Phosphoenolpyruvate carboxykinase oxaloacetate carboxylation

Finally, oxaloacetate is simultaneously decarboxylated andphosphorylated by phosphoenolpyruvate carboxykinase in the cytosol. The CO2 that vv as 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... 

Figure 21-1. Interconversion of phosphoenolpyruvate (PEP) and pymvate. The conversion of PEP to pyruvate is thermodynamically irreversible in the cell. To convert pyruvate back to PEP for gluconeogenesis, pyruvate must enter the mitochondrion, be carboxylated to oxaloacetate (OAA), and reduced to malate. After exiting the mitochondrion, malate is oxidized back to OAA and converted to PEP by the action of 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). 

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). 

Reaction 3.1, the key reaction of propionic acid fermentation, is catalyzed by pyruvate carboxytransphosphorylase, a unique biotin-dependent transcarboxylase (see below). There are other reactions of carboxyl group transfer catalyzed by phosphoenolpyruvate (PEP) carboxytransphosphorylase and phosphoenolpyruvate carboxykinase, but these (i) do not require biotin and (ii) use CO2 as the source of carboxyl groups. The actual species involved may be HCO3" (or H2CO3) rather than free CO2 (Cooper et al., 1968), since free CO2 is not evolved in the PEP carboxytransphosphorylase reaction (Swick and Wood, 1960). Propionic acid bacteria are able to decarboxylate succinate, producing CO2 in a biotin-dependent reaction (Delwiche,1948 Lichstein, 1958). If succinate is accumulated as the end product, then the cycle (see Fig. 3.1) is broken, and oxaloacetic acid is not supplied by reaction 3.1, but is formed primarily by CO2 fixation onto PEP catalyzed by PEP carboxytransphosphorylase (PEP-CTP). 

Anaplerotic reactions refer to C3-carboxylation and C4-decarboxylation around the phosphoenolpyruvate-pyruvate-oxaloacetate node, which interconnect the TCA cycle with glycolysis. These reactions result in direct oxaloacetate formation or depletion. Carboxylation of phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase and that of pyruvate by pyruvate carboxylase contribute to its formation. Accordingly, decarboxylation of oxaloacetate catalyzed by phosphoenolpyruvate carboxykinase and oxaloacetate decarboxylase form phosphoenolpyruvate and... 

In order to progress from pyruvate to glucose, pyruvate is first carboxylated in mitochondria to form oxaloac-etate. The action of malate dehydrogenase converts oxaloacetate to malate, which is able to leave the mitochondria and enter the cytosol where the reverse reaction regenerates oxaloacetate. The enzyme phosphoenolpyruvate carboxykinase then catalyses the phosphorylation and decarboxylation of oxaloacetate to form phosphoenolpyruvate (31a). 

Oxaloacetate is an intermediate of many metabolic pathways. It also plays a role in the malate-aspartate shuttle, which transfers high energy electrons into mitochondria. Citrate is formed by the condensation of oxaloacetate with acetyl CoA. A transamination reaction transfers an amino group from an amino acid to an a-keto acid. Transfer of the amino group from aspartate to a-ketoglutarate forms oxaloacetate and glutamate. In gluconeogenesis, pyruvate is carboxylated in mitochondria to form oxaloacetate. After transfer to the cytosol, the enzyme phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate to phosphoenolpyruvate. 

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). 

In this malate dismutation pathway, carbohydrates are degraded to phosphoenolpyruvate (PEP) via the classical glycolytic pathway. This PEP is then carboxylated by PEP carboxykinase (PEPCK) to oxaloacetate, which is subsequently reduced to malate. This malate is transported into the mitochondria and is degraded in a split pathway. A portion of the malate is oxidized to acetate and another portion is reduced to succinate, which can then be further metabolized to propionate (Fig. 20.1). 

Pathway of C02 in Gluconeogenesis In the first bypass step of gluconeogenesis, the conversion of pyruvate to phosphoenolpyruvate (PEP), pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate, which is subsequently decarboxylated to PEP by PEP carboxykinase (Chapter 14). Because the addition of C02 is directly followed by the loss of C02, you might expect that in tracer experiments, the 14C of 14C02 would not be incorporated into PEP, glucose, or any intermediates in gluconeogenesis. 

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... 

In mammalian tissues, excluding muscle, the most important anaplerotic reaction employs pyruvate carboxylase which contains a biotin prosthetic group (Figure 5.3b) responsible for the transfer of a carboxyl group. ATP provides the energy to bond covalently the carboxyl group from HCO, to the biotin which transfers it when pyruvate binds to the enzyme. In muscle cells, the major pathway utilizes phosphoenolpyruvate and phos-phoenolpyruvate carboxykinase which occurs both in the cytosol and mitochondrial matrix. Two routes are therefore possible in oxaloacetate syn-... 

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