3NAD + FAD +GDP + P i + Acetyl-CoA 3NADH + FADH 2 + GTP + CoA + 2CO 2. CONTROL, REGULATION AND ACTIVITY OF Pyruvate dehydrogenase, (Pdh).

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1 TCA cycle; companion to the TCA cycle ppt. THE starting substrate for the TCA cycle is acetylcoa, that can be derived from pyruvate, fatty acids through β-oxidation anf leucine and lysine. There are basic questions needed to be answered to understand the function of the TCA cycle are; Why is this called an amphibolic pathway? Is this pathway only found in aerobes? In aerobes it is an oxidative pathway, in anaerobes is it a reductive pathway? 3NAD + FAD +GDP + P i + Acetyl-CoA 3NADH + FADH 2 + GTP + CoA + 2CO 2 CONTROL, REGULATION AND ACTIVITY OF Pyruvate dehydrogenase, (Pdh). 1

2 Enzyme abbrev cofactors No. su Prok No. su Euk Pdh E1 TPP transacetylase E2 lipoamide, CoA Dihydrolipoyl dh E3 FAD +, NAD Factors regulating the activity of Pdh. Pdh activity is regulated by its state of phosphorylation, being most active in the dephosphorylated state. Phosphorylation of Pdh is catalyzed by a specific Pdh kinase. Of the keto acid dh only Pdh is controlled by phosphorylation and has an endogenous kinase ans phosphatase within it structure. The activity of the kinase is enhanced when cellular energy charge is high which is reflected by an increase in the level of ATP, NADH and acetyl-coa. Conversely, an increase in pyruvate strongly inhibits Pdh kinase. Additional negative effectors of Pdh kinase are ADP, NAD + and CoASH, the levels of which increase when energy levels fall. The regulation of Pdh phosphatase is not completely understood but it is known that Mg 2+ and Ca + activate the enzyme. In adipose tissue insulin increases Pdh activity and in cardiac muscle Pdh activity is increased by catecholamines. Two products of the complex, NADH and acetyl-coa, are negative allosteric effectors on Pdh-a, the non-phosphorylated, active form of Pdh. These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the Pdh complex. In addition, NADH and acetyl- CoA are powerful positive effectors on Pdh kinase, the enzyme that inactivates Pdh by converting it to the phosphorylated Pdh-b form. Since NADH and acetyl-coa accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate Pdh kinase activity, reinforcing down-regulation of Pdh activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on Pdh kinase, with the result that when pyruvate levels rise, Pdh-a will be favored even with high levels of NADH and acetyl-coa. Concentrations of pyruvate, which maintains Pdh in the active form (Pdh-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of Pdh is nonetheless capable of converting pyruvate to acetyl-coa. With large amounts of pyruvate in cells having a high energy charge and high NADH, the pyruvate carbons will be directed to the 2 main storage forms of 2

3 carbons---glycogen via gluconeogenesis and fat production via fatty acid synthesis---where acetyl-coa is the principal carbon donor. Although the regulation of Pdh-b phosphatase is not well understood, it is quite likely regulated to maximize pyruvate oxidation under energy-poor conditions and to minimize Pdh activity under energy-rich conditions. The enzyme complex then is the gate keeper for the entrance into the TCA cycle and can be considered one of the rate-limiting steps ultimately of aerobic respiration. The TCA cycle showing enzymes, substrates and products. The GTP generated during the succinate thiokinase (succinyl-coa synthetase) reaction is equivalent to a mole of ATP by virtue of the presence of nucleoside diphosphokinase. The 3 moles of NADH and 1 mole of FADH 2 generated during each round of the cycle feed into the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP and each mole of FADH 2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate, which enters the TCA cycle, 12 moles of ATP can be generated. Human cells contain almost equal amounts of mitochondrial and cytosolic PEPCK so this second reaction can occur in either cellular compartment. For gluconeogenesis to proceed, the OAA produced by PC needs to be transported to the cytosol. However, no transport mechanism exist for its' 3

4 direct transfer and OAA will not freely diffuse. Mitochondrial OAA can become cytosolic via three pathways, conversion to PEP (as indicated above through the action of the mitochondrial PEPCK), transamination to aspartate or reduction to malate, all of which are transported to the cytosol. If OAA is converted to PEP by mitochondrial PEPCK, it is transported to the cytosol where it is a direct substrate for gluconeogenesis and nothing further is required. Transamination of OAA to aspartate allows the aspartate to be transported to the cytosol where the reverse transamination occurs yielding cytosolic OAA. This transamination reaction requires continuous transport of glutamate into, and a-ketoglutarate out of, the mitochondrion. Therefore, this process is limited by the availability of these other substrates. Either of these latter two reactions will predominate when the substrate for gluconeogenesis is lactate. Whether mitochondrial decarboxylation or transamination occurs is a function of the availability of PEPCK or transamination intermediates. Mitochondrial OAA can also be reduced to malate in a reversal of the TCA cycle reaction catalyzed by malate dehydrogenase (Mdh). The reduction of OAA to malate requires NADH, which will be accumulating in the mitochondrion as the energy charge increases. The increased energy charge will allow cells to carry out the ATP costly process of gluconeogenesis. The resultant malate is transported to the cytosol where it is oxidized to OAA by cytosolic Mdh which requires NAD + and yields NADH. 4

5 The NADH produced during the cytosolic oxidation of malate to OAA. Conversion of pyruvate to PEP requires the action of two mitochondrial enzymes. The first is an ATP-requiring reaction catalyzed by pyruvate carboxylase, (PC). As the name of the enzyme implies, pyruvate is carboxylated to form OAA. The CO 2 in this reaction is in the form of bicarbonate (HCO 3 2- ). This reaction is an anaplerotic reaction since it can be used to fill-up the TCA cycle. The second enzyme in the conversion of pyruvate to PEP is PEP carboxykinase (PEPCK). PEPCK requires GTP in the decarboxylation of OAA to yield PEP. Since PC incorporated CO 2 into pyruvate and it is subsequently released in the PEPCK reaction, no net OAA is utilized during the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. The coupling of these two oxidation-reduction reactions is required to keep gluconeogenesis functional when pyruvate is the principal source of carbon atoms. The conversion of OAA to malate predominates when pyruvate (derived from glycolysis or amino acid catabolism) is the source of carbon atoms for gluconeogenesis. When in the cytoplasm, OAA is converted to PEP by the cytosolic version of PEPCK. Hormonal signals control the level of PEPCK protein as a means to regulate the flux through gluconeogenesis. A significant amount of metabolic energy can come from amino acid metabolism, particularly under conditions of starvation. The metabolism of amino acids occurs through common metabolic intermediates, many of them part of or linked to the Krebs cycle. The intermediates like pyruvate, oxaloacetate, fumarate, succinyl-coa and a-ketobutyrate all can contribute to the net synthesis of glucose through gluconeogenesis. The amino acids that are degraded into these intermediates are called glucogenic. Some amino acids are degraded directly to gluconeogenic intermediates, while others contribute to gluconeogenesis more indirectly, such as through conversion to oxaloacetate through the Krebs cycle. Other common intermediates in amino acid metabolism are acetyl-coa and acetoacetate. Animals do not have a metabolic pathway to convert these intermediates into glucose. Instead the metabolic fate of acetyl-coa and acetoacetate is the production of fatty acids or ketone bodies. Amino acids that are metabolized to produce acetyl-coa and acetoacetate are called ketogenic. Several of the amino acids do not fall cleanly into one group or another, but are both ketogenic and glucogenic. For example, isoleucine metabolism produces both acetyl-coa, which makes it ketogenic, but it also produces succinyl-coa, which contributes to glucose production. 5

6 Glyoxylate Cycle The glyoxylate cycle, a variation of the TCA cycle, is ananabolic pathway occurring in plants, bacteria, protists, and fungi. The glyoxylate cycle centers on the conversion of acetyl-coa to succinate for the synthesis of carbohydrates. In microorganisms, the glyoxylate cycle allows cells to utilize simple carbon compounds as a carbon source when complex sources such as glucose are not available. The glyoxylate cycle utilizes three of the five enzymes associated with the TCA cycle and shares many of its intermediate steps. The two cycles vary when, in the glyoxylate cycle, isocitrate lyase converts isocitrate into glyoxylate and succinate instead of α-ketoglutarate as seen in the TCA cycle. This bypasses the decarboxylation steps that take place in the TCA cycle, allowing simple carbon compounds to be used in the later synthesis of macromolecules, including glucose. The glyoxylate cycle then continues on, using glyoxylate and acetyl-coa to produce malate. 6

7 The isocitrate used in the first reaction of the glyoxylate cycle is restored by the action of three enzymes characteristic of Krebs cycle (malate dehydrogenase, citrate synthase and cis-aconitase) on L-malate, with the utilization of a second molecule of acetyl-coa. The glyoxylate cycle thus enables the synthesis of a mole of succinate from two moles of acetate (as acetyl-coa), being the overall net reaction: 2 acetyl-coa + 2H 2 O + NAD + succinate + 2CoA + NADH The glyoxylate cycle replenishes intermediates of the Krebs cycle and conserves carbon that would otherwise be oxidized and lost to biosynthetic pathways, with the final result of a net conversion of fats to carbohydrates. This replenishing function has been termed anaplerotic and probably plays an essential role in growth of microorganisms on fatty acids. Isocitrate lyase is an enzyme that functions at a branch point of carbon metabolism and diverts isocitrate through a carbon-conserving pathway, the glyoxylate cycle, bypassing the two decarboxylative steps of the tricarboxylic acid cycle that convert isocitrate to succinyl-coa. The E-D pathway is older than the E-M-P pathway. Reverse EMP has been found in archea. Leading to the possibility that glycolysis originally evolved as gluconeogenesis an anabolic pathway. What appears to beunique is the PFK and the formation of Fr. 1,6 BP which is not found in the archeon. In Thermoproteus (an archea) it has a PFK that uses Pi and not ATP. GLUCONBEOGENESIS in Pyrococcus furiosus has a PEP synthase and a fr 1,6 phosphatase. In this organism E-D pathway oxidation is linked to ferridoxin and not NAD(P). The citric acid cycle evolved first as a reductive anabolic pathway in anaerobes. It is suggested that it arose to accept electrons from fermentation in the formation of OAA malate fumarate succinate and the pyruvate:ferridoxin oxidoreductase which forms acetyl-coa+co 2 +H 2 led to the evolution of citrate synthase. These would form two arms of a cycle. OAA+acetyl-CoA citrate isocitrate αkg. The evolution of αkg:ferridoxin oxidoreductase would have close the cycle. This would have been similar to the reductive TCA cycle of Chlorobium limicola. 7

8 The Entner-Doudoroff pathway is found in prokaryotes exclusively. It is widespread amongst the gram negative aerobes. Not usually found in anaerobes since it only produces 1 ATP. Entner-Doudoroff pathway begins the same way as the pentose phosphate pathway to produce glyceraldehyde-3- phosphate Glyceraldehyde-3-phosphate catabolized to pyruvate via the 3-carbon stage of glycolysis. Total yield per glucose molecule; One ATP One NADH One NADPH (1) Pyruvate dehydrogenase. (2) Malate dehydrogenase. (3)α- Ketoglutarate dehydrogenase. (4). Succinyl CoA synthase. (5) Succinate dehydrogenase/fumarate reductase.(6) Phosphoenolpyruvate carboxykinase (PEPCK). (7) 2- Oxoglutarate/malate translocator. (8) Acetyl/propionyl CoA carboxylase( α-subunit) 8

9 Hydrogenosomal metabolism of Nyctotherus The hydrogenosomes perform a chimeric metabolism that exhibits mitochondrial and hydrogenosomal traits. The TCA cycle is incomplete, and it is likely that the TCA cycle is used in the reductive way in the hydrogenosomes. The function of complex I has been substantiated by the demonstration of a proton gradient that is sensitive against several inhibitors of a mitochondrial complex I and insensitive against cyanide and antimycin, which interfere with the pumping of mitochondrial complex III/IV. The excretion of succinate (besides acetate, lactate and ethanol) strongly suggests a 9

10 function of mitochondrial complex II as fumarate reductase, which is likely to accept electrons from complex I through a rhodoquinone. The Fehydrogenase and the fumarate respiration regulate the organellar NADH pool allowing reoxidation to NAD. The links to the fatty acid and amino acid metabolism are substantiated by the identification of a number of key genes. The presence of an active ATP synthase remains to be established by further experimentshydrogenosomal metabolism of Nyctotherus The hydrogenosomes perform a chimeric metabolism that exhibits mitochondrial and hydrogenosomal traits. The TCA cycle is incomplete, and it is likely that the TCA cycle is used in the reductive way in the hydrogenosomes. The function of complex I has been substantiated by the demonstration of a proton gradient that is sensitive against several inhibitors of a mitochondrial complex I and insensitive against cyanide and antimycin, which interfere with the pumping of mitochondrial complex III/IV. The excretion of succinate (besides acetate, lactate and ethanol) strongly suggests a function of mitochondrial complex II as fumarate reductase, which is likely to accept electrons from complex I through a rhodoquinone. The Fe-hydrogenase and the fumarate respiration regulate the organellar NADH pool allowing reoxidation to NAD. The links to the fatty acid and amino acid metabolism are substantiated by the identification of a number of key genes. The presence of an active ATP synthase remains to be established by further experiments 10