The Gluconeogenic Pathway

COMPARTMENTALIZED PYRUVATE CARBOXYLASE DEPENDS ON METABOLITE CONVERSION AND TRANSPORT The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol or in the mitochondria or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix, where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis see Figure 25.1). /Uternatively, it may be converted directly to 0/ A by pyruvate carboxylase and used in glu-... 

Answer 6. The negative AG value indicates the reaction is thermodynamically favorable (irreversible), requiring a different bypass reaction for conversion of FI, 6BP to F6P in the gluconeogenic pathway. 

In fact, these two processes are metabolically linked. The oxidation generates ATP whereas gluconeogenesis utilises this ATP. Consequently, in the well-fed human, gluconeogenesis is essential for oxidation of amino acids, otherwise oxidation is limited by the need to utilise the ATP (Chapter 8). The reactions in which amino acids are converted to compounds that can enter the gluconeogenic pathway are described in Chapter 8. The position in the gluconeogenic pathway where amino acids, via their metabolism (Chapter 8), enter the pathway is indicated in Figure 6.23. 

The pathway for gluconeogenesis is shown in Figures 6.23 and 6.24. Some of the reactions are catalysed by the glycolytic enzymes i.e. they are the near-equilibrium. The non-equilibrium reactions of glycolysis are those catalysed by hexokinase (or glucokinase, in the liver), phosphofructokinase and pyruvate kinase and, in order to reverse these steps, separate and distinct non-equilibrium reactions are required in the gluconeogenic pathway. These reactions are ... 

Figure 6.23 Positions in the gluconeogenic pathway where amino acids, fructose and glycerol enter the pathway. For details of the metabolism that provides the intermediates that actually enter the pathway from the amino acids, see Chapter 8. Not all of the carbon in some of the amino acids is incorporated into glucose (e.g. tryptophan). Two amino acids, leucine and lysine, do not give rise to glucose.

Figure 6.24 The gluconeogenic pathway indicating the glycolytic and gluconeogenic non-equilibrium reactions. The non-equilibrium reactions provide for the substrate cycles. (See Chapter 3 for a discussion of substrate cycles and their role in regulation.)...

Gluconeogenesis. The gluconeogenic pathway is present in the kidney, as in the liver. Thus, amino acids (and lactate) can be converted to glucose in the kidney but a major precursor, in acidotic conditions, is glutamine. 

The reaction involves biotin as a carrier of activated HCO3 (Fig. 14-18). The reaction mechanism is shown in Figure 16-16. Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. (Acetyl-CoA is produced by fatty acid oxidation (Chapter 17), and its accumulation signals the availability of fatty acids as fuel.) As we shall see in Chapter 16 (see Fig. 16-15), the pyruvate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle. 

Vertebrates cannot convert fatty acids, or the acetate derived from them, to carbohydrates. Conversion of phosphoenolpyruvate to pyruvate (p. 532) and of pyruvate to acetyl-CoA (Fig. 16-2) are so exergonic as to be essentially irreversible. If a cell cannot convert acetate into phosphoenolpyruvate, acetate cannot serve as the starting material for the gluconeogenic pathway, which leads from phosphoenolpyruvate to glucose (see Fig. 15-15). Without this capacity, then, a cell or organism is unable to convert fuels or metabolites that are degraded to acetate (fatty acids and certain amino acids) into carbohydrates. 

Mitochondrial oxaloacetate (OAA) is produced by the first step in the gluconeogenic pathway. 

Answer In the liver, lactate is converted to pyruvate and then to glucose by gluconeogenesis (see Figs 14-15, 14-16). This pathway includes the glycolytic bypass step catalyzed by fructose 1,6-bisphosphatase (FBPase-1). A defect in this enzyme would prevent the entry of lactate into the gluconeogenic pathway in hepatocytes, causing lactate to accumulate in the blood. 

The product of this reaction, oxaloacetate, can either enter the gluconeogenic pathway (Chap. 11) by way of malate or condense with acetyl-CoA to yield citrate. Pyruvate carboxylase is an allosteric enzyme, and it is activated by the heterotropic effector, acetyl-CoA. Thus, pyruvate in the mitochondria is the substrate for either pyruvate dehydrogenase or pyruvate carboxylase, the activities of which, in turn, are controlled by reactants associated with the citric acid cycle. The interplay among pyruvate dehydrogenase, pyruvate carboxylase, pyruvate, and the citric acid cycle is shown in Fig. 12-9. 

In most plants, the major products of photosynthesis are starch (formed in chloroplasts and sucrose (formed in the cytosol). Both of these products (collectively called photosynthate) are formed from photosynthetically generated dihydroxy-acetone phosphate (DHAP) via pathways that in some respects are similar to the gluconeogenic pathway of animal cells. In the first case, DHAP is converted to hexose phosphates, which, in turn, are converted to starch within the chloroplast. In sucrose synthesis, DHAP (or a derivative) is transported to the cytosol and there it is converted to sucrose. 

Thus, the equivalent of glucose 6-phosphate can be completely oxidized to CO 2 with the concomitant generation of NADPH. In essence, ribose 5-phosphate produced by the pentose phosphate pathway is recycled into glucose 6-phosphate by transketolase, transaldolase, and some of the enzymes of the gluconeogenic pathway. 

In fasting or diabetes, oxaloacetate is consumed to form glucose by the gluconeogenic pathway (Section 16,3.2) and hence is unavailable for condensation with acetyl CoA. Under these conditions, acetyl CoA is diverted to the formation of acetoacetate and d-3-hydroxybutyrate. Acetoacetate, d-3-hydroxyhutyrate, and acetone are often referred to as ketone bodies. Abnormally high levels of ketone bodies are present in the Wood of untreated diabetics (Section 22.3.6). 

Pyrophosphate is rapidly hydrolyzed, and so the equivalent of four molecules of ATP are consumed in these reactions to synthesize one molecule of urea. The synthesis of fumarate by the urea cycle is important because it links the urea cycle and the citric acid cycle (Figure 23.17). Fumarate is hydrated to malate, which is in turn oxidized to oxaloacetate. Oxaloacetate has several possible fates (1) transamination to aspartate, (2) conversion into glucose by the gluconeogenic pathway, (3) condensation with acetyl CoA to form citrate, or (4) conversion into pyruvate. 

Pyruvate carboxylase is an important enzyme in the gluconeogenic pathway converting pyruvate and bicarbonate into oxaloacetate. AcCoA is required for allosteric activation of the enzyme, and in the diabetic state where fatty acid oxidation is elevated, the high concentrations of AcCoA result in significant activation and gluconeogenesis overactivity. Phenylalkanoic... 

As shown in Figure 4-23, G-6-P can materialize within a tissue from two other sources. The G-l-P released during breakdown of glycogen, can be converted to G-6-P It can be produced via the gluconeogenic pathway involving conversion of pyruvate to OAA, to FEF to glyceraldehyde-3-phosphate, and finally to G-6-P. 

The third study involves the purified enzymes fmctose-l,6-bisphosphatase and phosphofmctokinase. This study brings discussion of "2-6" to a conclusion by demonstrating its effects on these enzymes. Fmctose-l,6-bisphosphatase, an enzyme of the gluconeogenic pathway, catalyzes the conversion of F-l,6-P2 to F-6-P. Ekdahl and Ekman (1984) showed that the activity of this enzyme is maximal in the absence of or presence of low concentrations of "2-6." These researchers showed that its activity is inhibited by "2-6" at concentrations of 10 micromolar and higher. The running rat experiment showed that the resting rat liver contained "2-6" at a concentration of about 12 [iM. Their data also indicated that this level of "2-6" is sufficient to inhibit the activity of the enzyme, but that the levels found in the running rat would be expected to permit near-maximal activity. 

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