Oxaloacetate to Phosphoenolpyruvate

Short-term regulation of this reaction is accomplished by changes in the relative proportions of substrates and products. Increased concentrations of oxaloacetate and GTP (or ITP) increase the rate, and accumulation of phosphoenolpyruvate and GDP (or IDP) decreases it. The cytosolic PEPCK is also under long-term regulation by hormones. Its synthesis is increased by corticosteroids. Starvation and diabetes mellitus increase the synthesis of cytosolic PEPCK, whereas refeeding and insulin have the opposite effect. 

The increase in glutamate favors transamination of oxaloacetate and limits oxaloacetate availability for phosphoenolpyruvate synthesis. When the [NADH]/[NAD+] ratio is low, malate formation occurs more readily. The cytosolic PEPCK is relatively unaffected by the mitochondrial [NADH]/[NAD+] ratio. Once malate and aspartate are transported to the cytosol and they are reconverted to oxaloacetate, cytosolic PEPCK can convert it to phosphoenolpyruvate. 

The concentration of fructose-2,6-bisphosphate is controlled by two competing enzyme activities in a single 

Phosphorylation is regulated by insulin and glucagon. Diabetes mellitus (in which the ratio of glucagon to insulin is increased) and glucagon therapy reduce the hepatic activity of PFK-2 and increase that of FBPase-2. The concentration of fructose-2,6-bisphosphate is thus reduced, thereby stimulating gluconeogenesis. 

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The following sequence will do the task a-ketoglutarate, the tricarboxylic acid cycle to oxaloacetate, to phosphoenolpyruvate, to pyruvate, to acetyl-CoA, into the tricarboxylic acid cycle. 

Figure 20-4. Biochemical pathways for gluconeogenesis in the liver. Alanine, a major gluconeogenic substrate, is used to synthesize oxaloacetate. The carbon skeletons of glutamine and other glucogenic amino acids feed into the TCA cycle as a-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate and thus also provide oxaloacetate. Conversion of oxaloacetate to phosphoenolpyruvate and ultimately to glucose limits the availability of oxaloacetate for citrate synthesis and thus greatly diminishes flux through the initial steps of the TCA cycle (dashed lines). Concurrent P-oxidation of fatty acids provides reducing equivalents (NADH and FADH2) for oxidative phosphorylation but results in accumulation of acetyl-CoA.

Four enzymes are unique to the process of gluconeogenesis and are required to circumvent the unidirectional steps of glycolysis (Fig. 13.1). Two of these are pyruvate carboxylase, which converts pyruvate to oxaloacetate, and PEP carboxykinase, which converts oxaloacetate to phosphoenolpyruvate. These enzymes are required to effectively reverse the action of pyruvate kinase. The other two are fructose-1,6-bisphosphatase and glucose-6-phosphatase, which effectively reverse the actions of PFK and hexokinase or glucokinase. 

PEPCK is an enzyme of gluconeogenesis. It catalyzes conversion of the 4-carbon compound, oxaloacetate, to phosphoenolpyruvate (PEP), releasing CO2. The reaction requires energy input from GTP and produces GDP. 

F. 31.7. The generation of PEP from gluconeogenic precursors. A. Conversion of oxaloacetate to phosphoenolpyruvate, using PEP carboxykinase. B. Interconversion of oxaloacetate and malate. C. Transamination of aspartate to form oxaloacetate. Note that the cytosolic reaction is the reverse of the mitochondrial reaction as shown in Eigure 31.5. 

The conversion of oxaloacetate to phosphoenolpyruvate is catalyzed by the enzyme phosphoenolpyruvate carboxykinase (PEPCK), which is found in the... 

Radioactive acetyl CoA can be generated by direct synthesis from C-acetate or from (3 oxidation of radioactive fatty acids, such as uniformly labeled palmitate. Examination of the reactions of the citric acid cycle reveals that neither of the two carbons that enter citrate horn acetate is removed as carbon dioxide during the first pass through the cycle. Labeled carbon from C-methyl-labeled acetate appears in C-2 and C-3 of oxaloacetate, because succinate is symmetrical, with either methylene carbon in that molecule labeling C-2 or C-3 of oxaloacetate. The conversion of oxaloacetate to phosphoenolpyruvate yields PEP labeled at C-2 or C-3 as well. Formation of glyceraldehyde 3-phosphate and its isomer dihydroxyacetone phosphate gives molecules, both labeled at carbons 2 and... 

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. 

The irreversible step of glycolysis catalysed by pyruvate kinase is bypassed in gluconeogenesis by conversion of pyruvate first to oxaloacetate, then conversion of oxaloacetate to phosphoenolpyruvate by phosphoenolpyru-vate carboxykinase. The transfer of the amino group from glutamate to oxaloacetate produces aspartate, catalysed by the enzyme aspartate aminotransferase. Malate dehydrogenase converts oxaloacetate to malate in the malate-aspartate shutde. 

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 succinate produced by the glyoxylate cycle is converted via malate and oxaloacetate to phosphoenolpyruvate this last reaction being accompanied by the loss of CO,— die only carbon lost during... 

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