Pyruvate mitochondria

Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cycle. Because, in eukaryotic ceils, glycolysis occurs in the cytoplasm, whereas the TCA cycle reactions and ail subsequent steps of aerobic metabolism take place in the mitochondria, pyruvate must first enter the mitochondria to enter the TCA cycle. The oxidative decarboxylation of pyruvate to acetyl-CoA,... 

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-... 

Pyruvate (CH3-CO-COOH) is an intermediate metabolite, the product of carbohydrate, fat or protein metabolism. Pyruvate is the end point of glycolysis. In mitochondria, pyruvate may be oxidized to C02 and H20, reduced to acetyl coenzyme A (acetyl CoA) by PDH, or carboxylated by PC into oxaloacetate (Fig. 1.4.2). 

The gluconeogenesis pathway is summarized in Figure 18.8. Note that both the mitochondria and cytosol are involved. In the mitochondria, pyruvate is carboxylated by PC to oxaloacetate, as shown in Equation (18.6) ... 

Pyruvate must first be transported into mitochondria by a specific carrier that cotransports a proton to maintain electrical neutrality. Inside mitochondria pyruvate undergoes oxidative decarboxylation by three enzymes that function sequentially and are present as a complex known as the pyruvate dehydrogenase complex. The overall reaction is physiologically irreversible, has a high negative AG° (—8.0 kcal/mol, or —33.5 kJ/mol), and commits pyruvate to the formation of acetyl-CoA ... 

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). 

Anaerobic parasites, such as T. vaginalis and E. histolytica, lack functional mitochondria. Pyruvate generated by the glycolytic pathway is further metabolized within the cytosol of Giardia and Entamoeba species and in the hydrogenosome of these parasites, a subcellular organelle unique to trichomonads (4). Under anaerobic conditions, pyruvate is converted into acetyl Co-A and COj by pyruvateferredoxin oxidoreductase with the concomitant reduction of ferredoxin. Metronidazole and other nitro-imidazole derivatives are toxic to these anaerobes after reduction of the nitro moiety by ferredoxin. As these compounds are not reduced in mamalian cells, selectivity of their antiparasitic effects is assured (see Chapter 3). 

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... 

FIGURE 3 Schematic outline of carbohydrate metabolism. Glucose is oxidized to two molecules of pyruvate by glycolysis in the cytoplasm. In mitochondria, pyruvate is oxidized by molecular oxygen to CO2 and water. The synthesis of ATP is coupled to pyruvate oxidation. 

Under aerobic conditions, the hydrogen atoms of NtUDH are oxidised within the mitochondrion pyruvate is also oxidised in the mitochondrion (Figure 9.15). However, NADH cannot be transported across the inner mitochondrial membrane, and neither can the hydrogen atoms themselves. This problem is overcome by means of a substrate shuttle. In principle, this involves a reaction between NADH and an oxidised substrate to produce a reduced product in the cytosol, followed by transport of the reduced product into the mitochondrion, where it is oxidised to produce hydrogen atoms or electrons, for entry into the electron transfer chain. Finally, the oxidised compound is transported back into the cytosol. The principle of the shuttle is shown in Figure 9.16. 

Summary diagram of the breakdown of glucose to carbon dioxide and water in a eukaryotic cell. As depicted here, the process starts with the absorption of glucose at the plasma membrane and its conversion into glucose-6-phosphate. In the cytosol, this six-carbon compound is then broken down by a sequence of enzyme-catalyzed reactions into two molecules of the three-carbon compound pyruvate. After absorption by the mitochondrion, pyruvate is broken down to carbon dioxide and water by a sequence of reactions that requires molecular oxygen. 

Although fatty acid synthesis occurs within the cytoplasm of most animal cells, liver is the major site for this process. (Recall, for example, that liver produces VLDL. See p. 349.) Fatty acids are synthesized when the diet is low in fat and/or high in carbohydrate or protein. Most fatty acids are synthesized from dietary glucose. As discussed, glucose is converted to pyruvate in the cytoplasm. After entering the mitochondrion, pyruvate is converted to acetyl-CoA, which condenses with oxaloacetate, a citric acid cycle intermediate, to form citrate. When mitochondrial citrate levels are sufficiently high (i.e., cellular energy requirements are low), citrate enters the cytoplasm, where it is cleaved to form acetyl-CoA and oxaloacetate. The net reaction for the synthesis of palmitic acid from acetyl-CoA is as follows ... 

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... 

Glycolysis, the pentose phosphate pathway, and fatty acid synthesis are all found in the cytosol. In gluconeo-genesis, substrates such as lactate and pyruvate, which are formed in the cytosol, enter the mitochondrion to yield oxaloacetate before formation of glucose. 

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). 

Answer C. Oxaloacetate, produced from pyruvate, exits the mitochondrion after conversion to malate. 

Figure 9.13 Examples of mitochondrial transport systems for anions. 0 The anb port system transfers malate into but oxo-glutarate out of the mitochondrion. The symport system transfers both pyruvate and protons into the mitochondrion across the inner membrane. Both transport processes are electroneutral.

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

Rotte C, Stejskal F, Zhu G, Keithly JS, Martin W (2001) Pyruvate NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists. Mol Biol Evol 18 710-720 Schnarrenberger C, Martin W (2002) Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer. Eur J Biochem 269 868-883... 

Fig. 4 Transmission electron microscopy of a longitudinal section of the posterior end of a Cryptosporidium parvum sporozoite showing immunogold localization of pyruvate NADP+ oxidoreductase (CpPNO). The mitochondrion-like organelle ( ) is posterior to the nucleus, and lies between the nucleus and the CB. It is labeled by mitochondrion-specific 15-nm gold anti- particles. Small -nm gold goat anti-CpPFO particles (arrows) show the localization of CpPNO. There are no 6-nm particles localized within the mitochondrion-like organelle (reprinted from Fig. 12 of Ctrnacta et al. 2006 with permission of the publishers)...

Haresh K, Suresh K, Khairul Anus A, Saminathan S (1999) Isolate resistance of Blastocystis hominis to metronidazole. Trop Med Int Health 4 274-277 Inui H, Ono K, Miyatake K, Nakano Y, Kitaoka S (1987) Purification and characterization of pyruvate NADP+ oxidoreductase in Euglena gracilis. J Biol Chem 262 9130-9135 Keithly JS, Langreth SG, Buttle KF, Mannella CA (2005) Electron tomographic and ultra-structural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles. J Eukaryot Microbiol 52 132-140 Kurland CG, Andersson SGE (2000) Origin and evolution of the mitochondrial proteome. Micro Mol Biol Rev 64 786-820... 

FIGURE 14-19 Alternative paths from pyruvate to phospho-enolpyruvate. The path that predominates depends on the glucogenic precursor (lactate or pyruvate). The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion (see text). The relative importance of the two pathways depends on the availability of lactate and the cytosolic requirements for NADH by gluconeogenesis. 

This three-step process for transferring fatty acids into the mitochondrion—esterification to CoA, transesterification to carnitine followed by transport, and transesterification back to CoA—links two separate pools of coenzyme A and of fatty acyl-CoA, one in the cytosol, the other in mitochondria These pools have different functions. Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids, and some amino acids, whereas cytosolic coenzyme A is used in the biosynthesis of fatty acids (see Fig. 21-10). Fatty acyl-CoA in the cytosolic pool can be used for membrane lipid synthesis or can be moved into the mitochondrial matrix for oxidation and ATP production. Conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate. 

In hepatocytes and adipocytes, cytosolic NADPH is largely generated by the pentose phosphate pathway (see Fig. 14-21) and by malic enzyme (Fig. 21-9a). The NADP-linked malic enzyme that operates in the carbon-assimilation pathway of C4 plants (see Fig. 20-23) is unrelated in function. The pyruvate produced in the reaction shown in Figure 21-9a reenters the mitochondrion. In hepatocytes and in the mammary gland of lactating animals, the NADPH required for fatty acid biosynthesis is supplied primarily by the pentose phosphate pathway (Fig. 21-9b). 

FIGURE 21-10 Shuttle for transfer of acetyl groups from mitochondria to the cytosol. The mitochondrial outer membrane is freely permeable to all these compounds. Pyruvate derived from amino acid catabolism in the mitochondrial matrix, or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the matrix. Acetyl groups pass out of the mitochondrion as citrate in the cytosol they are de-... 

Matrix of the mitochondrion This gel-like solution in the interior of mitochondria is fifty percent protein. These molecules include the enzymes responsible for the oxidation of pyruvate, amino acids, fatty acids (by p-oxidation), and those of the tricarboxylic acid (TCA) cycle. The synthesis of urea and heme occur partially in the matrix of mitochondria. In addition, the matrix contains NAD+and FAD (the oxidized forms of the two coenzymes that are required as hydrogen acceptors) and ADP and Pj, which are used to produce ATP. [Note The matrix also contains mitochondrial RNA and DNA (mtRNA and mtDNA) and mitochondrial ribosomes.]... 

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... 

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