Phosphoenolpyruvate carboxykinase, reaction catalyzed

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a critical reaction in gluconeogenesis, which under many conditions is the rate-limiting step in the pathway. A cAlVfP response element (CRE) and a glucocorticoid response element (GRE) are each located upstream from the transcription start site. 

Step 2 of Figure 29.13 Decarboxylation and Phosphorylation Decarboxylation of oxaloacetate, a jB-keto acid, occurs by the typical retro-aldol mechanism like that in step 3 in the citric acid cycle (Figure 29.12), and phosphorylation of the resultant pyruvate enolate ion by GTP occurs concurrently to give phosphoenol-pyruvate. The reaction is catalyzed by phosphoenolpyruvate carboxykinase. 

Phosphoenolpyruvate carboxykinase (GTP) [EC 4.1.1.32], also known as phosphoenolpyruvate carboxylase and phosphopyruvate carboxylase, catalyzes the reaction of GTP with oxaloacetate to produce GDP, phosphoenolpyruvate, and carbon dioxide. ITP can replace GTP as the phosphorylating substrate. 

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 ... 

If we add the equations for the reactions catalyzed by pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and nucleoside diphosphate kinase, we obtain the overall reaction for conversion of pyruvate to phosphoenolpyruvate. 

The cardinal requirement of any assay is its specificity. Since most enzyme assays monitor disappearance of a substrate or appearance of a product, care must be exercised to ensure that only one enzyme activity is contributing to the monitored effect. Consider, for example, assay of phosphoenolpyruvate (PEP) carboxykinase, which catalyzes the reaction... 

The first reaction is catalyzed by pyruvate carboxylase and the second by phosphoenolpyruvate carboxykinase. The sum of these reactions is ... 

PCK1 Phosphoenolpyruvate carboxykinase, key enzyme in gluconeogenesis, catalyzes early reaction in carbohydrate biosynthesis, glucose represses transcription and accelerates mRNA degradation, regulated by Mcmlp and Cat8p, located in the cytosol... 

Fig. 6. Proposed mechanism for the phosphoenolpyruvate carboxykinase-catalyzed reaction. The orientation of the substrate with respect to enzyme-bound Mn is shown. The second cation, which also takes part in the catalytic reactions, has not been included but is thought to form a /3,y-bidentate complex with GTP (SO).

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

Comparison of the reactions of glycolysis and gluconeogenesis. All the reactions of glycolysis occur in the cytoplasm of the cell. However, in many human cells, pyruvate carboxylase is found in the mitochondria and phosphoenolpyruvate carboxykinase is located in the cytoplasm. Oxaloacetate, the product of the reaction catalyzed by pyruvate carboxylase, is shuttled out of the mitochondria and into the cytoplasm by a complex set of reactions. 

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 CO2 that was added to pyruvate to form oxaloacetate is released in the reaction catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), which generates PEP (Fig. 31.7A). For this reaction, GTP provides a source of energy as well as the phosphate group of PEP. Pyruvate carboxylase is found in mitochondria. In various species, PEPCK is located either in the cytosol or in mitochondria, or it is distributed between these two compartments. In humans, the enzyme is distributed about equally in each compartment. 

Phosphoenolpyruvate carboxykinase is induced. Oxaloacetate produces PEP in a reaction catalyzed by PEPCK. Cytosolic PEPCK is an inducible enzyme, which means that the quantity of the enzyme in the cell increases because of increased transcription of its gene and increased translation of its mRNA. The major inducer is cyclic adenosine monophosphate (cAMP), which is increased by hormones that activate adenylate cyclase. Adenylate cyclase produces cAMP from ATP. Glucagon is the hormone that causes cAMP to rise during fasting, whereas epinephrine acts during exercise or stress. cAMP activates protein kinase A, which phosphorylates a set of specific transcription factors (CREB) that stimulate transcription of the PEPCK gene (see Chapter 16 and Pig. 16.18). Increased synthesis of mRNA for PEPCK results in increased synthesis of the enzyme. Cortisol, the major human glucocorticoid, also induces PEPCK. 

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... 

Enzymes present in the liver cytosol with short half-lives include ornithine decarboxylase, thymidine kinase, tyrosine aminotransferase, tryptophan oxygenase, hydroxymethylglutaryl-CoA reductase, serine dehydratase, and phosphoenolpyruvate carboxykinase. All of these enzymes have degradation rate constants greater than 0.1/h—more than 10 times more rapid than the average ka for liver cytosol proteins (Schimke, 1970). Perhaps a scrutiny of the group can provide information on the enzyme properties as well as the nature of reactions catalyzed by enzymes with rapid turnover rates. 

As is especially appropriate, for regulatory enzymes, all either catalyze the initial reaction in a metabolic pathway or the first reaction committed to a particular pathway (e.g., phosphoenolpyruvate carboxykinase and hydroxymethylglutaryl-CoA reductase). The enzymes are at points of regulatory control where an increase in activity should result in an increase in substrate throughput. In the basal state, each enzyme is present at a low activity. 

Native enzyme is inactivated initially without loss of antibody reactivity. The inactivation reaction is catalyzed by a membrane protein present in all liver membranes but at highest specific activity in plasma membranes and at lowest activity in lysosomal membranes. Inactivation is greatly accelerated in the presence of disulfides such as oxidized glutathione or cystine and retarded by thiols. Disulfides on the membrane protein are implicated because treatment of membranes with dithiothreitol in the presence of iodoacetamide destroys the capacity to inactivate phosphoenolpyruvate carboxykinase. This treatment would reduce and fix protein disulfides. Inactivation requires a membrane protein that shows some tissue specificity, since plasma membranes from reticulocytes or erythrocytes are not active, nor are liposomes prepared from the lipids of liver microsomes. 

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... 

This last pair of reactions is complicated by the fact that p)rruvate carboxylase is found in the mitochondria, whereas phosphoenolp)mivate carboxykinase is found in the cytoplasm. As we will see in Chapters 22 and 23, mitochondria are organelles in which the final oxidation of food molecules occurs and large amounts of ATP are produced. A complicated shuttle system transports the oxaloacetate produced in the mitochondria through the two mitochondrial membranes and into the cytoplasm. There, phosphoenolp)mivate carboxykinase catalyzes its conversion to phosphoenolpyruvate. 

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