Lecture 16: Pyruvate Dehydrogenase and the Citric Acid Cycle PDF

BioC 3021 Notes Robert Roon Lecture 16: Pyruvate Dehydrogenase and the Citric Acid Cycle Slide 1. Pyruvate Dehydrogenase and the Citric Acid Cycle In this lecture, we will first study the pyruvate dehydrogenase complex and then the citric acid cycle. Thes...

BioC 3021 Notes Robert Roon Lecture 16: Pyruvate Dehydrogenase and the Citric Acid Cycle Slide 1. Pyruvate Dehydrogenase and the Citric Acid Cycle In this lecture, we will first study the pyruvate dehydrogenase complex and then the citric acid cycle. These reactions collectively result in the conversion of pyruvate to carbon dioxide. Slide 2. Major Catabolic Pathways Converge on AcetylCoA The pyruvate dehydrogenase complex catalyzes the breakdown of pyruvate, the three carbon product of glycolysis. The pyruvate dehydrogenase reaction produces the two carbon intermediate acetylCoA plus carbon dioxide. The acetylCoA then enters the citric acid cycle, where it is oxidized into two molecules of carbon dioxide. Note that acetylCoA is also the end product of fatty acid metabolism and a major product of amino acid catabolism. Slide 3. Pyruvate Dehydrogenase and the Citric Acid Cycle are Located in the Mitochondria Glycolysis and the TCA cycle are metabolically linked by pyruvate. In contrast to glycolysis, which takes place in the cytoplasm, the citric acid cycle (TCA cycle) is localized in the mitochondria. The metabolic linkage between the two pathways is the pyruvate molecule. Under aerobic conditions, pyruvate is the endproduct of glycolysis. Pyruvate can diffuse into the mitochondria, where it is converted to acetylCoA by the action of the pyruvate dehydrogenase complex. The product of pyruvate dehydrogenase is acetylCoA, which can then enter the TCA cycle. Slide 4. Change in Free Energy for the Conversion of Glucose to Carbon Dioxide This figure shows the overall free energy change that occurs during the conversion of glucose to carbon dioxide. About 20 percent of that free energy change occurs in the glycolysis pathway when glucose is metabolized to pyruvate. Another 20% occurs when 1 BioC 3021 Notes Robert Roon pyruvate is converted to acetylCoA and carbon dioxide by the pyruvate dehydrogenase complex. The remaining 60% of the energy drop occurs as acetylCoA is converted to carbon dioxide by the TCA cycle. There is an interesting correlation between loss of free energy in these pathways and the synthesis of ATP equivalents. About 17% of the ATP equivalents are produced by aerobic glycolysis, with another 17% resulting from the conversion of pyruvate to acetylCoA, and the remaining 65% are produced during acetylCoA metabolism in the TCA cycle. Slide 5. Artist’s Representation of a Mitochondrion. Mitochondria are subcellular organelles that are found in eukaryotic cells. Mitochondria have a porous outer membrane and a convoluted inner membrane that is impermeable to most polar and ionic materials. The electron transport and oxidative phosphorylation systems are immersed in the inner membrane. Slide 6. Electron microscopy of a mitochondrion. An electron microscopy image of a mitochondrion reveals a structure with a double membrane, the inner membrane being very convoluted. The micrograph corresponds nicely with the artist’s rendition that we have just seen. Slide 7. Pyruvate Dehydrogenase Complex -Pyruvate dehydrogenase is a complex of three enzymes that transforms pyruvate into acetyl-CoA. -The acetyl-CoA can then be used in the citric acid cycle for cellular respiration. -The complex links the glycolysis pathway to the citric acid cycle. -In eukaryotes, the complex is located in the mitochondrial matrix. -It consists of a total of 96 subunits, containing three types of protein. 2 BioC 3021 Notes Robert Roon Slide 8. Overall Reaction of the Pyruvate Dehydrogenase Complex. This reaction scheme lists the reactants, cofactors, and products of the pyruvate dehydrogenase reaction. The NADH produced in the reaction serves as an electron donor for oxidative phosphorylation. The other product, acetylCoA, is the primary substrate for the TCA cycle. Slide 9. Pyruvate Dehydrogenase Reaction Mechanism. In the series of reactions catalyzed by pyruvate dehydrogenase, the pyruvate is first attached to thiamine pyrophosphate, where it is converted to a two-carbon hydroxyethyl intermediate with the release of carbon dioxide. That two-carbon intermediate is passed to lipoate and simultaneously oxidized to an acetyl derivative. The acetyl group is then transferred to coenzyme A. During the oxidation of the substrate, the cofactor lipoate is reduced. The reduced lipoate is reoxidized by FAD, which is converted to FADH2. The FADH2 then passes its electrons on to NAD+ to form NADH. Slide 10. Thiamine Pyrophosphate (TPP). The first cofactor involved in the pyruvate dehydrogenase reaction is thiamin pyrophosphate. The organic part of this cofactor is thiamine, which must be supplied in the diet. Humans have the capacity to add the pyrophosphate group to thiamine to produce the functional cofactor. The part of the coenzyme involved in catalysis is the thiazole ring, and the functional site of catalysis is the carbon atom at the top of the thiazole ring that lies between a nitrogen atom and a sulfur atom. The delocalized aromatic nature of the ring system makes the hydrogen atom on that carbon more acidic than usual. Slide 11. Pyruvate and Thiamine Pyrophosphate 3 BioC 3021 Notes Robert Roon In this figure, the pyruvate molecule approaches the carbon atom at the top of the thiazole ring (shown in red). It is that thiazole carbon that will become the point of attachment between the thiazole ring and the carbonyl group of pyruvate. Slide 12. Pyruvyl-Thiamine Pyrophosphate. When that upper thiazole carbon is deprotonated, it has two unpaired electrons that can attack the carbonyl carbon of pyruvate. That attack results in the formation of a covalent bond between the thiazole carbon and pyruvate. Two electrons from between the carbonyl carbon and oxygen move onto the carbonyl oxygen, making it negatively charged. A proton from the solvent can neutralize that charge, thereby producing a hydroxyl group. The structure of the resulting pyruvyl-thiamine pyrophosphate intermediate is shown on this slide. Electrons from the negative oxygen of the carboxylate anion of this intermediate move between that oxygen and the adjacent carbon. Electrons between the carboxylate carbon and C-2 move down between the C-2 carbon and the thiazole carbon. This releases carbon dioxide. An electron pair from the double bond between the thiazole carbon and nitrogen moves onto the thiazole nitrogen and neutralizes its positive charge. Slide 13. Release of Carbon Dioxide from Pyruvyl-Thiamine Pyrophosphate. In the next step, the carboxyl group of pyruvate is released as carbon dioxide. An electron pair from that carboxyl group moves between the hydroxyl carbon and the thiazole carbon to form a double bond. Slide 14. Hydroxyethyl-Thiamine Pyrophosphate. This hydroxyethyl intermediate is formed when pyruvyl-thiamine pyrophosphate is decarboxylated. It is one form of hydroxyethyl- Mayo Clinic 7/22/11 4:53 PM thiamine pyrophosphate. In the next step of the pyruvate Comment: This term is a little vague 4 BioC 3021 Notes Robert Roon dehydrogenase mechanism, this two carbon intermediate is transferred to lipoamide. Slide 15. Pyruvate Dehydrogenase Mechanism. This diagram shows that the hydroxyethyl group is passed from thiamine pyrophosphate to lipoamide. At the same time, it is oxidized. Although the hydroxyethyl derivative looks like an alcohol, because of its attachment to the thiamine pyrophosphate, it is at the oxidation level of an aldehyde. When the hydroxyethyl group is passed to lipoamide, it is simultaneously oxidized to an acetyl group, which is at the oxidation level of a carboxylic acid. The reaction intermediate at this point is an acetylthioester derivative. Slide 16. Lipoamide in Oxidized and Reduced Form. The lipoamide coenzyme is covalently bound to a protein of the pyruvate dehydrogenase complex. The lipoamide can exist in an oxidized or reduced form. The oxidized form has a disulfide ring structure, and the reduced form is an open chain structure with two reduced sulfur groups. The conversion to the reduced form involves the addition of two electrons and two protons to the oxidized form. In the pyruvate dehydrogenase reaction, that conversion occurs when the acetylthioester is formed. When the hydroxyethyl intermediate is passed from thiamine pyrophosphate, it loses two electrons and two protons that are passed to lipoamide. The hydroxyethyl group is oxidized to an acetyl group and the lipoamide is reduced to dihydrolipoamide. That reduced dihydrolipoamide intermediate looks different than what you see here, because there is an acetyl group attached to one of its sulfur atoms. Slide 17. Pyruvate Dehydrogenase Mechanism. The final step in the pyruvate dehydrogenase mechanism involves the transfer of the acetyl group from lipoamide to coenzyme A. That reaction, which is catalyzed by dihydrolipoyltransacetylase, 5 BioC 3021 Notes Robert Roon produces acetylCoA, which is the end product of the whole reaction sequence. The acetylCoA is a thioester derivative with a very high energy of hydrolysis. This permits the acetyl group to be transferred to a number of different types of carbon acceptor molecules with a favorable thermodynamic potential. Sometimes, acetylCoA is referred to as “active acetate”. Slide 18. Coenzyme A Structure. Let’s take a minute to look at coenzyme A. This structure contains the elements of adenosine diphosphate connected to pantothenic acid. The pantothenic acid portion of the molecule is a vitamin that must be provided in the human diet. Humans have the metabolic pathways to construct coenzyme A from pantothenic acid. The working part of coenzyme A is the sulfhydryl group at the very end of the molecule. That sulfur becomes connected to and activates various acyl groups by means of a thioester bond. Slide 19. AcetylCoA Structure. Here is acetylCoA with the acetyl group in thioester linkage to the sulfur group of the coenzyme A. Slide 20. Pyruvate Dehydrogenase Mechanism. There is one more component of the reaction to be considered before we proceed to the TCA cycle. That is, the reoxidation of dihydrolipoamide to lipoamide. The dihydrolipoamide is produced when the hydroxyethyl group is oxidized to an acetyl group. It must be converted back to oxidized lipoamide before the oxidation-reduction reaction can be repeated. That conversion is catalyzed by dihydrolipoyl dehydrogenase. In the reoxidation of dihydrolipoamide, two electrons and two protons are transferred to FAD to form FADH2. The FADH2 is then converted back to FAD when two electrons and one proton are transferred to NAD+ to form NADH (with the other proton being released into the solvent). Slide 21. Flavine Adenine Dinucleotide Structure. 6 BioC 3021 Notes Robert Roon Here is a diagram of FAD for your review. The FAD coenzyme contains the elements of adenosine diphosphate connected to ribitol, which in turn is connected to an isoalloxazine ring system. (The ribitol isoalloxazine pair, collectively known as riboflavin, constitutes one of the B vitamins.) It is the isoalloxazine ring that is involved in catalysis. Slide 22. Conversion of FAD to FADH2. During the oxidation of dihydrolipoamide, FAD is converted to FADH2. This reaction involves two electrons and two protons being transferred to FAD. The two electrons enter the isoalloxazine ring and the two protons sit on the two nitrogen atoms coded in blue. Slide 23. Citric Acid Cycle We are now ready to turn our attention to the citric acid cycle. It carries this name because citric acid is the first product. It is also called the tricarboxylic acid cycle because citrate and isocitrate have three carboxyl groups. Finally, it is called the Krebs cycle after its discoverer, Sir Hans Krebs. Slide 24. Sir Hans Krebs. Here is a photograph of the distinguished researcher who not only discovered the TCA cycle, but also discovered the urea cycle. Those were quite remarkable accomplishments, especially at a time when radioisotopes were not yet available. Slide 25. Acetate Ion Compared with Acetyl Group AcetylCoA is the intermediate that enters the TCA cycle. This slide should reacquaint you with some nomenclature used for acetic acid and its derivatives. The term acetic acid refers to the protonated form of the two carbon carboxylic acid. In its deprotonated, negatively charged, anionic form, the molecule is referred to as acetate ion. When the acetate is attached to some organic or inorganic group other than a hydroxyl group, it is called 7 BioC 3021 Notes Robert Roon an acetyl group. An acetyl group is the two carbon member of the more general class of acyl groups. Slide 26. TCA Cycle Preview The TCA cycle converts the two carbons of acetylCoA into two molecules of carbon dioxide. The net products of the TCA cycle are 2 carbon dioxide, 8 electrons (6 as NADH and 2 as FADH2) and 1 GTP. Slide 27. The Chemical Logic of the TCA Cycle Pyruvate decarboxylation yields acetylCoA, which contains two carbon molecules. Each carbon will be oxidized all the way to carbon dioxide in the TCA cycle, but it is very hard to cleave between the two carbons of acetylCoA. However, it is easier to cleave between two carbons that are α and β to a carbonyl group. So, acetylCoA is first condensed with oxaloacetete to make the six carbon compound citrate. The citrate is then rearranged in order to perform a β-cleavage reaction. Slide 28. Sequence of TCA Cycle Product Formation. A more detailed diagram of the TCA cycle shows the relative sequence in which the various products are synthesized. NADH (two electrons) and carbon dioxide are removed in the conversion of a C6 compound to a C5 compound. NADH (two electrons) and carbon dioxide are removed in the subsequent conversion of a C5 compound to a C4 compound. The cycle is completed by oxidation reactions involving C4 intermediates, which produce one NADH (two electrons), one FADH2 (two electrons), and one GTP. Slide 29. Loss of Carbons in the TCA Cycle If you follow the carbon atoms from acetylCoA through a single round of the TCA cycle, you will see that the two carbons lost as carbon dioxide in each round are NOT the two that entered the cycle in that round. The carbons that are lost come initially from 8 BioC 3021 Notes Robert Roon oxaloacetate. In succeeding rounds, the acetylCoA carbons will be lost. Slide 30. Citrate Synthase The first reaction of the TCA cycle is catalyzed by citrate synthase. In the reaction, the methyl carbon of acetylCoA condenses with the carbonyl carbon of oxaloacetate. In the course of the reaction, the CoA group is released. The cleavage of the thioester linkage of acetylCoA releases energy and helps to drive the reaction toward product formation. The reaction product is citrate. In addition to its role in the TCA Cycle, citrate is found in high levels in citrus fruits and is a ubiquitous ingredient in processed foods and drinks. The presence of citrate imparts a pleasing tartness to many beverages and foods. Slide 31. Mechanism of Citrate Synthase The citrate synthase reaction involves a nucleophylic attack of a pair of electrons from the methyl group of acetylCoA on the carbonyl carbon of oxaloacetate. A bond is formed between the two compounds, creating the six carbon tricarboxylic acid, citrate. During the reaction, two electrons from the carbonyl double bond jump onto the carbonyl oxygen. That oxygen is protonated by a hydrogen ion from the solvent, producing a hydroxyl group. Sometime during the reaction, the CoA group is cleaved, giving the overall process a favorable thermodynamic balance. The two carbons that originated with acetylCoA are colored red so that you can follow their fate as the cycle progresses. Slide 32. Aconitase. The second reaction of the TCA Cycle is catalyzed by aconitase. This enzyme catalyzes a two step process with relatively low transition state energies to move the hydroxyl group of citrate down from the middle carbon to the carbon below it. The reaction involves a dehydration reaction, which removes the elements of water to create a double bond, followed by a hydration reaction 9 BioC 3021 Notes Robert Roon that adds the elements of water back into the double bond and establishes a hydroxyl group on the lower carbon. The product of this isomerization sequence is called isocitrate. If this reaction were conducted chemically in the absence of an enzyme, the new hydroxyl group would have a significant chance of ending up on the carbon above or the carbon below the middle carbon. Citrate is a symmetrical compound, and chemical reactions generally would not distinguish between the upper and lower part of the molecule. However, because enzymes have binding sites that are asymmetric, they often discriminate between two parts of a symmetrical molecule. That selectivity also has something to do with the number of points of attachment between substrate and enzyme—enzymes that have at least three points of attachment are able to discriminate between different parts of symmetrical compounds. So the result is that, although the starting material, citrate, is a symmetrical compound, the reaction product, isocitrate, is asymmetric with a chiral center. The upper part of isocitrate that originated from acetylCoA does not carry the hydroxyl group. Slide 33. Isocitrate Dehydrogenase. The next reaction, catalyzed by isocitrate dehydrogenase, involves an oxidative decarboxylation. (Any time you see the name dehydrogenase, you can be certain that the reaction involves oxidation-reduction.) In the first step of the reaction, the hydroxyl group of isocitrate is oxidized to a ketone, coupled to the reduction on NAD+ to NADH. That produces oxalosuccinate, which has a carboxyl group that is located β to a carbonyl group. It turns out that β-keto acids are relatively unstable and can be easily decarboxylated. That decarboxylation reaction releases carbon dioxide and yields a five carbon product, α-ketoglutarate. This is the first reaction in the TCA cycle in which NADH is produced. The NADH will donate two electrons to the electron transport system that will lead to the production of ATP. 10 BioC 3021 Notes Robert Roon Slide 34. α-Ketoglutarate Dehydrogenase. The next reaction is another oxidative decarboxylation, but in this reaction the product is also condensed with Coenzyme A. This step, which is catalyzed by α-ketoglutarate dehydrogenase, should remind you of pyruvate dehydrogenase reaction. In fact, the mechanism and cofactors are very similar for both reactions. If you look at the structure of α-ketoglutarate, you will see that the lower three carbons mirror the structure of pyruvate. In the decarboxylation phase of the reaction sequence, carbon dioxide is released, and the product succinyl-CoA now has four carbons. This is the second reaction in the TCA cycle that produces NADH. Slide 35. Succinyl CoA Synthase. Next, we have a clever little reaction that manages to use the energy from hydrolysis of a thioester bond to squeeze out an ATP equivalent. SuccinylCoA is a thioester, and such compounds have a very high standard state free energy of hydrolysis (ΔG0’). The energy of hydrolysis from the release of CoA is coupled to the production of a GTP molecule. (Keep in mind that GTP is energetically equivalent to ATP.) The other product of the reaction is succinate, a four carbon dicarboxylic acid. Slide 36. Conversion of Succinate to Oxaloacetate. The next series of reactions all involve four carbon intermediates and lead to the conversion of succinate back to the starting material, oxaloacetate. In the first step, succinate is oxidized to fumarate by succinate dehydrogenase. This is the only reaction in the TCA cycle that is catalyzed by a membrane bound enzyme complex. We will come back to this point later when we consider the electron transport chain. This is the third oxidation-reduction reaction in the TCA cycle, but it differs from the previous oxidation-reduction reactions in two ways. First, it involves the removal of two protons and two 11 BioC 3021 Notes Robert Roon electrons from a carbon-carbon single bond to create a double bond. Second, it uses FAD as the oxidant instead of NAD+. The oxidation of a carbon-carbon single bond has a lower reducing potential than the previous oxidation reactions in the TCA cycle. We will see later that the product, FADH2, has a correspondingly lower potential than NADH for yielding ATP in oxidative phosphorylation. The product of the succinate dehydrogenase reaction is fumarate, which is a four carbon dicarboxylic acid with a trans double bond between the central carbon atoms. (If this double bond were in the cis configuration, we would have a different compound with a different name.) The fumarate is hydrated by the enzyme fumarase to produce L-malate. The addition of water adds a hydroxyl group to one carbon and a hydrogen atom to the other carbon. Fumarase is classified as a lyase because it can add water to a double bond. The product is designated as L-malate because it has a center of asymmetry at the carbon atom bearing the hydroxyl group, and that chiral center is in the L-configuration. The final reaction in the cycle is another dehydrogenase—this one is L-malate dehydrogenase, which catalyzes the oxidation of the alcohol group of L-malate to a form the ketone carbonyl group of oxaloacetate. This reaction produces the third and final NADH of the cycle. At this point, the reaction series has returned to the starting four carbon intermediate, oxaloacetate, and is ready to begin another cycle. Slide 37. TCA Cycle Compounds. Let’s use this slide to look at the carbon balance of the TCA cycle. The first reaction catalyzed by citrate synthase results in the condensation of a two carbon compound, AcetylCoA, and a four carbon compound, oxaloacetate, to yield a six carbon product, citrate. Citrate is then converted to isocitrate, whose decarboxylation yields the five carbon intermediate, α- 12 BioC 3021 Notes Robert Roon ketoglutarate plus carbon dioxide. The α-ketoglutarate is decarboxylated to the four carbon compound succinylCoA plus a second carbon dioxide. A series of reactions then gets the four carbon intermediate, succinylCoA, back to the starting four carbon compound, oxaloacetate. So the net carbon input into the cycle is--two carbons in as acetylCoA and four carbons in as oxaloacetate. The products of the cycle are two carbons released as carbon dioxide and four carbons regenerated as oxaloacetate—six carbons in and six carbons out! Slide 38. TCA Cycle Cofactors. Next, we look at the energy balance of the TCA cycle. One round of the TCA cycle uses 3 NAD+, 1 FAD, 1GDP and produces 3 NADH, 1 FADH2, 1 GTP. At this point, most of the energy from the acetylCoA is tied up in the reduced cofactors. In the next lectures, we will consider oxidative phosphorylation, in which that potential energy of NADH and FADH2 is converted into ATP. Slide 39. TCA Cycle Enzymes. For review, we have added the TCA cycle enzymes. There are eight of them. You should commit all of their names to memory, because this is a core reaction sequence in biochemistry, and your instructors just love these enzymes. Slide 40. Electrons from the TCA Cycle Energize Oxidative Phosphorylation. The electrons removed in the TCA cycle as NADH and FADH2 are channeled into oxidative phosphorylation. As the electrons pass through the electron transport chain, they drive the transmembrane flow of protons out of the mitochondrial matrix. This creates a proton gradient between the intermembrane space and the matrix. The downhill flow of electrons back into the matrix through the ATP synthase drives the formation of ATP 13 BioC 3021 Notes Robert Roon from ADP and Pi. Slide 41. TCA Cycle Provides Substrates for Biosynthetic Pathways. In addition to producing energy, the TCA cycle provides starting materials for a variety of biosynthetic pathway--for amino acid, purine, pyrimidine biosynthesis, etc. 14