Phosphoenolpyruvate carboxykinase synthesis

J. M. Gunn, S. M. Tilghman, R. W. Hanson, L. Reshef, and F. J. Ballard, Effects of cyclic adenosine monophosphate, dexamethasone and insulin on phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 hepatoma cells. Biochemistry 14 2350-2357 (1975). 

The glucocorticoids have important dose-related effects on carbohydrate, protein, and fat metabolism. The same effects are responsible for some of the serious adverse effects associated with their use in therapeutic doses. Glucocorticoids stimulate and are required for gluconeogenesis and glycogen synthesis in the fasting state. They stimulate phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and glycogen synthase and the release of amino acids in the course of muscle catabolism. 

Uptake of Ca2+ into cells, or release of this ion from intracellular stores, is a major regulatory mechanism in many if not all cells (see Section E). Mn2+ activates phosphoenolpyruvate carboxykinase (Eq. 13-46) and maybe a regulator of gluconeogenesis.142 Iron controls the synthesis of ferritin and of transferrin receptors137 (Chapter 16). The specific metal ions present in many biological macromolecules are likely to participate in additional regulatory processes. 

In addition to providing amino acids and glycerol as carbon sources, glucocorticoids promote gluconeogenesis by inducing synthesis of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). 

Phosphoenolpyruvate carboxykinase (PEPCK) - This enzyme has no known allosteric regulators. The enzyme is controlled by hormonal regulation of its synthesis. The hormone, glucagon, activates transcription of the structural gene for the enzyme. Insulin, on the other hand, inhibits transcription of the gene. 

Glucagon brings about the inhibition of pyruvate kinase (PK) in the liver, causing phosphoenolpyruvate (PEP) to accumulate. The level of pyruvate decreases, both because its synthesis from PEP is blocked and because it continues to be converted to PEP, via the pyruvate carboxylase and phosphoenolpyruvate carboxykinase reactions. Accumulation of PEP promotes gluconeogenesis, while the inhibition of pyruvate kinase diminishes the glycolytic flux rate. 

Some effects of insulin occur within seconds or minutes, including the activation of glucose and ion transport systems, the covalent modification of enzymes (i.e., phosphorylation or dephosphorylation), and some effects on gene transcription (i.e., inhibition of the phosphoenolpyruvate carboxykinase gene). Effects on protein synthesis and gene transcription require hours, while those on cell proliferation and differentiation may take days. 

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). 

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. 

Gluconeogenesis is stimulated because the synthesis of phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose 6-phosphatase is induced and because there is an increased availability of precursors. Fructose 1,6-bisphos-phatase is also activated because the levels of its inhibitor, fructose 2,6-bisphos-phate, are low (Fig. 36.9). During fasting, the activity of the corresponding enzymes of glycolysis is decreased. 

Iron is also an essential constituent of several non-porphyrin enzymes, e.g. aconitase, aldolase, and succinic dehydrogenase. Inhibition of the synthesis of glucose by tryptophan in animal cells depends on chelation. The tryptophan is metabolized to pyridine-2,3-dicarboxylic acid, which complexes the divalent iron necessary for the action of phosphoenolpyruvate carboxykinase (a key enzyme in the neogenesis of glucose) (Veneziale et al., 1967). 

Figure 3. Appearance of phosphoenolpyruvate carboxykinase in developing rat liver, (a) The activity increase is expressed as units of enzyme per total liver, (b) Enzyme synthesis ( ) is expressed as the percent of radioactivity in the enzyme pool as compared to radioactivity in cytosol protein after injections of radioactive leucine. Degradation (o), in the same terms, is shown at various times after a leucine chase was given. The half-lives at each age are indicated. Details are given by Philippidis et al. (1972). Values are means SEM. T, term.

The values in Table 5 are at steady states. During the transition from one steady state to another there can be transient but dramatic changes in either kg or For example, refeeding starved rats decreases the synthesis rate for hepatic phosphoenolpyruvate carboxykinase to... 

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). 

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