Citrate Cycle Supplemental Reading

Citrate Cycle Supplemental Reading Key Concepts - The Citrate Cycle captures energy using redox reactions - Eight enzymatic reactions of the Citrate Cycle - Key control points in the citrate cycle regulate metabolic flux KEY CONCEPT QUESTIONS IN THE CITRATE CYCLE: What role does NADH and FADH 2 have in connecting the citrate cycle to ATP synthesis? Why is the citrate cycle considered the hub of metabolism? Biochemical Applications of the Citrate Cycle: Fluoroacetate is found in poisonous plants and it is the active ingredient in "compound 1080" which is used by ranchers to kill coyotes and foxes. Cells convert fluoracetate to fluorocitrate, a potent inhibitor of the citrate cycle enzyme mitochondrial aconitase. The Citrate Cycle Captures Energy Using Redox Reactions The citrate cycle is central to aerobic metabolism and ATP production because it contains four dehydrogenase reactions that generate NADH and FADH 2 that are reoxidized by the electron transport chain to generate large amounts of ATP by oxidative phosphorylation (lecture 30). The citrate cycle is considered the "hub" of cellular metabolism because it not only links the oxidation of metabolic fuels (carbohydrate, fatty acids and proteins) to ATP synthesis, but it also provides shared metabolites for numerous other metabolic pathways as shown in figure 1. Unlike glycolysis which occurs in the cytosol, all of the enzymes in the citrate cycle, electron transport chain and oxidative phosphorylation reside in the mitochondrial matrix where pyruvate is converted to acetyl-coa by the enzyme pyruvate dehydrogenase. As illustrated in figure 2, pyruvate can also be derived from amino acid catabolism, and acetyl-coa can be derived from both amino a acids and fatty acids. The primary function of the citrate cycle in terms of energy conversion reactions is to oxidize acetyl-coa, and in the process, transfer four pairs of electrons from citrate cycle intermediates to 3 moles of NADH and 1 mole of FADH 2 during each turn of the cycle. As shown in figure 3, the citrate cycle is a "metabolic Figure 1. engine" in which all eight of the cycle intermediates are continually replenished to maintain a smooth-running energy conversion process. The fuel for this metabolic engine is acetyl-coa, the exhaust is two molecules of CO 2, and the energy output is eight electrons (redox energy) and one GTP that is converted to ATP (phosphoryl transfer energy). The electron transport system and oxidative phosphorylation convert the available redox energy into ATP through a series of reactions that reoxidize NADH and FADH 2 in the presence of O 2. 1 of 9 pages

Figure 2, Figure 3. The "currency exchange" for redox energy and ATP synthesis in the mitochondria is such that for every 2 NADH molecules that are reoxidized in the electron transport chain, there is a production of ~5 molecules of ATP by oxidative phosphorylation (~2.5 ATP/ NADH). Oxidation of 2 FADH 2 molecules by the electron transport chain results in only ~3 molecules of ATP (~1.5 ATP/FADH 2 ) because of differences in where these two coenzymes enter the electron transport chain. Based on this ATP currency exchange ratio, and the one substrate level phosphorylation reaction, each turn of the cycle produces ~10 ATP for every acetyl-coa that is oxidized. Since regeneration of NAD + and FAD inside the mitochondrial matrix is required to maintain flux through the citrate cycle (four of the enzymes require NAD + or FAD as coenzymes), this metabolic engine is dependent on a constant supply of O 2 just like a combustion engine. The reliance on redox reactions in the electron transport chain to maintain metabolic flux in the citrate cycle is similar to the way that the glyceraldehyde-3-p dehydrogenase reaction in glycolysis requires the regeneration of NAD + by lactate dehydrogenase under anaerobic conditions. Hans Krebs, a biochemist who fled Nazi Germany for England in 1933, first described the citrate cycle in 1937. The citrate cycle is sometimes called the Krebs cycle or the tricarboxylic acid cycle (citrate is a tricarboxylate), although we will refer to it as the citrate cycle because citrate is the first product of the pathway and the unprotonated form of citric acid is the predominant species at physiological ph (the pk a values of the three carboxylate groups are 3.1, 4.7 and 6.4). Figure 4 shows the eight citrate cycle reactions. The citrate cycle is distinguished from linear metabolic pathways in that oxaloacetate is both the substrate for the first reaction (citrate synthase) and the product of the last reaction (malate dehydrogenase), which means that it must be regenerated from citrate after each turn of the cycle. 2 of 9 pages

Figure 4. 1. What does the citrate cycle accomplish for the cell? Transfers 8 electrons from acetyl-coa to the coenzymes NAD + and FAD to form 3 NADH and 1 FADH 2 which are then re-oxidized by the electron transport chain to produce ATP by the process of oxidative phosphorylation. Generates 2 CO 2 as waste products and uses substrate level phosphorylation to generate 1 GTP which is converted to ATP by nucleoside diphosphate kinase. Supplies metabolic intermediates for amino acid and porphyrin biosynthesis. 2. What is the overall net reaction of the citrate cycle? Acetyl-CoA + 3 NAD + + FAD + GDP + P i + 2 H 2 O CoA + 2 CO 2 + 3 NADH + 2 H + + FADH 2 + GTP ΔGº = -57.3 kj/mol 3 of 9 pages

3. What are the key enzymes in the citrate acid cycle? Pyruvate dehydrogenase not a citrate cycle enzyme but it is critical to flux of acetyl-coa through the cycle; this multisubunit enzyme complex requires five coenzymes, is activated by NAD +, CoA and Ca 2+ (in muscle cells), and inhibited by acetyl-coa, ATP and NADH. Citrate synthase catalyzes the first reaction in the pathway and can be inhibited by citrate, succinyl-coa, NADH and ATP; inhibition by ATP is reversed by ADP. Isocitrate dehydrogenase - catalyzes the oxidative decarboxylation of isocitrate by transferring two electrons to NAD + to form NADH, and in the process, releasing CO 2, it is activated by ADP and Ca 2+ and inhibited by NADH and ATP. α-ketoglutarate dehydrogenase - is functionally similar to pyruvate dehydrogenase in that it is a multisubunit complex, requires the same five coenzymes and catalyzes an oxidative decarboxylation reaction that produces CO 2, NADH and succinyl-coa; it is activated by Ca 2+ and AMP and it is inhibited by NADH, succinyl-coa and ATP. 4. What are examples of the citrate cycle in real life? Citrate is produced commercially by fermentation methods using the microorganism Aspergillus niger. Every year almost a half of million tonnes (5 x 10 8 kg) of citrate are produced worldwide by exploiting the citrate synthase reaction. Citrate is used as a flavor enhancer and food preservative. Eight Reactions Of The Citrate Cycle In the first four reactions, the two carbon acetate group of acetyl-coa is linked to the four carbon oxaloacetate substrate to form a six carbon citrate molecule. Citrate is then transformed to isocitrate to set up two decarboxylation reactions yielding two NADH and the high energy four carbon cycle intermediate succinyl-coa. In the second half of the cycle, oxaloacetate is regenerated from succinyl-coa by four successive reactions that lead to the formation of one GTP (ATP), one FADH 2 and one NADH. Reaction 1: Condensation of oxaloacetate and acetyl-coa by citrate synthase to form citrate The purpose of the first reaction in the citrate cycle is to commit the acetate unit of acetyl-coa to oxidative decarboxylation. The citrate synthase reaction follows an ordered mechanism in which oxaloacetate binds first, inducing a conformational change in the enzyme that facilitates acetyl- CoA binding. Formation of the transient intermediate, citryl-coa, is followed by a rapid hydrolysis reaction that releases CoA-SH and citrate as shown in figure 5. Figure 5. 4 of 9 pages

Reaction 2: Isomerization of citrate by aconitase to form isocitrate This reversible isomerization reaction involves a two step mechanism in which the intermediate, cis-aconitate, is formed by a dehydration reaction that requires the participation of an iron-sulfur cluster (4Fe-4S) in the enzyme active site. Next, water is added back to convert the double bond in cis-aconitate to a single bond with a hydroxyl group on the terminal carbon (figure 6). Figure 6. Aconitase is one of the targets of the toxic compound fluorocitrate which is derived from fluoroacetate, a naturally occurring poison found in native Australian and African plants (and compound 1080). Fluoroacetate-containing plants are so deadly that Australian sheep herders have reported finding sheep with their heads still in the bush they were feeding on when they died. Reaction 3: Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase to form α- ketoglutarate, CO 2 and NADH Reaction 3 is the first of two decarboxylation steps in the citrate cycle, and also the first reaction to generate NADH which is used for energy conversion reactions in the electron transport system. Eukaryotic cells actually contain two isocitrate dehydrogenases, the mitochondrial NAD + - dependent enzyme that functions in the citrate cycle, and a second NADP + -dependent enzyme that is present in both the cytosol and mitochondria. As shown in figure 7, NAD + catalyzes an oxidation reaction that generates the transient intermediate oxalosuccinate. In the presence of the divalent cations Mg 2+ or Mn 2+, oxalosuccinate is decarboxylated to form α-ketoglutarate. Figure 7. Reaction 4: Oxidative decarboxylation of α-ketoglutarate by α-ketoglutarate dehydrogenase to form succinyl-coa, CO 2 and NADH In this second oxidative decarboxylation reaction, the α-ketoglutarate dehydrogenase complex utilizes essentially the same catalytic mechanism we have already described for the pyruvate dehydrogenase reaction. This includes the binding of substrate to an E1 subunit (α-ketoglutarate dehydrogenase), followed by decarboxylation and formation of a TPP-linked intermediate. Figure 8 on the next page summarizes the α-ketoglutarate dehydrogenase reaction, which not surprisingly, is similar to pyruvate dehydrogenase in that it has a large change in free energy (ΔGº' = -33.5 kj/mol) and is allosterically-regulated by changes in the energy charge of the cell. 5 of 9 pages

Reaction 5: Conversion of succinyl-coa to succinate by succinyl-coa synthetase in a substrate level phosphorylation reaction that generates GTP The available free energy in the thioester bond of succinyl-coa Figure 8. Bioc 460 - Dr. Miesfeld Spring 2008 (ΔGº' = -32.6 kj/mol) is used in the succinyl-coa synthetase reaction to carry out a phosphoryl transfer reaction (ΔGº' = +30.5 kj/mol) that leads to the production of GTP (or ATP) and succinate (figure 9). The enzyme nucleoside diphosphate kinase interconverts GTP and ATP by a readily reversible phosphoryl transfer reaction: GTP + ADP <===> GDP + ATP (ΔGº' = 0 kj/mol). Figure 9. Reaction 6: Oxidation of succinate by succinate dehydrogenase to form fumarate This coupled redox reaction directly links the Figure 10. citrate cycle to the electron transport system through the redox conjugate pair FAD/FADH 2 which is covalently linked to the enzyme succinate dehydrogenase, an inner mitochondrial membrane protein. As shown in figure 10, oxidation of succinate results in the transfer of an electron pair to the FAD moiety, which in turn, passes the two electrons to the electron carrier coenzyme Q in complex II of the electron transport system. Reaction 7: Hydration of fumarate by fumarase to form malate Fumarase is a highly stereo-specific enzyme that catalyzes the hydration of the C=C double bond in fumarate to generate the L-isomer of malate. This reversible reaction is shown in figure 11. Figure 11. Reaction 8: Oxidation of malate by malate dehydrogenase to form oxaloacetate The final reaction in the citrate cycle involves the oxidation of the hydroxyl group of malate to form oxaloacetate in the presence of NAD + as shown in figure 12. This leads to the production of the last of 3 NADH molecules generated by the citrate cycle. The two carbon atoms Figure 12. 6 of 9 pages

Figure 13. Bioc 460 - Dr. Miesfeld Spring 2008 originating from acetyl- CoA in reaction 1 are incorporated into oxaloacetate and not lost as CO 2 until subsequent turns. While the change in standard free energy for this reaction is unfavorable (ΔGº' = +29.7 kj/mol), the extremely low concentration of oxaloacetate inside the mitochondrial matrix ensures that the reaction is pulled to the right under physiological conditions (as a result of actual free energy ΔG). Now that we have looked at all eight citrate cycle reactions individually, let's review the bioenergetics of the pathway by summing up the ΔGº' values as shown above in figure 13. By combining the glycolytic pathway, the pyruvate dehydrogenase reaction and the citrate cycle into one complete circuit we obtain the following net reaction: Glucose + 2 H 2 O + 10 NAD + + 2 FAD + 4 ADP + 4 P i ---> 6 CO 2 + 10 NADH + 6 H + + 2 FADH 2 + 4 ATP The reducing power of the 10 NADH and 2 FADH 2 can be converted to ATP equivalents using the currency exchange ratios of ~2.5 ATP/NADH and ~1.5 ATP/FADH 2 to yield ~28 ATP, which when combined with the 4 ATP synthesized by substrate phosphorylation, generates a maximum of ~32 ATP as shown in figure 14 (~30 ATP in muscle cells). The complete oxidation of glucose by the pyruvate dehydrogenase complex and the citrate cycle leads to the production of 6 CO 2 molecules as waste. When Krebs first described the citrate cycle in 1937, it wasn't possible to follow the fate of individual carbon atoms throughout the cycle, and therefore it wasn't until techniques were developed using radioactively-labeled metabolic intermediates that it became clear exactly how each enzyme reaction functioned. Based on the results of these 14 C-labeling experiments, figure 15 shows how a game of "follow the bouncing carbon" can be played to see what Figure 14. 7 of 9 pages

happens to the two carbon atoms that enter the citrate cycle as acetyl-coa. In the first turn of the cycle, both carbons are incorporated into oxaloacetate, and it isn't until the second cycle that the first carbon is released as CO 2 by the isocitrate dehydrogenase reaction. The second carbon is not lost as CO 2 until the α-ketoglutarate reaction in the fourth turn of the cycle. This is a good way to demonstrate that intermediates must be continually regenerated in a cyclic pathway to maintain metabolic flux. Figure 15. Bioc 460 - Dr. Miesfeld Spring 2008 REGULATION OF THE CITRATE CYCLE The citrate cycle is quite different than glycolysis in that the end product of the pathway, oxaloacetate, is the substrate for the first reaction, meaning that flux through the pathway is continuously monitored by resetting the level of available substrate after each turn of the cycle. In addition to substrate availability, the two other regulatory mechanisms at play in the citrate cycle are product inhibition and feedback control of key enzymes. Figure 16 illustrates that the three main control points within the citrate cycle are regulation of citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase activities. Figure 16 also shows the factors that regulate pyruvate dehydrogenase and pyruvate carboxylase activity as these two enzymes control the rate of citrate cycle flux by providing acetyl CoA and Figure 16. oxaloacetate, respectively. Importantly, pyruvate carboxylase is allosterically activated by acetyl-coa to increase production of oxaloacetate and thereby maintain flux through the citrate cycle. Pyruvate carboxylase plays a key role in providing intermediates for the citrate cycle by being the "tag team partner" for pyruvate dehydrogenase by balancing the input of oxaloacetate with that of acetyl-coa. For example, when flux through the citrate cycle is decreased due to loss of cycle intermediates to anabolic pathways, acetyl-coa accumulates in the mitochondrial matrix and allosterically activates pyruvate carboxylase. 8 of 9 pages

ANSWERS TO KEY CONCEPT QUESTIONS IN THE CITRATE CYCLE: The coenzymes NADH and FADH 2 function as carrier molecules that transport electron pairs from redox reactions in the citrate cycle to the electron transport system where they are reoxidized to provide redox energy that can be harnessed for ATP synthesis. The primary role of the citrate cycle is to strip 4 electron pairs (8 e-) from the acetate group of acetyl-coa and transfer them to 3 NAD + and 1 FAD molecules to form 3 NADH and 1 FADH 2, respectively. Reoxidation of NADH and FADH 2 by the electron transport system generates a proton gradient across the inner mitochondrial membrane that drives ATP synthesis inside the mitochondrial matrix through a process called oxidative phosphorylation. The ATP currency exchange for reoxidation of NADH by the coupled electron transport system/oxidative phosphorylation reactions is ~2.5 ATP/NADH and ~1.5 ATP/FADH 2. Based on the yield of NADH and FADH 2 from the citrate cycle, and the ATP currency exchange, each turn of the citrate cycle produces ~10 ATP for every acetyl-coa that enters. Moreover, complete oxidation of glucose to 6 CO 2 and 6 H 2 O by the combined reactions of glycolysis, the pyruvate dehydrogenase complex, and the citrate cycle, results in the maximum production of ~32 ATP/glucose (only ~30 ATP are produced per glucose in muscle cells because of the glycerol-3p shuttle as described in lecture 30). The citrate cycle is considered the hub of metabolism because it provides the crucial link between oxidation of acetyl-coa and ATP synthesis, and in addition, provides metabolic precursors for the biosynthesis of amino acids, fatty acids, cholesterol, heme and glucose. The citrate cycle intermediates α-ketoglutarate and oxaloacetate provide carbon skeletons for amino acid biosynthesis, whereas, succinyl-coa combines with glycine to from δ-aminolevulinic acid, a precursor for heme biosynthesis. Citrate is transported out of the mitochondria and converted back to acetyl-coa and oxaloacetate in the cytosol by the enzyme citrate lyase. Cytosolic acetyl- CoA is used for fatty acid and cholesterol biosynthesis, and oxaloacetate can be converted to phosphoenolpyruvate by the enzyme phosphoenolpyruvate carboxykinase (PEPCK) for use in glucose synthesis. Another citrate cycle intermediate, malate, can also be used for gluconeogenesis following export to the cytosol where it is converted first to oxaloacetate by cytosolic malate dehydrogenase, and converted to phosphoenolypyruvate by the enzyme PEPCK. 9 of 9 pages