Mitochondria pyruvate carboxylase

ADP -t- Pi. This reaction, catalyzed by pyruvate carboxylase, is the most important anaplerotic reaction in animal tissues and occurs in mitochondria. Pyruvate carboxylase is an allosteric enzyme that requires acetyl-CoA for activity (see gluconeogenesis Chapter 15). 

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... 

As can be seen in Figure 6.42, pyruvate is very much a focal point in GNG. Normally, once pyruvate has entered a mitochondrion, it is converted into acetyl-CoA by pyruvate dehydrogenase complex, but for GNG the pyruvate is diverted in to oxaloacetate (OAA) by pyruvate carboxylase (see Figure 6.43). 

In bacteria and green plants PEP carboxylase (Eq. 13-53), a highly regulated enzyme, is responsible for synthesizing oxaloacetate. In animal tissues pyruvate carboxylase (Eq. 14-3) plays the same role. The latter enzyme is almost inactive in the absence of the allosteric effector acetyl-CoA. For this reason, it went undetected for many years. In the presence of high concentrations of acetyl-CoA the enzyme is fully activated and provides for synthesis of a high enough concentration of oxaloacetate to permit the cycle to function. Even so, the oxaloacetate concentration in mitochondria is low, only 0.1 to 0.4 x 10-6 M (10-40 molecules per mitochondrion), and is relatively constant.65 79... 

Pyruvate carboxylase is a mitochondrial matrix enzyme whereas the other enzymes of gluconeogenesis are located outside the mitochondrion. Thus oxaloacetate, produced by pyruvate carboxylase, needs to exit the mitochondrion. However the inner mitochondrial membrane is not permeable to this compound. Thus oxaloacetate is converted to malate inside the mitochondrion... 

OAA by pyruvate carboxylase (EC 6.4.1.1), thereby completing the net transport of the C2 unit (acetate) from the mitochondrion to the cytosol with the added advantage of having converted a reducing equivalent as NADH + H+ to NADPH + H+. This mechanism of C2 transport provides up to 50% of the NADPH + H+ for fatty acid synthesis in nonruminants. 

Step A, the conversion of pyruvate to phosphoenolpyruvate, is accomplished by a circuitous process commencing with pyruvate entering the mitochondrion, which for gluconeogenesis to occur must be in a high-energy state. Under these conditions, the mitochondrial enzyme pyruvate carboxylase catalyzes the conversion of pyruvate to oxaloacetate-. 

Pyruvate carboxylase is a mitochondrial enzyme, vhereas the other enzymes of gluconeogenesis are cytoplasmic. Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol (Figure 16.28). 

Pyruvate carboxylase converts pyruvate to oxaloacetate in the mitochondrion. Oxaloacetate is converted to malate or aspartate, which travels to the cytosol and is reconverted to oxaloacetate. 

A. In mitochondria, C02 is added to pyruvate to form oxaloacetate (OAA). The enzyme is pyruvate carboxylase, which requires biotin and ATP. OAA leaves the mitochondrion as malate or aspartate and is regenerated in the cytosol. OAA is converted to phosphoenolpyru-vate by a reaction that utilizes GTP and releases the same C02 that was added in the mitochondrion. The remainder of the reactions occur in the cytosol. 

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a mitochondrion in the form of malate oxaloacetate is reduced to malate inside the mitochondrion by an NADH-linked malate dehydrogenase. After malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD -linked malate dehydrogenase in the cytoplasm (Figure 16.26). The formation of oxaloacetate from malate also provides NADH for use in subsequent steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated and phospho-ry lated by phosphoenolpyruvate carboxy kinase to generate phosphoenol pyruvate. The phosphoryl donor is GTP. The GO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. 

Fig. 31.5. Conversion of pyruvate to phosphoenolpyruvate (PEP). Follow the shaded circled numbers on the diagram, starting with the precursors alanine and lactate. The first step is the conversion of alanine and lactate to pyruvate. Pyruvate then enters the mitochondria and is converted to OAA (circle 2) by pyruvate carboxylase. Pyruvate dehydrogenase has been inactivated by both the NADH and acetyl-CoA generated from fatty acid oxidation, which allows oxaloacetate production for gluconeogenesis. The oxaloacetate formed in the mitochondria is converted to either malate or aspartate to enter the cytoplasm via the malate/aspartate shuttle. Once in the cytoplasm the malate or aspartate is converted back into oxaloacetate (circle 3), and phosphoenolpyruvate carboxykinase will convert it to PEP (circle 4). The white circled numbers are alternate routes for exit of carbon from the mitochondrion using the malate/aspartate shuttle. OAA = oxaloacetate FA = fatty acid TG = triacylglycerol.

Pyruvate carboxylase in the mitochondrion needs ATP and biotin, is induced by cortisol and is stimulated by acetyl CoA. 

Lactate and alanine enter as pyruvate following the activities of lactate dehydrogenase (Figure 11.4) and alanine aminotransferase (Section 16.3). The first of the bypass reactions, the objective of which is to overcome the unfavourable energetics of a reversal of the pyruvate kinase reaction, seems a tortuous route (Figure 11.11). The reaction sequence relies on two important enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Since pyruvate carboxylase is located exclusively in the mitochondrion, pyruvate must cross the inner mitochondrial membrane (Section 12.2). Oxaloacetate produced by pyruvate carboxylase cannot traverse the inner membrane and is reduced by malate dehydrogenase into l-malate. This step is the reversal of the tricarboxylate cycle reaction (Section 12.4). Malate may, of... 

The carbon used for fatty-acid synthesis typically derives from the products of glycolysis. The end product of glycolysis, pyruvate, enters the mitochondria and becomes the substrate for two separate reactions. In one, pyruvate is decarboxylated via the pyruvate dehydrogenase complex, yielding acetyl-CoA. Lipogenic tissues also contain another mitochondrial enzyme, pyruvate carboxylase, which converts pyruvate to the four-carbon acid oxaloace-tate (OAA). Acetyl-CoA and oxaloacetate condense to form the six-carbon acid citrate. As citrate accumulates within the mitochondrion, it is exported to the cytoplasm, where it is converted back to oxaloacetate and acetyl-CoA. Cytoplasmic acetyl-CoA is the fundamental building block for de novo synthesis of fatty acids. 

Unlike glycolysis, which occurs strictly in the cell cytosol, gluconeogen-esis involves a complex interaction between the mitochondrion and the cytosol. This interaction is necessitated by the irreversibility of the pyruvate kinase reaction, by the relative impermeability of the inner mitochondrial membrane to oxaloacetate, and by the specific mitochondrial location of pyruvate carboxylase. Compartmentation within the cell has led to the distribution of a number of enzymes (aspartate and alanine aminotransferases, and NAD -malate dehydrogenase) in both the mitochondria and the cytosol. In the classical situation represented by the rat, mouse, or hamster hepatocyte, the indirect "translocation" of oxaloacetate—the product of the pyruvate carboxylase reaction—into the cytosol is effected by the concerted action of these enzymes. Within the mitochondria oxaloacetate is converted either to malate or aspartate, or both. Following the exit of these metabolites from the mitochondria, oxaloacetate is regenerated by essentially similar reactions in the cytosol and is subsequently decarboxylated to P-enolpyruvate by P-enol-pyruvate carboxykinase. Thus the presence of a membrane barrier to oxaloacetate leads to the functioning of the malate-aspartate shuttle as an important element in gluconeogenesis. 

For the conversion of pyruvate to oxaloacetate and the formation of citrate in the mitochondrion, see Chap. 12. Acetyl-CoA for fatty acid synthesis is converted to malonyl-CoA this reaction is catalyzed by acetyl-CoA carboxylase. Seven molecules of acetyl-CoA are converted to malonyl-CoA for the synthesis of one molecule of palmitic acid. 

The control of fatty-acid oxidation is related to the availability of circulating fatty acids and the activity of palmitoyl carnitine transferase 1. When circulating fatty acids are elevated, considerable fatty-acyl CoA is formed in a number of tissues, including the liver, which is sufficient to inhibit both acetyl CoA carboxylase in the cytosol and, indirectly, pyruvate dehydrogenase in the mitochondrion. Under this condition, neither malonyl CoA nor citrate would accumulate thus, there would be a diminution of fatty-acid synthesis. When large amounts of fatty... 

Fatty acids are synthesized mainly in the liver in humans, with dietary glucose serving as the major source of carbon. Glucose is converted through glycolysis to pyruvate, which enters the mitochondrion and forms both acetyl CoA and oxaloacetate (Fig. 33.1). These two compounds condense, forming citrate. Citrate is transported to the cytosol, where it is cleaved to form acetyl CoA, the source of carbon for the reactions that occur on the fatty acid synthase complex. The key regulatory enzyme for the process, acetyl CoA carboxylase, produces malonyl CoA from acetyl CoA. 

---