BioC 3021 Notes - Lipid Metabolism PDF

BioC 3021 Notes Robert Roon Lecture 18: Lipid Metabolism Slide 1. Overview of Catabolism. We are now going to consider the left wing of the catabolism diagram. (I always like to start on the left rather than the right for reasons I will not expound upon...

BioC 3021 Notes Robert Roon Lecture 18: Lipid Metabolism Slide 1. Overview of Catabolism. We are now going to consider the left wing of the catabolism diagram. (I always like to start on the left rather than the right for reasons I will not expound upon here.) We are going to look at lipid catabolism, starting with triacylglycerols. The triacylglycerols provide another source of energy for living cells. Quantitatively, triacylglycerols constitute the most important energy store in humans. Remember that carbohydrates stored as glycogen constitutes less than one day’s supply of energy. In contrast, the fats stored as triacylglycerols contain on an average enough energy to fuel basal metabolism in humans for one or two months. Slide 2. Lipid Metabolism Summary The general pattern of lipid metabolism is summarized on this slide. When energy is needed, lipid catabolism occurs. The triacylglycerols from the diet or from lipid stores can be broken down by lipases to fatty acids and glycerol. The fatty acid components of triacylglycerols are converted to acetylCoA by the b-oxidation pathway. The acetylCoA can enter the TCA cycle or can serve as a starting material for a number of biosynthetic pathways. The glycerol component is integrated into glycolysis or gluconeogenesis by a short series of reactions that feed into these pathways. On the anabolism side, lipid biosynthesis occurs when there is excess energy. The fatty acids can be synthesized from acetylCoA, and glycerol can be produced from glycolysis intermediates. The two components can be combined in ATP-dependent reactions to form triacylglycerols. Slide 3. Lipid Digestion in the Lumen of the Intestine. Most dietary lipids are ingested as triacylglycerol compounds. The 1 BioC 3021 Notes Robert Roon dietary lipids are emulsified in the intestine by bile acids secreted from the gall bladder. Pancreatic lipases hydrolyze the triacylglycerols into free fatty acids and 2-monoglycerides in the lumen of the intestine. Slide 4. The Digestion and Absorption of Triacylglycerols. This diagram shows the pathway of dietary triacylglycerol digestion from the lumen of the intestine to the lymph system. -The triacylglycerols are hydrolyzed into free fatty acids and 2- monoglycerides in the lumen of the intestine. -The 2-monoglycerides and fatty acids are transported into the enterocytes, where they are reassembled into triacylglycerols and packaged into large chylomicron complexes. -The chylomicrons are exported from the cell and transported in the lymph system to adipocyte cells. -The adipocytes store the lipid as triacylglycerols until there is a need for energy. Slide 5. Photomicrograph of an Adipocyte Cell. A photomicrograph of an adipocyte cell reveals that the lipids are stored in large vacuoles, which take up the majority of cell volume. The vacuoles are so large that they sometimes displace the nucleus to the periphery of the cell. Slide 6. Scanning Electron Micrograph of an Adipocyte Cell. Another look at an adipocyte using a scanning electron microscope reveals the same spherical shape with a bulging nucleus on one side of the cell. Slide 7. Lipid Mobilization in Adipose Tissue. In adipose tissue, the hydrolysis of stored triacylglycerol molecules is initiated by hormones such as adrenaline, glucagon, and ACTH. The triacylglycerol lipase is activated by an enzymatic cascade that is triggered by a hormone binding to the appropriate receptor. 2 BioC 3021 Notes Robert Roon After cleavage of the first fatty acid by the triacylglycerol lipase, the second and third fatty acids are also removed from the diacyl- and monoacylglycerol molecules. If this reaction takes place in adipose tissue, the fatty acids are released into the blood and are carried to other tissues in a complex that includes the blood protein albumin. Some of the glycerol is also released into the blood and can feed into glycolysis or gluconeogenesis in other tissues. Slide 8. Glycerol Is Converted to Glycolysis or Gluconeogenesis Intermediates. The glycerol that is released from adipose tissue is taken up in other tissues, where it is integrated into glycolysis or gluconeogenesis by a two step pathway. First, an ATP-dependent kinase reaction yields glycerol 3-phosphate. Then, the glycerol 3- phosphate is oxidized to dihydroxyacetone phosphate by an NAD+ dependent dehydrogenase. Slide 9. Acyl vs Acetyl Group This is a good time to distinguish between two terms that are used to designate functional groups that are derived from carboxylic acids. The term acetyl specifically refers to a two carbon group with carbonyl and methyl function. Acetyl groups can be viewed as derivatives of acetic acid. The term acyl is more broadly defined and encompasses a range of homologous carboxylic acid derivatives. This includes the acetyl group plus other carboxylic acid derivatives that have one or many methylene groups. Slide 10. Fatty Acid Activation. Fatty acids are carried in the blood from adipose tissues in association with serum albumin protein. The fatty acids are taken into the cytosol of target cells, where they are activated by being converted to acylCoA derivatives. The conversion of a fatty acid into an acylCoA intermediate is a thermodynamically unfavorable process, because the acylCoA is a thioester derivative. Thioesters have a very high energy of hydrolysis, which means it takes a lot 3 BioC 3021 Notes Robert Roon of energy to make them starting with a carboxylic acid. To make the acylCoA derivative, the fatty acid is first activated using ATP. The intermediates in the process are inorganic pyrophosphate and a fatty acyl adenylate. (The pyrophosphate is cleaved to two inorganic phosphates with the release of energy, a pattern that we have previously noted, which contributes to the overall thermodynamic favorability of the reaction.) The acyl adenylate intermediate is a mixed acid anhydride with a high energy of hydrolysis. When it is swapped for CoA in the next step, the reaction is thermodynamically neutral. The formation of the acyl adenylate intermediate thus provides a way of using ATP to facilitate the synthesis of a high energy CoA product from a free fatty acid and CoA. Slide 11. Fatty Acyl Transport from Cytosol to Mitochondria. Fatty acylCoA derivatives are synthesized in the cytosol of the cell, but the oxidation of such derivatives takes place in the matrix of the mitochondria. An elaborate little dance must take place to get the fatty acylCoA into the mitochondrial matrix. In the cytosol, the activated CoA derivative is first transferred to carnitine, a polar carrier molecule. The fatty acyl carnitine derivative is then transported through the mitochondrial inner membrane into the matrix. In the matrix, the fatty acyl group is transferred from carnitine back to CoA. Slide 12. Fatty Acyl Transfer from CoA to Carnitine. The transfer of a fatty acyl group from fatty acylCoA to carnitine occurs in the cytosol and is catalyzed by an acyl transferase enzyme. The reaction is energetically neutral, and thus is reversible. The fatty acyl group is transferred from the thiol group of CoA to a hydroxyl group (noted in red) on carnitine. Slide 13. Carnitine Cycle. The overall carnitine cycle is outlined on this slide. The activated 4 BioC 3021 Notes Robert Roon CoA derivative is first transferred to carnitine. The fatty acyl carnitine derivative is then transported through the mitochondrial membrane into the matrix. In the matrix, the fatty acyl group is transferred from carnitine back to CoA. Then the carnitine is transported back into the cytosol. The transporter that accomplishes this exchange process is an integral membrane protein. It is an antiporter that exchanges an acyl carnitine for a free carnitine—the acyl carnitine comes into the matrix, and the free carnitine goes back out into the cytosol on the same transport protein. Slide 14. The β-Oxidation Cycle. One whole cycle of β-oxidation is illustrated on this slide. The process begins with a fatty acylCoA derivative with n carbon atoms, and it ends with a fatty acyl derivative with n minus two carbon atoms. The two missing carbon atoms are released as acetylCoA. These oxidation cycles are repeated—each cycle shortening the fatty acylCoA by two carbons and releasing an acetylCoA. In the final cycle, a four carbon acylCoA (butyrylCoA) is converted to two molecules of acetylCoA. The β-oxidation process starts with an oxidation-reduction reaction. In the reaction, catalyzed by an acylCoA dehydrogenase, a saturated acylCoA is converted to an unsaturated product. The new double bond, which is in the trans configuration, is introduced between the α and β carbons (coded in red). The cofactor in the reaction is FAD, which is reduced to FADH2. The second reaction involves the addition of water to the double bond, forming a hydroxyl group on the β carbon. The reaction is catalyzed by enol-CoA hydratase. Note that the carbon carrying the hydroxyl group is now a new chiral center in the L-form. In the third step, the hydroxyl group is oxidized to a ketone 5 BioC 3021 Notes Robert Roon coupled to the reduction of NAD+ to NADH. The enzyme that catalyzes the reaction is L-3-hydroxyacyl CoA dehydrogenase. The final step in the cycle is catalyzed by β-ketothiolase. In this reaction, a CoA molecule (coded in green) attacks the β carbon of the fatty acylCoA. The fatty acylCoA loses two carbons that are released as acetylCoA. The thiolytic cleavage is facilitated by the presence of the carbonyl group on the β carbon of the acylCoA. That β carbon becomes the site of attachment of the incoming CoA molecule to the shortened fatty acylCoA. The major fate of the acetylCoA from β -oxidation is entry into the TCA cycle. Because the acetylCoA is produced in the matrix of the mitochondria and is used by the TCA cycle in the same place, that process is facilitated. Slide 15. Compare β-Oxidation and TCA cycle. Here is another example of the potential of “learning by analogy”. The β-oxidation pathway and the TCA cycle both use common reactions that occur in the same sequence. That sequence is: -AcylCoA dehydrogenase for β-oxidation vs. succinate dehydrogenase for the TCA cycle. Both reactions use FAD as a cofactor and convert a single bond into a double bond. -EnolCoA hydratase vs. fumarase. Both reactions add water to a double bond to produce a hydroxyl group. -3-L-hydroxy-acylCoA dehydrogenase vs. L-malate dehydrogenase. Both reactions use NAD+ to convert a hydroxyl group to a ketone. The point is, that nature has given students of biochemistry a gift here. You do not have to learn two completely different pathways. If you memorized the TCA cycle, then learning β-oxidation should be “duck soup”, because it is essentially the same pathway with 6 BioC 3021 Notes Robert Roon slightly different surrounding structures. Slide 16. ATP Yield from Fatty Acid Oxidation. The oxidation of fatty acids is a catabolic process that has the goal of producing energy. So the question arises, “How much ATP can you get from the oxidation of a fatty acid?” And as luck would have it, we are here to answer our own question. We can divide the answer into two parts. -One cycle of β-oxidation yields 4 ATP’s--1.5 ATP’s for the FADH2 and 2.5 ATP’s for the NADH. In a round of β-oxidation, there is one acetylCoA produced. The acetylCoA enters the TCA cycle and yields 10 ATP’s--7.5 ATP’s from the 3 NADH’s, 1.5 ATP’s from the FADH2, and one ATP equivalent from GTP. Slide 17. Total ATP Produced from Stearate. Let’s look at the complete oxidation of a fatty acid to carbon dioxide. We arbitrarily pick stearic acid (18 carbons, no double bonds) as the starting material. -It costs 2 ATP equivalents to activate stearic acid to the acylCoA derivative. (Remember that in that reaction, ATP is converted to AMP and inorganic pyrophosphate. That is the energy equivalent of 2 ATP’s going to ADP and Pi.) Those two ATP equivalents constitute the only energy expended in the process. -There are 8 rounds of β-oxidation, with each round producing 4 ATP’s, so this phase yields a total of 32 ATP’s. (You might be tempted to think that there are 9 rounds of β-oxidation from a C-18 fatty acid, but remember that the eighth round, starting with a four carbon acylCoA, produces two acetylCoA’s.) 7 BioC 3021 Notes Robert Roon -There are 9 acetylCoA’s fed into the TCA cycle, and each one produces 10 ATP’s for a total of 90 ATP’s. Minus 2, plus 32, plus 90 gives you a net yield of 120 ATP’s. WOW! Slide 18. Caloric Content of Carbohydrate vs. Fat. Now you will probably be asking another question (And if you don’t, we will again ask it for you.). That is, how do carbohydrates and fats compare as sources of energy? The answer is that fats are more energy rich than carbohydrates. When we calculate the moles of ATP produced per gram, it turns out that glucose produces 0.17 mol/g, and stearate produces 0.42 mol/g. That converts to 4 kcal/g for glucose and 9 kcal/g for stearate. In general, on a per gram basis, fats produce more than twice the energy as carbohydrates. This reflects the fact that fats are more highly reduced than carbohydrates, and a more reduced compound typically yields more energy in biological oxidation reactions. Slide 19. Enoyl-CoA Isomerase. Up until now, we have only considered the β-oxidation of saturated fatty acids. There are some minor complications in the oxidation of unsaturated fatty acids. We will use the oxidation of oleic acid (an 18-carbon fatty acid with one cis double bond) as an example. This fatty acid has its double bond between carbons 9 and 10. The first three rounds of β-oxidation proceed normally, because all the bonds are saturated. However, on round four, cis- Δ3-enoyl-CoA is the substrate. The problem with cis- Δ3-enoyl- CoA is that its cis- Δ3 double bond should be trans- Δ2 in order for oxidation to continue. The enzyme enoyl-CoA isomerase makes that conversion, and from there on β-oxidation can proceed normally. 8 BioC 3021 Notes Robert Roon Because the oleate was already unsaturated, there will be one less FADH2 produced than for the equivalent saturated fatty acid. The consequence is that the unsaturated compound will yield 1.5 less ATP’s than its saturated analog. Slide 20. Isomerization of a Cis-Δ 4 Double Bond. Another complication arises in the oxidation of linoleic acid, a C- 18 fatty acid that has two double bonds. The first double bond occurs between carbons 9 and 10 and is treated to the same isomerization sequence as shown on the previous slide. At the second double bond, a cis- Δ4-enoyl-CoA is the substrate. The conversion of the cis- Δ4 into the trans- Δ2 bond takes three reactions. Two reactions convert the cis- Δ4 double bond to a trans- Δ3. Then an enol-CoA isomerase converts the trans- Δ3 double bond to a trans- Δ2. And with that, we will leave fatty acid oxidation. Slide 21. Structure of Propionyl CoA. Most fatty acids contain an even number of carbon atoms, and produce the two carbon compound acetyl CoA as the only oxidation product. However, there are a significant number of fatty acids that contain an odd number of carbons. During the β- oxidation process, these odd-chain fatty acids produce acetyl CoA until the last cycle. In the last cycle, the products are one molecule of acetyl CoA and one molecule of the three carbon compound propionyl CoA. There is a special metabolic pathway for the breakdown of propionyl CoA. Slide 22. Propionyl CoA (C3) is Converted to Succinyl CoA (C4). The propionyl CoA is integrated into mainstream metabolism by conversion to an intermediate of the TCA cycle, succinyl CoA. This process requires three metabolic steps. 9 BioC 3021 Notes Robert Roon -In the first step, propionyl CoA is carboxylated by a biotin- dependent enzyme to yield D-methylmalonyl CoA. -The second reaction converts the carboxylated product from D- to L-methylmalonyl CoA. -In the third reaction, a vitamin B12 dependent enzyme catalyzes the isomerization of L-methylmalonyl CoA to succinyl CoA. Slide 23. The Structure of Coenzyme B12. This figure shows the structure of coenzyme B12. The cofactor has a polyporphyrin ring system similar to that of the hemes, but the central metal ion in B12 is cobalt. Vitamin B12 dependent enzymes generally catalyze very exotic isomerization reactions, in which carbon atoms are moved from one place to another. Slide 24. Ketone Body Formation. Ketone bodies are produced when there is a need for energy in various tissues, or when the rate of the TCA cycle is inadequate to utilize all of the acetylCoA produced by fatty acid oxidation. Slide 25. Production of Ketone Bodies from Acetyl CoA. Under most conditions, acetyl CoA is produced from glycolysis and fatty acid oxidation at a rate that can be accommodated by the TCA cycle. Under these circumstances, most of the acetyl CoA is converted to carbon dioxide. The acetyl CoA is diverted from the TCA cycle to the formation of ketone bodies (short chain fatty acids) as a normal part of metabolism, when there is a specific need for extra energy in various tissues, such as heart muscle. The production of ketone bodies also occurs when the rate of the TCA cycle is inadequate to utilize all of the acetylCoA produced by fatty acid oxidation. Such conditions include starvation, uncontrolled diabetes, or as a result of some low carbohydrate diets in which fatty acids are released very rapidly. 10 BioC 3021 Notes Robert Roon Slide 26. Conversion of Acetyl CoA to Ketone Bodies. There are three products of acetyl CoA metabolism that are collectively referred to as ketone bodies. These metabolites are acetoacetate, β-hydroxybutyrate, and acetone. Slide 27. Ketone Body Synthesis Steps 1 and 2. In the first two steps of ketone body synthesis, two units of acetyl CoA react to form acetoacetylCoA, and a third acetyl CoA unit reacts to produce HMG-CoA. These are also the first two steps in cholesterol biosynthesis. Slide 28. Ketone Body Synthesis Steps 3 and 4. In the third step of ketone body synthesis, an acetyl CoA unit is then removed from HMG-CoA, releasing acetoacetate. The acetoacetate is converted enzymatically into β-hydroxybutyrate, and is decarboxylated nonenzymatically to give acetone. Slide 29. Overview of Catabolism. As we start our study of the synthesis of fatty acids, we take one last look at this chart of catabolism. We use this figure to remind you that every pathway has to release energy, or it would not proceed in the forward direction. We have just looked at β- oxidation of fatty acids, and we know that the catabolic pathway that converts fatty acids to acetylCoA releases energy. Now, some of the energy in fatty acids gets trapped as FADH2 and NADH, and those reduced nucleotides eventually yield ATP. However, in addition to forming reduced nucleotides, some energy is released as heat. That is the consequence of the pathway being thermodynamically favorable (having a negative DG). OK! So, if the β-oxidation pathway is thermodynamically favorable (as it has to be), and the pathway for fatty acid biosynthesis is also thermodynamically favorable (as it also has to 11 BioC 3021 Notes Robert Roon be), and the two pathways connect the same two compounds... THEN the two pathways have to have some differences. That is, there must be some way to introduce extra energy into the biosynthetic pathway so that it is energy releasing, which it would not be if it were the exact reversal of the β-oxidation pathway. Keep this in mind as we look at the biosynthesis of fatty acids. Slide 30. Comparison of Fatty Acid Biosynthesis vs. β- Oxidation. This table shows a number of differences between fatty acid biosynthesis and β-oxidation. Three of these differences contribute to the favorable thermodynamics of both pathways. The differences that change the energetics are: - Fatty acid synthesis involves a biotin dependent carboxylation reaction, but β-oxidation does not. - A malonic acid derivative is a participant in fatty acid biosynthesis, but not in β-oxidation. - All of the oxidation-reduction reactions in fatty acid biosynthesis use NADH, but one of the oxidation-reduction steps in β-oxidation uses FAD+. In addition to the energy related differences, there are other contrasts between the two systems. - As the name implies, β-oxidation involves oxidation, while fatty acid synthesis involves reduction. - The acyl carrier for β-oxidation is CoA, but for fatty acid synthesis, it is an acyl carrier protein (ACP). - The hydroxyl intermediates in the two processes have a different chirality. Slide 31. The β-Oxidation Cycle. 12 BioC 3021 Notes Robert Roon Take another look at the β-oxidation cycle. If we were to run the β- oxidation cycle backwards, there would be a sequence in which: -AcetylCoA condenses with an acylCoA to form a β-ketone. -The β-ketone is reduced to a hydroxyl group by a dehydrogenase. -The hydroxyl group is dehydrated to form a double bond by a (de)hydratase. -The double bond is reduced to a single bond by a dehydrogenase. That is essentially the sequence that we will now see in fatty acid biosynthesis. Slide 32. Fatty Acid Biosynthesis. The sequence of reactions for the β-oxidation cycle running backwards is the same as the sequence for fatty acid biosynthesis running in the forward direction. In the biosynthesis process: -MalonylACP (the replacement for acetylCoA) condenses with an acylACP derivative to form a β-ketone. -The β-ketone is reduced to a hydroxyl group by a reductase. -The hydroxyl group is dehydrated to form a double bond by a dehydratase. -The double bond is reduced to a single bond by a reductase. Slide 33. Comparison of CoA and Acyl Carrier Protein. On this slide, we compare the structure of CoA with the acyl carrier protein. The interesting thing is, that the working ends of both molecules, near the thiol groups, are identical. At the opposite end away from the thiol group, CoA has an ADP residue, whereas the acyl carrier protein has a small protein (molecular weight about 10,000 daltons.) The attachment to the protein portion of ACP is through a serine hydroxyl group. 13 BioC 3021 Notes Robert Roon Slide 34. AcetylCoA Carboxylase. In the first step of fatty acid biosynthesis, acetylCoA is carboxylated to form malonylCoA. The reaction is catalyzed by acetylCoA carboxylase. The acetylCoA carboxylase enzyme contains a covalently bound coenzyme, biotin, which serves as a carrier of an activated bicarbonate molecule. The reaction takes place in two stages. In the first stage, bicarbonate becomes covalently attached to the coenzyme to form a carboxybiotin intermediate. The attachment of bicarbonate to the biotin coenzyme is coupled to the cleavage of ATP to ADP and Pi. In the second stage of the reaction, the activated carboxyl group is transferred from biotin to acetylCoA to form malonylCoA. The formation of malonylCoA contributes toward making fatty acid biosynthesis an energy releasing process. The carboxyl group that is added to acetylCoA to form malonylCoA will be released during a subsequent step in the biosynthetic pathway. That decarboxylation is an energy releasing reaction, and it will help make the overall pathway thermodynamically favorable. Slide 35. Conversion of CoA Derivatives to ACP Derivatives. The second step of fatty acid biosynthesis actually consists of two parallel reactions. In one of these reactions, an acetyl group is transferred from acetylCoA to an acyl carrier protein by an acetyl transacylase enzyme. In the other reaction, a malonyl group is transferred from malonylCoA to an acyl carrier protein by a malonyl transacylase enzyme. The products of these reactions, acetylACP and malonylACP, will react with each other in the next step. Slide 36. Fatty Acid Synthesis. The complete cycle of fatty acid biosynthesis is outlined on this slide. There are four steps in this biosynthetic cycle, and as we saw before, these steps mimic the reversal of the β-oxidation cycle. 14 BioC 3021 Notes Robert Roon In the first reaction, catalyzed by a condensing enzyme, malonylACP reacts with acetylACP to give a four carbon β- ketoacylACP product. There are a lot of things happening in this seemingly simple reaction. First, a two carbon compound and a three carbon compound are condensing with each other to form a four carbon compound. Wait! Two and three equal five, not four. What happened to the fifth carbon atom? It turns out that, in the process of forming the condensation product, a carbon dioxide was released from the malonylACP. That not only accounts for the fifth carbon, but also explains where some of the energy comes from to make this reaction proceed toward product. The release of carbon dioxide is usually thermodynamically favorable, and this reaction is no exception to that rule. So, carbon dioxide release helps to drive this reaction toward completion. That explains why the acetylCoA was carboxylated to malonylCoA in the first place—not to add an extra carbon to the pathway, but to provide some additional energy, giving the overall pathway a negative DG. There is one additional feature of the first reaction that contributes to its favorable thermodynamics. In the process of forming the four carbon product, an ACP molecule is released. That ACP was attached to the acetyl group by a thioester bond, which has a high energy of hydrolysis. When the thioester bond is broken, energy is released as heat. Thus, that bond cleavage contributes towards the negative ΔG of the overall reaction. In the second reaction, the β-ketone group is reduced to an alcohol with NADPH serving as the reductant. That reaction is catalyzed by β-ketoacyl-ACP reductase. The use of NADPH in this reaction is typical of what occurs in anabolic oxidation-reduction reactions. Remember that NADH is generally used to generate ATP, and NADPH is used as a reductant in biosynthetic reactions. The hydroxyl group that is produced in this reaction is in the D- configuration. In β-oxidation cycle, the equivalent hydroxyl 15 BioC 3021 Notes Robert Roon intermediate was of the L-configuration. The third step involves a dehydration reaction. The elements of water are removed, creating a trans carbon-carbon double bond. The enzyme catalyzing this reaction is 3-hydroxyacyl-ACP dehydratase. In the final step of the cycle, enoyl-ACP reductase uses NADPH to reduce the double bond to a single bond. The product is a four carbon butyryl-ACP. That four carbon acyl-ACP can go on to condense with a malonylACP to initiate a second round of biosynthesis—the product of which would be a six carbon acyl- ACP. Each succeeding round of biosynthesis elongates the acyl- ACP by two carbon atoms. Because fatty acid biosynthesis occurs two carbons at a time, most naturally occurring fatty acids have an even number of carbon atoms. Slide 37. The Glyoxylate Cycle For the most part, we are focusing on mammalian biochemistry in this course, but we will now consider a process that does not take place in mammals. It is called the glyoxylate cycle. The glyoxylate cycle is an anabolic metabolic pathway occurring in plants, and several microorganisms, such as E. coli and yeast. This cycle enables organisms to use acetylCoA for the net synthesis of carbohydrates. It is absent in animals including humans. Its absence explains why humans can synthesize glucose from pyruvate and TCA cycle intermediates, but not from acetylCoA. Slide 38. Glyoxylate Cycle Reactions with Complete TCA Cycle Intermediates. The glyoxylate cycle makes use of many of the reactions of the TCA cycle, but it adds two new reactions. Those two additional reactions are shown here in the middle of the complete TCA cycle. 16 BioC 3021 Notes Robert Roon Slide 39. Glyoxylate Cycle Reactions. On this slide, we have removed the TCA cycle reactions that are not involved in the glyoxylate cycle. The first reaction that is unique to the glyoxylate cycle is catalyzed by isocitrate lyase. That reaction cleaves isocitrate into glyoxylate and succinate. The second unique reaction is catalyzed by malate synthase. In that reaction, glyoxylate condenses with acetylCoA to produce malate. When we combine these two reactions with other reactions of the TCA cycle, we get two four carbon products—succinate and malate—and both of these products can go on in the TCA cycle to produce any of the other four, or five, or six carbon intermediates. The genius of the glyoxylate pathway is that it provides a mechanism by which two-carbon acetylCoA units can produce four-carbon products. The glyoxylate cycle facilitates the net fixation of two-carbon reactants into four-carbon products— something that cannot be accomplished by the TCA cycle acting alone. Furthermore, one of the TCA cycle intermediates, oxaloacetate, can be used to produce phosphoenolpyruvate, and phosphoenolpyruvate can yield glucose via gluconeogenesis. The bottom line is, that bacteria can make glucose from acetylCoA. Mammals do not have the enzymes of the glyoxylate cycle. Without the two glyoxylate cycle enzymes, the TCA cycle absorbs the two carbons of acetylCoA and releases them both as carbon dioxide—there is no net fixation of the two carbons of acetate into four carbon products. That means there is something that can be achieved by a lowly bacterium, but not by a mighty mammal. We and all other mammals are unable to carry out the net synthesis of TCA cycle intermediates from acetylCoA. Furthermore, and of extreme importance, is the fact that mammals cannot accomplish the net synthesis of glucose from acetylCoA. In fact, any compound that yields acetate or acetylCoA as its only product cannot be used as a net source of glucose. That can cause problems during fasting or starvation, because the major energy 17 BioC 3021 Notes Robert Roon storage compounds in mammals are triacylglycerols. Critical levels of glucose must be maintained during starvation, but the fatty acids released from triacylglycerols cannot contribute to glucose synthesis. They can only produce acetylCoA. Slide 40 Cholesterol Biosynthesis We now turn to a consideration of cholesterol, which is the good guy/bad guy of lipids. Cholesterol has many necessary functions, including serving as an essential membrane component and as a precursor of steroid hormones and bile acids. On the other hand, cholesterol esters that accumulate in the arteries of old guys like me are the villains that cause atherosclerosis, leading to heart attacks and strokes. Slide 41. Origin of the Carbon Atoms of Cholesterol. The diagram shows that all of the carbons of cholesterol are derived from acetylCoA—the red carbons from the carboxyl group, and the black carbons from the methyl group. Cholesterol has three six-membered rings and one five-membered ring, none of which are aromatic. Slide 42. Summary of Cholesterol Biosynthesis. This diagram breaks the pathway of cholesterol biosynthesis into four arbitrary stages. Although it is not shown here, each of those stages involves multiple steps catalyzed by many different enzymes. The first stage begins with the two carbon compound, acetylCoA, and ends with a six carbon intermediate, mevalonate. The second stage begins with mevalonate and ends with a five carbon activated isoprene analog, isopentenyl-pyrophosphate. Next, in stage three, six isopentenyl-pyrophosphate units are polymerized to give a 30-carbon intermediate, squalene. And finally in stage four, squalene is converted to cholesterol by a series of rearrangements and ring closure reactions. Slide 43. Pathway from AcetylCoA to Mevalonate. 18 BioC 3021 Notes Robert Roon We mentioned before that acetylCoA occurs at a branch point in metabolism. In this case, it serves as the precursor of cholesterol. In the first reaction, two molecules of acetylCoA condense to form acetoacetylCoA. The acetoacetylCoA can then react with a third molecule of acetylCoA to give 3-hydroxy-3-methylglutaryl-CoA. The thioester carbonyl group of 3-hydroxy-3-methylglutaryl-CoA is then reduced to an alcohol yielding mevalonate. Notice that it takes two molecules of NADPH to convert the thioester (which is the equivalent of a carboxyl group) to an alcohol. That is a four electron reduction, hence the need for two molecules of NADPH. In the process, a CoA molecule is released. The driving force for this series of reactions is the release of two CoA units and the conversion of two NADPH molecules to NADP+. Slide 44. Conversion of Mevalonate to Geranyl Pyrophosphate. In the next series of reactions, the mevalonate is first converted to a pyrophosphate derivative. That takes two ATP’s going to ADP. The next reaction involves an ATP dependent dehydration and decarboxylation. Wow! All that stuff happening in one reaction— I am not going to burden you with the mechanism, primarily because I don’t know how it works—else I would happily burden you, and it would have very little effect on my conscience. The end result is a nice little five-carbon compound isopentenyl pyrophosphate, which has a carbon-carbon double bond at one end. Next, there is an isomerization of that new double bond. At that point, two five-carbon dimethylallyl pyrophosphate molecules condense to form a ten-carbon product, geranyl pyrophosphate, with the release of an inorganic pyrophosphate. This series of reactions is energized by the cleavage of three molecules of ATP, a decarboxylation reaction, and the release of a pyrophosphate. I am sure that the pyrophosphate is promptly cleaved to two inorganic phosphates to give the pathway a further boost. Slide 45. Formation of Farnesyl Pyrophosphate. In the next reaction, a ten carbon geranyl pyrophosphate condenses 19 BioC 3021 Notes Robert Roon with a five-carbon isopentenyl pyrophosphate to give a fifteen- carbon farnesyl pyrophosphate. An inorganic pyrophosphate molecule is released in the process, and its cleavage would again promote the forward reaction. Slide 46. Conversion of Farnesyl Pyrophosphate to Cholesterol. This slide summarizes the rest of the pathway, and thank goodness, most of the action takes place off the slide. I am really sick of this pathway by this point. In essence, two fifteen-carbon farnesyl pyrophosphate molecules condense to give a thirty-carbon product, squalene. Then there are a series of reactions that bond various carbon atoms together to form three fused six-membered rings and a fused five membered ring. Somewhere along the line, three carbons are lost as carbon dioxide, a double bond is introduced into one of the six-membered rings, and one of the rings is hydroxylated. We are left with the final 27 carbons of cholesterol—three fused six membered rings, one fused five membered ring, and an eight-carbon hydrocarbon side chain. This final phase of the pathway is energized by NADPH oxidation, pyrophosphate release, and some other stuff which will remain anonymous. Slide 47. Steroid Hormones Derived from Cholesterol. Here, we see some of the good guy features of cholesterol. It is a necessary precursor of steroid hormones including progesterone, testosterone, and estradiol. 20