas), other sources such as alkaline and earth alkaline carbonates can provide the necessary CO2 for the fixation reaction (Guettler et ak, 1996 Van et al., 1997). Combinations of gas and carbonates are also used (Lin et al., 2008). 

This enzyme, similar to all C02 assimilating enzymes, contains biotin for a cofactor. Oxaloacetate is released from the mitochondria into the cytoplasm to enter gluconeogenesis. In the cytoplasm, oxaloacetate converts to phosphoenolpyruvate via a reaction catalyzed by phosphoenolpyruvate carboxylase ... 

Pyruvate is derived from phosphoenolpyruvate may be inter-converted into lactate, alanine, oxaloacetate. Formation ofacetyl-CoA from pyruvate is essentially irreversible... 

A problem for gluconeogenesis is that pyruvate carboxylase, which produces oxaloacetate from pyravate, is present in the mitochondria but phosphoenolpyruvate carboxylase, at least in human liver, is present in the cytosol. For reasons given in Chapter 9, oxaloacetate cannot cross the mitochondrial membrane and so a transporter is not present in any cells. Hence, oxaloacetate is converted to phosphoenolpyruvate which is transported across the membrane (Figure 6.25). 

C4 plants incorporate CO2 by the carboxylation of phosphoenolpyruvate (PEP) via the enzyme PEP carboxylase to make the molecule oxaloacetate which has 4 carbon atoms (hence C4). The carboxylation product is transported from the outer layer of mesophyll cells to the inner layer of bundle sheath cells, which are able to concentrate CO2, so that most of the CO2 is fixed with relatively little carbon fractionation. 

Table 16-2 shows the most common anaplerotic reactions, all of which, in various tissues and organisms, convert either pyruvate or phosphoenolpyruvate to ox-aloacetate or malate. The most important anaplerotic reaction in mammalian liver and kidney is the reversible carboxylation of pyruvate by C02 to form oxaloacetate, catalyzed by pyruvate carboxylase. When the citric acid cycle is deficient in oxaloacetate or any other intermediates, pyruvate is carboxylated to produce more oxaloacetate. The enzymatic addition of a carboxyl group to pyruvate requires energy, which is supplied by ATP—the free energy required to attach a carboxyl group to pyruvate is about equal to the free energy available from ATP. 

Because the carbon atoms of acetate molecules that enter the citric acid cycle appear eight steps later in oxaloacetate, it might seem that this pathway could generate oxaloacetate from acetate and thus generate phosphoenolpyruvate for gluconeogenesis. However, as an examination of the stoichiometry of the citric acid cycle shows, there is no net conversion of acetate to ox-... 

The first "roadblock" to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of pyruvate to phosphoenolpyruvate (PEP) by pyruvate kinase. In gluconeogenesis, pyruvate is first carboxylated by pyruvate carboxylase to oxaloacetate (OAA), which is then converted to PEP by the action of PEP-carboxykinase (Figure 10.3). 

Phosphoenolpyruvate, a key metabolic intermediate. A compound of central importance in metabolism is the phosphate ester of the enol form of pyruvate, commonly known simply as phosphoenolpyruvate (PEP).249 It is formed in the glycolysis pathway by dehydration of 2-phosphoglycerate (Eq. 13-15) or by decarboxylation of oxaloacetate. Serving as a preformed enol from which a reactive enolate anion can be released for condensation reactions,250 251 PEP... 

The carboxylation of pyruvate supplies a significant portion of the thermodynamic push for the next step in the sequence. This is because the free energy change for decarboxylation of /3-keto carboxylic acids such as oxaloacetate is large and negative. The oxaloacetate formed from pyruvate by carboxylation is converted to phosphoenolpyruvate in a reaction catalyzed by phosphoenolpyruvate carboxyki-nase. In many species, including mammals, this reaction involves a GTP-to-GDP conversion. 

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. 

After PEP carboxylase makes the oxaloacetate, it is transported to the bundle sheath cells. First, NADPH reduces it to malate, and it is then transported to the bundle sheath cells. In the bundle sheath cells, malic enzyme cleaves the malate to pyruvate and C02 for Rubisco. This generates NADPH as well, so the C4 cycle consumes no reducing equivalents. Pyruvate is transported from the bundle sheath back to the mesophyll cells where it is rephosphorylated to phosphoenolpyruvate, expending the equivalent of two ATP high-energy phosphates. ... 

The preceding two reactions are necessary to achieve the overall result of transporting oxaloacetate to the cytoplasm from the mitochondria, as there is no direct mechanism for this. Cytoplasmic oxaloacetate is then converted irreversibly to phosphoenolpyruvate by way of phosphoenolpyruvate carboxykinase, a cytoplasmic enzyme that operates only when the ATP concentration is high ... 

The enzymes are, respectively (1) mitochondrial malate dehydrogenase (2) cytoplasmic malate dehydrogenase (3) phosphoenolpyruvate carboxykinase (4) pyruvate kinase and pyruvate dehydrogenase. The acetyl-CoA could then condense with oxaloacetate (produced from a second molecule of aspartate) to yield citrate. Aspartate could, therefore, continue to supply acctyl-CoA, which would continue to fuel the citric acid cycle. 

Today the metabolic network of the central metabolism of C. glutamicum involving glycolysis, pentose phosphate pathway (PPP), TCA cycle as well as anaplerotic and gluconeogenetic reactions is well known (Fig. 1). Different enzymes are involved in the interconversion of carbon between TCA cycle (malate/oxaloacetate) and glycolysis (pyruvate/phosphoenolpyruvate). For anaplerotic replenishment of the TCA cycle, C. glutamicum exhibits pyruvate carboxylase [20] and phosphoenol-pyruvate (PEP) carboxylase as carboxylating enzymes. Malic enzyme [21] and PEP carboxykinase [22,23] catalyze decarboxylation reactions from the TCA cycle... 

Fig. 13 Relative changes in metabolic fluxes. Relative changes in the metabolic fluxes of B. megaterium WH323 carrying TFH encoding pYYBm9 after induction of recombinant protein production with either glucose (a) or pyruvate (b) as the sole carbon source are given. AcCoA acetyl-coenzyme A Activ activation F6P fructose 6-phosphate G6P glucose 6-phosphate MAL malate OAA oxaloacetate PEP phosphoenolpyruvate PPP pentose phosphate pathway PYR pyruvate TCA tricarboxylic acid. Data adapted from [88]...

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