Gluconeogenesis, biotin pyruvate carboxylase

Pyruvate carboxylase is the most important of the anaplerotie reactions. It exists in the mitochondria of animal cells but not in plants, and it provides a direct link between glycolysis and the TCA cycle. The enzyme is tetrameric and contains covalently bound biotin and an Mg site on each subunit. (It is examined in greater detail in our discussion of gluconeogenesis in Chapter 23.) Pyruvate carboxylase has an absolute allosteric requirement for acetyl-CoA. Thus, when acetyl-CoA levels exceed the oxaloacetate supply, allosteric activation of pyruvate carboxylase by acetyl-CoA raises oxaloacetate levels, so that the excess acetyl-CoA can enter the TCA cycle. 

Step 1 of Figure 29.13 Carboxylation Gluconeogenesis begins with the carboxyl-afion of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis (Figure 29.6), in which acetyl CoA is carboxylated to yield malonyl CoA. 

Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is a biotin-dependent reaction (see Figure 8.24). This reaction is important because it replenishes the citric acid cycle intermediates, and provides substrate for gluconeogenesis (see p. 116). 

Eight enzyme-catalyzed reactions are involved in the conversion of acetyl-CoA into fatty acids. The first reaction is catalyzed by acetyl-CoA carboxylase and requires ATP. This is the reaction that supplies the energy that drives the biosynthesis of fatty acids. The properties of acetyl-CoA carboxylase are similar to those of pyruvate carboxylase, which is important in the gluconeogenesis pathway (see chapter 12). Both enzymes contain the coenzyme biotin covalently linked to a lysine residue of the protein via its e-amino group. In the last section of this chapter we show that the activity of acetyl-CoA carboxylase plays an important role in the control of fatty acid biosynthesis in animals. Regulation of the first enzyme in a biosynthetic pathway is a strategy widely used in metabolism. 

Thus pyruvate carboxylase generates oxaloacetate for gluconeogenesis but also must maintain oxaloacetate levels for citric acid cycle function. For the latter reason, the activity of pyruvate carboxylase depends absolutely on the presence of acetyl CoA the biotin prosthetic group of the enzyme cannot be carboxy-lated unless acetyl CoA is bound to the enzyme. This allosteric activation by acetyl CoA ensures that more oxaloacetate is made when excess acetyl CoA is present. In this role of maintaining the level of citric acid cycle intermediates, the pyruvate carboxylase reaction is said to be anaplerotic, that is filling up. ... 

Biotin, an essential water-soluble B-complex vitamin, is the coenzyme for four human carboxylases (Fig. 12-2) These include the three mitochondrial enzymes pyruvate carboxylase, which converts pyruvate to oxaloacetate and is the initial step of gluconeogenesis propionyl-CoA carboxylase, which catabolizes several branched-chain amino acids and odd-chain fatty acids and 3-methylcrotonyl-CoA carboxylase, which is involved in the catabolism of leucine and the principally cytosolic enzyme, acetyl-CoA carboxylase, which is responsible for the... 

The activities of biotin-dependent carboxylases fall in deficiency, resulting in impaired gluconeogenesis, with accumulation of lactate, pyruvate, and alanine, and impaired lipogenesis, with accumulation of acetyl CoA, resulting in ketosis. There are also changes in the fatty acid composition of membrane lipids. A variety of abnormal organic acids are excreted by bothbiotin-deficient patients and experimental animals (as shown in Table 11.1). 

Bannister DW (1976a) The biochemistry of fatty liver and kidney syndrome. Biotin-mediated restoration of hepatic gluconeogenesis in vitro and its relationship to pyruvate carboxylase activity. Biochemical Journal 156, 167-73. 

In order for pyruvate carboxylase to be ready to function, it requires biotin. Mg", and Mn" ". It is allosterically activated by acetyl CoA. The biotin is not carboxylated until acetyl CoA binds the enzyme. By this means, high levels of acetyl CoA signal the need for more oxaloacetate. When ATP levels are high, the oxaloacetate is consumed in gluconeogenesis. When ATP levels are low, the oxaloacetate enters the citric acid cycle. Gluconeogenesis only occurs in the liver and kidneys. 

Pyruvate carboxylase is activated allosterically by acetyl-CoA. The enzyme is a tetrameric protein carrying four molecules of biotin, each bound covalently through an amide bond involving the -amino group of a lysine residue. In animals the reaction catalyzed by pyruvate carboxylase is the most important anaplerotic reaction, particularly in liver and kidney. Pyruvate carboxylase is the only enzyme of gluconeogenesis in the mitochondria, requiring pyruvate and oxaloacetate to be transported across the mitochondrial membrane for gluconeogenesis to occur. 

In glycolysis, PEP is converted to pyruvate by pyruvate kinase. In gluconeogenesis, a series of steps are required to accomplish the reversal of this reaction (Fig. 31.5). Pyruvate is carboxylated by pyruvate carboxylase to form oxaloacetate. This enzyme, which requires biotin, is the catalyst of an anaplerotic (refilling) reaction of the TCA cycle (see Chapter 20). In gluconeogenesis, this reaction replenishes the oxaloacetate that is used for the synthesis of glucose (Fig. 31.6). 

The enzyme that catalyzes this reaction is pyruvate carboxylase, an allosteric enzyme found in the mitochondria. Acetyl-CoA is an allosteric effector that activates pyruvate carboxylase. If high levels of acetyl-GoA are present (in other words, if there is more acetyl-GoA than is needed to supply the citric acid cycle), pyruvate (a precursor of acetyl-GoA) can be diverted to gluconeogenesis. (Oxaloacetate from the citric acid cycle can frequendy be a starting point for gluconeogenesis as well.) Magnesium ion (Mg +) and biotin are also required for effective catalysis. We have seen Mg + as a cofactor before, but we have not seen biotin, which requires some discussion. 

Of the three processes—glycogen formation, gluconeogenesis, and the pentose phosphate pathway—only one, gluconeogenesis, involves an enzyme that requires biotin. The enzyme in question is pyruvate carboxylase, which catalyzes the conversion of pyruvate to oxaloacetate, an early step in gluconeogenesis. 

Oxaloacetate is formed from pyruvate by pyruvate carboxylase, located in the mitochondria. Pyruvate carboxylase is a biotin-requiring enzyme its kinetic mechanism has been studied in detail using the enzyme purified from domestic fowl liver (Attwood Graneri, 1992). In tissues where PEPCK is located in the cytosol, its substrate oxaloacetate is required in the cytosol for the formation of PEP for gluconeogenesis. The malate-aspartate shuttle is required for gluconeogenesis in avian kidney, according to the scheme in Fig. 3.3, but whether or not it is also required for gluconeogenesis in avian liver is unresolved. There is evidence for the existence of a... 

Pyruvate carboxylase (EC 6.4.1.1) a biotin-dependent ligase, in animals and plants, which catalyses the addition of CO2 to pyruvate Pyruvate + CO2 + ATP + H2O v Oxaloacetate + ADP + Pj. The enzyme is Mn -dependent, and it is practically inactive in the absence of its positive allosteric elector, ace-tyl-CoA. This reaction is an important early stage of Gluconeogenesis (see), and is an example of CO2 fixation in the animal organism. For the mode of attachment of the coenzyme, biotin, and the mechanism of CO2 transfer, see Biotin enzymes. The active form of Pc. is a tetramer, M, 600,000 (yeast), 650,000... 

Pyruvate carboxylase is a mitochondrial enzyme which requires Mg and biotin as a carrier of activated carbon dioxide. In addition, it has an absolute requirement for acetyl-CoA, which acts as an allosteric activator (page 340). In this way the accumulation of acetyl-CoA normally triggers the formation of oxaloacetate. If ATP is in short supply, oxaloacetate will combine with some of the acetyl-CoA to form citrate, and so be drawn into the citrate cycle. On the other hand, if there is a surplus of ATP, both this and the oxaloacetate will be used for gluconeogenesis. 

Biotin functions to transfer carbon dioxide in a small number of carboxylation reactions. The reactive intermediate is 1-N-carboxybiocytin (Figure 11.25), formed from bicarbonate in an ATP-dependent reaction. A single holocarboxylase synthetase acts on the apoenzymes of acetyl CoA carboxylase (a key enzyme in fatty acid synthesis section 5.6.1), pyruvate carboxylase (a key enzyme in gluconeogenesis section 5.7), propionyl CoA carboxylase and methylcrotonyl CoA carboxylase to form the active holoenzymes from (inactive) apoenzymes and free biotin. 

Biotin. Biotin deficiency blocks pyruvate carboxylase resulting in an accumulation of pyruvate and lactate. It suppresses gluconeogenesis and causes fasting hypoglycaemia. 

Pyruvate may re-enter the gluconeogenic pathway only via conversion to oxaloacetate by the action of the mitochondrial biotin-dependent pyruvate carboxylase (EC 6.4.1.1) (see also Chapter 10), and thence to phosphoenolpyruvate (PEP) by he action of GTP-requiring mitochondrial and cytosolic PEP carboxykinase (EC 4.1.1.32). Thus further regulation of pyruvate metabolism may occur in gluconeogenesis by the requirement to transport the oxaloacetate out of the mitochondria into the cytoplasm, this being achieved by the mitochondrial malate shuttle , after conversion of the mitochondrial oxaloacetate to malate, since oxaloacetate itself cannot be transported through the mitochondrial membrane. Phosphoenolpyruvate is also produced within the mitochondria in man and is transported into the... 

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