Phosphoenolpyruvate carboxylase, pyruvate decarboxylation

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. 

The Q pathway for the transport of CO2 starts in a mesophyll cell with the condensation of CO2 and phosphoenolpyruvate to form oxaloacetate, in a reaction catalyzed by phosphoenolpyruvate carboxylase. In some species, oxaloacetate is converted into malate by an NADP+-linked malate dehydrogenase. Malate goes into the bundle-sheath cell and is oxidatively decarboxylated within the chloroplasts by an NADP+-linked malate dehydrogenase. The released CO2 enters the Calvin cycle in the usual way by condensing with ribulose 1,5-bisphosphate. Pyruvate formed in this decarboxylation reaction returns to the mesophyll cell. Finally, phosphoenolpyruvate is formed from pyruvate by pyruvate-Pi dikinase. 

Fig. 6.3. Carbon dioxide-fixing pathway in the C4-plants (prepared mainly on the basis of Hatch et al., 1967). Circled numbers 1, phosphoenolpyruvate carboxylase 2, malate dehydrogenase (NADP ) 3, malate dehydrogenase (NADP ) (OAA decarboxylating) (= malic enzyme) 4, Rubisco 5, pyruvate orthophosphate dikinase. Pi, phosphate PPi, diphosphate...

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). 

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... 

Pyruvate is converted to oxaloacetate (by pyruvate carboxylase). The oxaloacetate is decarboxylated and phosphorylated to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase (PEP carboxykinase). PEP is converted to fructose 1,6-bisphosphate by a direct reversal of several reactions in glycolysis. Next, fructose 1,6-bisphosphate is dephosphorylated to fructose 6-phosphate (by fructose 1,6-bisphosphatase) and this is then converted to glucose 6-phosphate (by phosphoglucoisomerase). Finally, glucose 6-phosphate is dephosphorylated (by glucose 6-phosphatase) to yield glucose. 

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... 

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... 

Pyruvate carboxylase is followed by the Phosphoenolpyruvate carboxykinase (PEPCK) reaction. In this reaction oxaloacetate is decarboxylated with a simultaneous phosphorylation by GTP to give GDP ... 

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a mitochondrion in the form of malate oxaloacetate is reduced to malate inside the mitochondrion by an NADH-linked malate dehydrogenase. After malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD -linked malate dehydrogenase in the cytoplasm (Figure 16.26). The formation of oxaloacetate from malate also provides NADH for use in subsequent steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated and phospho-ry lated by phosphoenolpyruvate carboxy kinase to generate phosphoenol pyruvate. The phosphoryl donor is GTP. The GO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. 

Once in the cytosol, the oxaloacetate is decarboxylated and phos-phorylated by the enzyme pyruvate carboxykinase (see Figure 12-8). (The nomenclature is confusing try to remember that pyruvate carboxylase only adds CO2, whereas pyruvate carboxykinase removes CO2 and adds phosphate. In the pyruvate carboxykinase reaction, the CO2 added to make oxaloacetate is removed, so the only net reaction in the series is the addition of a phosphate to pyruvate to make phosphoenolpyruvate). 

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... 

The reverse of the last irreversible reaction in glycolysis is actually two successive enzyme-catalyzed reactions. First pyruvate is converted to oxaloacetate by pyruvate carboxylase, a biotin-dependent enzyme whose mechanism we looked at in Section 24.4. Oxaloacetate is Ihen converted to phosphoenolpyruvate. In this reaction, the 3-oxocarboxyhc acid is decarboxylated (Section 18.17) and the oxygen of the enolate ion attacks the y-phosphorus of GTP (see page 1194). 

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