Biochemistry 9e - Lipids & Metabolism - Campbell PDF

Chapter Outline 21-1 Lipids Are Involved in the Generation 12 21 and Storage of Energy 636 21-2 Catabolism of Lipids 636 Fatty Acid Transport...

Chapter Outline 21-1 Lipids Are Involved in the Generation 12 21 and Storage of Energy 636 21-2 Catabolism of Lipids 636 Fatty Acid Transport into Mitochondria 637 Lipid Metabolism Oxidation of Saturated Fatty Acids 639 21-3 The Energy Yield from the Oxidation of Fatty Acids 641 21-4 Catabolism of Unsaturated Fatty Acids 21-1 Lipids Are Involved in the Generation and Odd-Carbon Fatty Acids 643 and Storage of Energy Oxidation of Monounsaturated Fatty Acids 643 Oxidation of Polyunsaturated Fatty Acids 644 In the past few chapters we have seen how energy can be released 21-5 Ketone Bodies 646 by the catabolic breakdown of carbohydrates in aerobic and anaer- obic processes. In Chapter 16, we saw that there are carbohydrate 21-6 Fatty Acid Biosynthesis 647 polymers (such as starch in plants and glycogen in animals) that rep- 21A BIOCHEMICAL CONNECTIONS GENE resent stored energy, in the sense that these carbohydrates can be EXPRESSION Transcription Activators in Lipid hydrolyzed to monomers and then oxidized to provide energy in re- Biosynthesis 647 sponse to the needs of an organism. In this chapter, we shall see how First Steps in Fatty Acid Biosynthesis 648 the metabolic oxidation of lipids releases large quantities of energy 21B BIOCHEMICAL CONNECTIONS through production of acetyl-CoA, NADH, and FADH2 and how lip- NUTRITION Acetyl-CoA Carboxylase—A New Target in the Fight against Obesity? 650 ids represent an even more efficient way of storing chemical energy. Two-Carbon Addition by Fatty Acid Synthase 650 21C BIOCHEMICAL CONNECTIONS GENETICS A Gene for Obesity 655 21-2 Catabolism of Lipids 21-7 Synthesis of Acylglycerols and Compound The oxidation of fatty acids is the chief source of energy in the ca- Lipids 655 tabolism of lipids; in fact, lipids that are sterols (steroids that have a Triacylglycerols 655 hydroxyl group as part of their structure; Section 8-2) are not catab- Phosphoacylglycerols 656 olized as a source of energy but are excreted. Both triacylglycerols, Sphingolipids 658 which are the main storage form of the chemical energy of lipids, 21-8 Cholesterol Biosynthesis 659 and phosphoacylglycerols, which are important components of bio- From Acetyl-CoA to Cholesterol 659 logical membranes, have fatty acids as part of their covalently bonded HMG-CoA in Cholesterol Biosynthesis 660 Cholesterol as a Precursor to Other Steroids 661 structures. In both types of compounds, the bond between the fatty The Role of Cholesterol in Heart Disease: LDL and acid and the rest of the molecule can be hydrolyzed (Figure 21.1), HDL 664 with the reaction catalyzed by suitable groups of enzymes—lipases, in 21D BIOCHEMICAL CONNECTIONS ALLIED the case of triacylglycerols, and phospholipases, in the case of phos- HEALTH Atherosclerosis 667 phoacylglycerols (Section 8-2). 21-9 Hormonal Control of Appetite 669 Several different phospholipases can be distinguished on the ba- sis of the site at which they hydrolyze phospholipids (Figure 21.2). Phospholipase A2 is widely distributed in nature; it is also being ac- tively studied by biochemists interested in its structure and mode of action, which involves hydrolysis of phospholipids at the surface of micelles (Section 2-1). Phospholipase D occurs in spider venom and is responsible for the tissue damage that accompanies spider bites. Snake venoms also contain phospholipases; the concentration of phospholipases is particularly high in venoms with comparatively low concentrations of the toxins (usually small peptides) that are charac- teristic of some kinds of venom. The lipid products of hydrolysis lyse red blood cells, preventing clot formation. Snakebite victims bleed to death in this situation. 636 Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-2 Catabolism of Lipids 637 O O CH2OCR CH2OCR O H2O O H2O O Lipases Phospholipases CHOCR RCO– CHOCR Free O Glycerol fatty Glycerylphosphorylcholine O acids + CH2OCR CH2OPOCH2CH2N (CH3)3 Triacylglycerol O – Reuse Phosphatidylcholine or oxidation Figure 21.1 The release of fatty acids for future use. The source of fatty acids can be a triacylglycerol (left) or a phospholipid such as phosphatidylcholine (right). The release of fatty acids from triacylglycerols in adipocytes is controlled by lipases enzymes that hydrolyze lipids hormones. In a scheme that will look familiar from our discussions of carbohy- phospholipases enzymes that hydrolyze drate metabolism, a hormone binds to a receptor on the plasma membrane of phospholipids the adipocyte (Figure 21.3). This hormone binding activates adenylate cyclase, which leads to production of active protein kinase A (cAMP-dependent pro- tein kinase). Protein kinase phosphorylates triacylglycerol lipase, which cleaves the fatty acids from the glycerol backbone. The main hormone that has this effect is epinephrine. Caffeine also mimics epinephrine in this regard, which is one reason competitive runners often drink caffeine the morning of a race. Distance runners want to burn fat more efficiently to spare their carbohydrate stores for the later stages of the race. Fatty Acid Transport into Mitochondria Fatty acid oxidation begins with activation of the molecule. Activation in lipid metabolism involves the formation of a thioester bond between the activation in lipid metabolism the formation of a carboxyl group of the fatty acid and the thiol group of coenzyme A (CoA- thioester bond between a fatty acid and acetyl-CoA SH). The activated form of the fatty acid is an acyl-CoA, the exact nature of which depends on the nature of the fatty acid itself. Keep in mind through- out this discussion that all acyl-CoA molecules are thioesters, since the fatty acid is esterified to the thiol group of CoA. The enzyme that catalyzes formation of the ester bond, an acyl-CoA syn- A1 thetase, requires ATP for its action. In the course of the reaction, an acyl O adenylate intermediate is formed. The acyl group is then transferred to CoA- O H2C O C R2 SH. ATP is converted to AMP and PPi, rather than to ADP and Pi. The PPi R1 C O C O is hydrolyzed to two Pi; the hydrolysis of two high-energy phosphate bonds provides energy for the activation of the fatty acid and is equivalent to A2 H2C O P O R3 the use of two ATP. The formation of the acyl-CoA is endergonic without O – the energy provided by the hydrolysis of the two high-energy bonds. Note also that the hydrolysis of ATP to AMP and two P i represents an increase in C D entropy (Figure 21.4). There are several enzymes of this type, some specific A phosphoacylglycerol for longer-chain fatty acids and some for shorter-chain fatty acids. Both satu- Figure 21.2 Several phospholipases hydrolyze rated and unsaturated fatty acids can serve as substrates for these enzymes. phosphoacylglycerols. They are designated A1, A2, The esterification takes place in the cytosol, but the rest of the reactions of C, and D. Their sites of action are shown. The site fatty acid oxidation occur in the mitochondrial matrix. The activated fatty of action of phospholipase A2 is the B site, and the acid must be transported into the mitochondrion so that the rest of the name phospholipase A2 is the result of historical oxidation process can proceed. accident. Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 638 CHAPTER 21 Lipid Metabolism Hormone Receptor Plasma membrane Adenylate cyclase Adipose cell P P ATP cAMP Protein kinase Protein kinase (inactive) (active) ATP ADP Triacylglycerol Triacylglycerol lipase (inactive) Triacylglycerol lipase (active) Fatty acid P P Diacylglycerol Phosphatase DAG lipase Fatty acid Monoacylglycerol MAG lipase Fatty acid Glycerol Figure 21.3 Liberation of fatty acids from triacylglycerols in adipose tissue is hormone- dependent. P P O – Step 1. RCOO + ATP RC AMP Acyl adenylate intermediate O O Step 2. RC AMP + CoA-SH RC S CoA + AMP Thioester (activated acyl group) Acyl-CoA O Overall Reaction: synthetase RCOO– + ATP + CoA-SH RC S CoA + AMP + P P Figure 21.4 The formation of an acyl-CoA. Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-2 Catabolism of Lipids 639 O RC S CoA CoA-SH Acyl-CoA Cytosol Outer membrane Intermembrane space O CoA-SH RC S CoA + + O CH2N(CH3)3 CH2N(CH3)3 RC O CHCH2COO– CHOHCH2COO– Acyl-carnitine Carnitine E Inner carnitine membrane acyltransferase E Matrix CoA-SH O + + O CH2N(CH3)3 CH2N(CH3)3 RC S CoA RC O CHCH2COO– CHOHCH2COO– Acyl-carnitine b-Oxidation Carnitine Figure 21.5 The role of carnitine in the transfer of acyl groups to the mitochondrial matrix. The acyl-CoA can cross the outer mitochondrial membrane but not the carnitine a molecule used in fatty acid inner membrane (Figure 21.5). In the intermembrane space, the acyl group metabolism to shuttle acyl groups across the inner mitochondrial membrane is transferred to carnitine by transesterification; this reaction is catalyzed by the enzyme carnitine acyltransferase, which is located in the inner mem- carnitine acyltransferase an enzyme that transfers brane. Acyl-carnitine, a compound that can cross the inner mitochondrial a fatty acyl group to carnitine membrane, is formed. This enzyme has a specificity for acyl groups between carnitine palmitoyltransferase (CPT-I) 14 and 18 carbons long and is often called carnitine palmitoyltransferase the primary form of carnitine acyltransferase, found (CPT-I) for this reason. The acyl-carnitine passes through the inner mem- on the cytosol side of the inner mitochondrial membrane; it functions to transfer long-chain fatty brane via a specific carnitine/acyl-carnitine transporter called carnitine acyl groups from Coenzyme A to carnitine translocase. Once in the matrix, the acyl group is transferred from carnitine to mitochondrial CoA-SH by another transesterification reaction, involving a carnitine translocase the enzyme that moves acyl-carnitines across the inner mitochondrial second carnitine palmitoyltransferase (CPT-II) located on the inner face of membrane the membrane. carnitine palmitoyltransferase (CPT-II) another form of carnitine acyltransferase, found in Oxidation of Saturated Fatty Acids the mitochondrial matrix; it functions to transfer In the matrix, a repeated sequence of reactions successively cleaves two-carbon long-chain fatty acyl groups from carnitine to units from the fatty acid, starting from the carboxyl end. This process is called Coenzyme A Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 640 CHAPTER 21 Lipid Metabolism b-oxidation the main pathway of catabolism for b-oxidation, since the oxidative cleavage takes place at the -carbon of the acyl fatty acids group esterified to CoA. The -carbon of the original fatty acid becomes the carboxyl carbon in the next stage of degradation. The whole cycle requires four reactions (Figure 21.6). 1. The acyl-CoA is oxidized to an ,  unsaturated acyl-CoA (also called a -enoyl-CoA). The product has the trans arrangement at the double bond. This reaction is catalyzed by an FAD-dependent acyl-CoA dehydrogenase. 2. The unsaturated acyl-CoA is hydrated to produce a -hydroxyacyl-CoA. This reaction is catalyzed by the enzyme enoyl-CoA hydratase. 3. A second oxidation reaction is catalyzed by -hydroxyacyl-CoA dehydroge- nase, an NAD1-dependent enzyme. The product is a -ketoacyl-CoA. 4. The enzyme thiolase catalyzes the cleavage of the -ketoacyl-CoA; a molecule of CoA is required for the reaction. The products are acetyl-CoA and an acyl-CoA that is two carbons shorter than the original molecule that entered the -oxidation cycle. The CoA is needed in this reaction to form the new thioester bond in the smaller acyl-CoA molecule. This smaller molecule then undergoes another round of the -oxidation cycle. When a fatty acid with an even number of carbon atoms undergoes succes- sive rounds of the -oxidation cycle, the product is acetyl-CoA. (Fatty acids with even numbers of carbon atoms are the ones normally found in nature, so acetyl- CoA is the usual product of fatty acid catabolism.) The number of molecules of H H O R CH2 C C C S CoA H H Fatty acyl-CoA H O FAD O H C C S CoA R CH2 C S CoA Acyl-CoA Acetyl-CoA dehydrogenase H Fatty acyl-CoA 4 shortened by two carbons FADH2 Thiolase 1 CoA-SH Cleavage Oxidation Successive cycles O H O H O R CH2 C C C S CoA R CH2 C C C S CoA H H -Ketoacyl-CoA trans-Δ2-Enoyl-CoA Figure 21.6 The b-oxidation of saturated fatty acids involves a 3 cycle of four enzyme-catalyzed NADH + H+ Oxidation Hydration H2O reactions. Each cycle produces one FADH2 and one NADH, and L-Hydroxyacyl-CoA Enoyl-CoA it liberates acetyl-CoA, resulting dehydrogenase 2 hydratase in a fatty acid that is two carbons H H O shorter. The D symbol represents NAD+ a double bond, and the number R CH2 C C C S CoA associated with it is the location of the double bond (based on counting HO H the carbonyl group as carbon 1). L- -Hydroxyacyl-CoA Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-3 The Energy Yield from the Oxidation of Fatty Acids 641 9th 8th 7th 6th 5th 4th 3rd 2nd 1st Two-carbon units O C 8th 7th 6th 5th 4th 3rd 2nd 1st S CoA Cycles of b-oxidation Figure 21.7 Stearic acid (18 carbons) gives rise to nine 2-carbon units after eight cycles of b-oxidation. The ninth 2-carbon unit remains esterified to CoA after eight cycles of b-oxidation have removed eight successive 2-carbon units, starting at the carboxyl end on the right. Thus, it takes only eight rounds of b-oxidation to completely process an 18-carbon fatty acid to acetyl-CoA. acetyl-CoA produced is equal to half the number of carbon atoms in the origi- nal fatty acid. For example, stearic acid contains 18 carbon atoms and gives rise to 9 molecules of acetyl-CoA. Note that the conversion of one 18-carbon stearic acid molecule to nine 2-carbon acetyl units requires eight, not nine, cycles of - oxidation (Figure 21.7). The acetyl-CoA enters the citric acid cycle, with the rest of the oxidation of fatty acids to carbon dioxide and water taking place through the citric acid cycle and electron transport. Recall that most of the enzymes of the citric acid cycle are located in the mitochondrial matrix, and we have just seen that the -oxidation cycle takes place in the matrix as well. In addition to mitochondria, other sites of -oxidation are known. Peroxisomes and gly- oxysomes (Section 1-5), organelles that carry out oxidation reactions, are also sites of -oxidation, albeit to a far lesser extent than the mitochondria. Certain drugs, called hypolipidemic drugs, are used in an attempt to control obesity. Some of these work by stimulating -oxidation in peroxisomes. 21-3 The Energy Yield from the Oxidation of Fatty Acids In carbohydrate metabolism, the energy released by oxidation reactions is used to drive the production of ATP, with most of the ATP produced in aero- bic processes. In the same aerobic processes—namely, the citric acid cycle and oxidative phosphorylation—the energy released by the oxidation of acetyl-CoA formed by -oxidation of fatty acids can also be used to produce ATP. There are two sources of ATP to keep in mind when calculating the overall yield of ATP. The first source is the reoxidation of the NADH and FADH2 produced by the -oxidation of the fatty acid to acetyl-CoA. The second source is ATP produc- tion from the processing of the acetyl-CoA through the citric acid cycle and oxidative phosphorylation. We shall use the oxidation of stearic acid, which contains 18 carbon atoms, as our example. Eight cycles of -oxidation are required to convert one mole of stearic acid to nine moles of acetyl-CoA; in the process eight moles of FAD are reduced to FADH2, and eight moles of NAD1 are reduced to NADH. O CH3(CH2)16C S CoA 1 8 FAD 1 8 NAD1 1 8 H2O 1 8 CoA-SH O 9 CH3 C S CoA 1 8 FADH2 1 8 NADH 1 8 H1 The nine moles of acetyl-CoA produced from each mole of stearic acid en- ter the citric acid cycle. One mole of FADH2 and three moles of NADH are produced for each mole of acetyl-CoA that enters the citric acid cycle. At the same time, one mole of GDP is phosphorylated to produce GTP for each turn of the citric acid cycle. Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 642 CHAPTER 21 Lipid Metabolism O 9 CH3C S CoA 1 9 FAD 1 27 NAD1 1 9 GDP 1 9 Pi 1 27 H2O 18 CO2 1 9 CoA-SH 1 9 FADH2 1 27 NADH 1 9 GTP 1 27 H1 The FADH2 and NADH produced by -oxidation and by the citric acid cycle enter the electron transport chain, and ATP is produced by oxidative phos- phorylation. In our example, there are 17 moles of FADH2 (8 from -oxidation and 9 from the citric acid cycle); there are also 35 moles of NADH (8 from -oxidation and 27 from the citric acid cycle). Recall that 2.5 moles of ATP are produced for each mole of NADH that enters the electron transport chain, and 1.5 moles of ATP result from each mole of FADH2. Because 17 3 1.5 5 25.5 and 35 3 2.5 5 87.5, we can write the following equations: 17FADH2 1 8.5O2 1 25.5ADP 1 25.5Pi S 17FAD 1 25.5ATP 1 17H2O 35NADH 1 35H1 1 17.5O2 1 87.5ADP 1 87.5Pi S 35NAD1 1 87.5ATP 1 35H2O The overall yield of ATP from the oxidation of stearic acid can be obtained by add- ing the equations for -oxidation, for the citric acid cycle, and for oxidative phos- phorylation. In this calculation, we take GDP as equivalent to ADP and GTP as equivalent to ATP, which means that the equivalent of nine ATP must be added to those produced in the reoxidation of FADH2 and NADH. There are 9 ATP equiv- alent to the 9 GTP from the citric acid cycle, 25.5 ATP from the reoxidation of FADH2, and 87.5 ATP from the reoxidation of NADH, for a grand total of 122 ATP. O CH3(CH2)16C S CoA 1 26 O2 1 122 ADP 1 122 Pi 18 CO2 1 17 H2O 1 122 ATP 1 CoA-SH The activation step in which stearyl-CoA was formed is not included in this calculation, and we must subtract the ATP that was required for that step. Even though only one ATP was required, two high-energy phosphate bonds are lost because of the production of AMP and PPi. The pyrophosphate must be hydro- lyzed to phosphate (Pi) before it can be recycled in metabolic intermediates. As a result, we must subtract the equivalent of two ATP for the activation step. The net yield of ATP becomes 120 moles of ATP for each mole of stearic acid that is completely oxidized. See Table 21.1 for a balance sheet. Keep in mind that these values are theoretical consensus values that not all cells attain. Table 21.1 The Balance Sheet for Oxidation of One Molecule of Stearic Acid NADH Molecules FADH2 Molecules ATP Molecules Reaction 1. Stearic acid S Stearyl-CoA (activation step) 18 18 22 2. Stearyl-CoA S 9 Acetyl-CoA (8 cycles of -oxidation) 127 19 3. 9 Acetyl-CoA S 18 CO2 (citric acid cycle); GDP S GTP (9 molecules) 19 4. Reoxidation of NADH from -oxidation cycle 28 120 5. Reoxidation of NADH from citric acid cycle 227 167.5 6. Reoxidation of FADH2 from -oxidation cycle 28 112 7. Reoxidation of FADH2 from citric acid cycle 29 113.5 Net 0 0 1120 Note that there is no net change in the number of molecules of NADH or FADH2. Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-4 Catabolism of Unsaturated Fatty Acids and Odd-Carbon Fatty Acids 643 As a comparison, note that 32 moles of ATP can be obtained from the com- plete oxidation of one mole of glucose; but glucose contains 6, rather than 18, carbon atoms. Three glucose molecules contain 18 carbon atoms, and a more interesting comparison is the ATP yield from the oxidation of three glucose molecules, which is 3 3 32 5 96 ATP for the same number of carbon atoms. The yield of ATP from the oxidation of the lipid is still higher than that from the carbohydrate, even for the same number of carbon atoms. The reason is that a fatty acid is all hydrocarbon except for the carboxyl group; that is, it ex- ists in a highly reduced state. A sugar is already partly oxidized because of the presence of its oxygen-containing groups. Because the oxidation of a fuel leads to the reduced electron carriers used in the electron transport chain, a more reduced fuel, such as a fatty acid, can be oxidized further than a partially oxi- dized fuel, such as a carbohydrate. Another point of interest is that water is produced in the oxidation of fatty acids. We have already seen that water is also produced in the complete oxidation of carbohydrates. The production of metabolic water is a common metabolic water the water produced as a result of feature of aerobic metabolism. This process can be a source of water for or- complete oxidation of nutrients; sometimes it is the only water source of desert-dwelling organisms ganisms that live in desert environments. Camels are a well-known example; the stored lipids in their humps are a source of both energy and water dur- ing long trips through the desert. Kangaroo rats provide an even more strik- ing example of adaptation to an arid environment. These animals have been observed to live indefinitely without having to drink water. They live on a diet of seeds, which are rich in lipids but contain little water. The metabolic water that kangaroo rats produce is adequate for all their water needs. This metabolic response to arid conditions is usually accompanied by a reduced output of urine. 21-4 Catabolism of Unsaturated Fatty Acids and Odd-Carbon Fatty Acids Fatty acids with odd numbers of carbon atoms are not as frequently en- countered in nature as are the ones with even numbers of carbon atoms. Odd-numbered fatty acids also undergo -oxidation (Figure 21.8). The last cycle of -oxidation produces one molecule of propionyl-CoA. An enzymatic pathway exists to convert propionyl-CoA to succinyl-CoA, which then enters the citric acid cycle. In this pathway, propionyl-CoA is first carboxylated to methyl malonyl-CoA in a reaction catalyzed by propionyl-CoA carboxylase, which then undergoes rearrangement to form succinyl-CoA. Because pro- pionyl-CoA is also a product of the catabolism of several amino acids, the conversion of propionyl-CoA to succinyl-CoA also plays a role in amino acid metabolism (see Chapter 23). The conversion of methyl malonyl-CoA to succinyl-CoA requires vitamin B12 (cyanocobalamin), which has a cobalt(III) ion in its active state. Oxidation of Monounsaturated Fatty Acids c How does the oxidation of unsaturated fatty acids differ from that of saturated fatty acids? The conversion of a monounsaturated fatty acid to acetyl-CoA requires a reac- tion that is not encountered in the oxidation of saturated acids, a cis–trans isom- erization (Figure 21.9). Successive rounds of -oxidation of oleic acid (18:1) provide an example of these reactions. The process of -oxidation gives rise to unsaturated fatty acids in which the double bond is in the trans arrangement, whereas the double bonds in most naturally occurring fatty acids are in the cis arrangement. In the case of oleic acid, there is a cis double bond between Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 644 CHAPTER 21 Lipid Metabolism O CH 3 (CH 2 )n C S CoA where n is odd O (n –1) b-Oxidation CH 3 C S CoA 2 COO – CH 3 ATP ADP + P CH 3 CH 2 CH 2 HC COO – Rearrangement CH 2 CO S CoA CO2 CO S CoA CO S CoA Propionyl-S-CoA Methyl malonyl-S-CoA Succinyl-S-CoA Citric acid O cycle H H CH3(CH2)7 C C CH2(CH2)6C S CoA Thr Ile Met Val Oleoyl-CoA The catabolism of some amino acids also O yields propionyl-CoA and methyl malonyl-CoA b-oxidation 3 CH3 C S—CoA (three cycles) Figure 21.8 The oxidation of a fatty acid containing an odd number of carbon atoms. O H H CH3(CH2)7 C C CH2 C S CoA carbons 9 and 10. Three rounds of -oxidation produce a 12-carbon unsatu- cis -D3-Dodecenoyl-CoA rated fatty acid with a cis double bond between carbons 3 and 4. The hydratase of the -oxidation cycle requires a trans double bond between carbon atoms Enoyl-CoA isomerase 2 and 3 as a substrate. A cis–trans isomerase produces a trans double bond be- tween carbons 2 and 3 from the cis double bond between carbons 3 and 4. O From this point forward, the fatty acid is metabolized the same as for saturated H fatty acids. When oleic acid is -oxidized, the first step (fatty acyl-CoA dehydro- CH3(CH2)7CH2 C C C S CoA H genase) is skipped, and the isomerase deals with the cis double bond, putting it trans -D2-Dodecenoyl-CoA into the proper position and orientation to continue the pathway. H2O Oxidation of Polyunsaturated Fatty Acids Enoyl-CoA hydratase When polyunsaturated fatty acids are -oxidized, another enzyme is needed to handle the second double bond. Let’s consider how linoleic acid (18:2) would H O be metabolized (Figure 21.10). This fatty acid has cis double bonds at positions CH3(CH2)7CH2 C CH2 C S CoA 9 and 12 as shown in Figure 21.10, which are indicated as cis-D9 and cis-D12. Three normal cycles of -oxidation occur, as in our example with oleic acid, OH before the isomerase must switch the position and orientation of the double bond. The cycle of -oxidation continues until a 10-carbon fatty acyl-CoA is Continuation of b-oxidation attained that has one cis double bond on its carbon 4 (cis-D4). Then the first step of -oxidation occurs, putting in a trans double bond between carbons 2 O and 3 ( and ). Normal -oxidation cannot continue at this point because the fatty acid with the two double bonds so close together is a poor substrate for 6 CH3C S CoA the hydratase. Therefore, a second new enzyme, 2,4-dienoyl-CoA reductase, uses NADPH to reduce this intermediate. The result is a fatty acyl-CoA with a trans Figure 21.9 b-oxidation of unsaturated fatty acids. In the case of oleoyl-CoA, three -oxidation double bond between carbons 3 and 4. The isomerase then switches the trans cycles produce three molecules of acetyl-CoA and double from carbon 3 to carbon 2, and -oxidation continues. leave cis-D3-dodecenoyl-CoA. Rearrangement of A molecule with three double bonds, such as linolenic acid (18:3), would enoyl-CoA isomerase gives the trans-D2 species, use the same two enzymes to handle the double bonds. The first double bond which then proceeds normally through the requires the isomerase. The second one requires the reductase and the isom- -oxidation pathway. erase, and the third requires the isomerase. For practice, you can diagram the Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-4 Catabolism of Unsaturated Fatty Acids and Odd-Carbon Fatty Acids 645 O H H H H CH3(CH2)4 C C CH2 C C CH2(CH2)6C CoA cis-D12, cis-D9 b-oxidation (three cycles) O O H H H H CH3(CH2)4 C C CH2 C C CH2 C CoA + 3 CH3 C CoA cis-D6, cis-D3 Enoyl-CoA isomerase O H H H CH3(CH2)4 C C CH2 CH2 C C C CoA H cis-D6, trans-D2 One cycle of -oxidation O O H H CH3(CH2)4 C C CH2 CH2 C CoA + CH3 C CoA cis-D4 Acyl-CoA dehydrogenase O H H H CH3(CH2)4 C C C C C CoA H cis-D4, trans-D2 NADPH + H+ 2,4-Dienoyl-CoA reductase NADP+ O H CH3(CH2)4CH2 C C CH2 C CoA H trans-D3 Figure 21.10 The oxidation pathway for polyunsaturated fatty acids, illustrated for Enoyl-CoA isomerase linoleic acid. Three cycles of -oxidation O on linoleoyl-CoA yield the cis-D3, cis-D6 H CH3(CH2)4CH2 CH2 C C C CoA intermediate, which is converted to a trans-D2, H cis-D6 intermediate. An additional round of trans -D2 b-oxidation gives cis-D4 enoyl-CoA, which is oxidized to the trans-D2, cis-D4 species by b-oxidation acyl-CoA dehydrogenase. The subsequent (four cycles) action of 2,4-dienoyl-CoA reductase yields the O trans-D3 product, which is converted by enoyl- CoA isomerase to the trans-D2 form. Normal 5 CH3 C CoA -oxidation then produces five molecules of Acetyl-CoA acetyl-CoA. -oxidation of an 18-carbon molecule with cis double bonds at positions 9, 12, and 15 to see that this is true. Unsaturated fatty acids make up a large enough portion of the fatty acids in storage fat (40% for oleic acid alone) to make the reactions of the cis–trans isomerase and the epimerase of particular importance. Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 646 CHAPTER 21 Lipid Metabolism The oxidation of unsaturated fatty acids does not generate as many ATPs as it would for a saturated fatty acid with the same number of carbons. This is be- cause the presence of a double bond means that the acyl-CoA dehydrogenase step will be skipped. Thus, fewer FADH2 will be produced. 21-5 Ketone Bodies ketone bodies several ketone-based molecules Substances related to acetone (“ketone bodies”) are produced when an excess produced in the liver during overutilization of fatty of acetyl-CoA arises from -oxidation. This condition occurs when not enough acids when carbohydrates are limited oxaloacetate is available to react with the large amounts of acetyl-CoA that could enter the citric acid cycle. Oxaloacetate in turn arises from glycolysis be- cause it is formed from pyruvate in a reaction catalyzed by pyruvate carboxylase. A situation like this can come about when an organism has a high intake of lipids and a low intake of carbohydrates, but there are also other possible causes, such as starvation and diabetes. Starvation conditions cause an organ- O ism to break down fats for energy, leading to the production of large amounts of acetyl-CoA by -oxidation. The amount of acetyl-CoA is excessive by com- 2 CH3C CoA parison with the amount of oxaloacetate available to react with it. In the case of people with diabetes, the cause of the imbalance is not inadequate intake of Thiolase carbohydrates but rather the inability to metabolize them. CoA CoA c Do acetone and acetyl-CoA have a connection in lipid metabolism? O O The reactions that result in ketone bodies start with the condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA. Acetoacetate is produced CH3C CH2 C CoA from acetoacetyl-CoA through condensation with another acetyl-CoA to form Acetoacetyl-CoA O -hydroxy--methylglutaryl-CoA (HMG-CoA), a compound we will see again H2O + when we look at cholesterol synthesis (Figure 21.11). HMG-CoA lyase then re- CH3C CoA leases acetyl-CoA to give acetoacetate. Acetoacetate can then have two fates. A HMG -CoA synthase CoA reduction reaction can produce -hydroxybutyrate from acetoacetate. The other possible reaction is the spontaneous decarboxylation of acetoacetate to give acetone. The odor of acetone can frequently be detected on the breath of peo- O OH O ple with diabetes whose disease is not controlled by suitable treatment. The –O C CH2 C CH2 C CoA excess of acetoacetate, and consequently of acetone, is a pathological condi- tion known as ketosis. Because acetoacetate and -hydroxybutyrate are acidic, CH3 their presence at high concentration overwhelms the buffering capacity of the -Hydroxy-b-methylglutaryl-CoA (HMG-CoA) blood. The body deals with the consequent lowering of blood pH (ketoacido- sis) by excreting H1 into the urine, accompanied by excretion of Na1, K1, and HMG-CoA lyase O water. Severe dehydration can result (excessive thirst is a classic symptom of diabetes); diabetic coma is another possible danger. CH3C CoA The principal site of synthesis of ketone bodies is liver mitochondria, but they are not used there because the liver lacks the enzymes necessary to recover ace- O O tyl-CoA from ketone bodies. It is easy to transport ketone bodies in the blood- CH3C CH2 C O– stream because, unlike fatty acids, they are water-soluble and do not need to be Acetoacetate bound to proteins, such as serum albumin. Organs other than the liver can use -Hydroxybutyrate ketone bodies, particularly acetoacetate. Even though glucose is the usual fuel dehydrogenase in most tissues and organs, acetoacetate can be used as a fuel. In heart muscle CO2 NADH and the renal cortex, acetoacetate is the preferred source of energy. + H+ NAD+ Even in organs such as the brain, in which glucose is the preferred fuel, starvation conditions can lead to the use of acetoacetate for energy. In this situ- O H O ation, acetoacetate is converted to two molecules of acetyl-CoA, which can then CH3 C CH3 CH3 C CH2 C O– enter the citric acid cycle. The key point here is that starvation gives rise to Acetone long-term, rather than short-term, regulation over a period of hours to days OH rather than minutes. The decreased level of glucose in the blood over a pe- -Hydroxybutyrate riod of days changes the hormone balance in the body, particularly involving Figure 21.11 The formation of ketone bodies, insulin and glucagon (see Section 24-5). (Short-term regulation, such as allo- synthesized primarily in the liver. steric interactions or covalent modification, can occur in a matter of minutes.) Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-6 Fatty Acid Biosynthesis 647 The rates of protein synthesis and breakdown are subject to change under these conditions. The specific enzymes involved are those involved in fatty acid oxida- tion (increase in levels) and those for lipid biosynthesis (decrease in levels). 21-6 Fatty Acid Biosynthesis The anabolism of fatty acids is not simply a reversal of the reactions of -oxidation. Anabolism and catabolism are not, in general, the exact reverse of each other; for instance, gluconeogenesis (Section 18-2) is not simply a re- versal of the reactions of glycolysis. A first example of the differences between the degradation and the biosynthesis of fatty acids is that the anabolic reactions take place in the cytosol. We have just seen that the degradative reactions of -oxidation take place in the mitochondrial matrix. The first step in fatty acid biosynthesis is transport of acetyl-CoA to the cytosol. Acetyl-CoA can be formed either by -oxidation of fatty acids or by decarbox- ylation of pyruvate. (Degradation of certain amino acids also produces acetyl- CoA; see Section 23-6.) Most of these reactions take place in the mitochondria, requiring a transport mechanism to export acetyl-CoA to the cytosol for fatty acid biosynthesis. The transport mechanism is based on the fact that citrate can cross the mitochondrial membrane. Acetyl-CoA condenses with oxaloacetate, which cannot cross the mitochondrial membrane, to form citrate (recall that this is the first reaction of the citric acid cycle). BIOCHEMICAL CONNECTIONS 21A describes another form of control in lipid biosynthesis. 21A BIOCHEMICAL CONNECTIONS Gene Expression Transcription Activators in Lipid Biosynthesis A s we saw in Chapter 11, transcription factors can do double duty in cells. An example that directly deals with the material in this chapter is the action of the transcription factor XBP1 in mouse liver. into the Golgi apparatus. Processing of the proteins involved takes place in the Golgi before the proteins enter the nucleus. The role of XBP1 in regulating lipid biosynthesis follows a different pathway. As XBP1 regulates genes that deal with improperly folded proteins and shown in Figure 21.12, stress factors trigger a response by binding to genes that control lipid synthesis (Figure 21.12). It was already well IRE-1 in the endoplasmic reticulum, leading to the release of XBP1 established that other transcription factors, called SREBPs, played a into the Golgi. Another transcription factor, ATF6, enters the Golgi role in regulating gene expression leading to lipid synthesis. The sig- in similar fashion. After processing, all these transcription factors nal for regulation by SREBPs is low cholesterol in the cytoplasm, with play roles in regulating lipid synthesis. What is new in this picture is the signal being passed into the endoplasmic reticulum and then the hitherto unknown involvement of XBP1. ◗ Stress Low cholesterol CYTOPLASM NUCLEUS IRE-1a ATF6 SREBP ENDOPLASMIC Figure 21.12 Transcription factors in lipid RETICULUM XBP1 synthesis. XBP1, ATF6, and SREBP all undergo processing in the Golgi before they enter the nucleus. ATF6 activates XBP1. In turn, XBP1 and Regulate gene expression for lipid synthesis SREBPs regulate the expression of the genes CYTOPLASM S2P S1P for lipid synthesis. The regulation of SREBPs by cholesterol is a different process from the activation of ATF6 and XBP1 in response LGI to stress. (Horton, J. D. (2008). Science, GO 320(5882), 1434.) Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 648 CHAPTER 21 Lipid Metabolism First Steps in Fatty Acid Biosynthesis c How do the first steps of fatty acid synthesis take place? The citrate that is exported to the cytosol can undergo the reverse reaction, producing oxaloacetate and acetyl-CoA (Figure 21.13). Acetyl-CoA enters the pathway for fatty acid biosynthesis, while oxaloacetate undergoes a series of re- actions in which NADPH is substituted for NADH (see the discussion of lipid anabolism in Section 19-8). This substitution controls the pathway because NADPH is required for fatty acid anabolism. malonyl-CoA a three-carbon intermediate In the cytosol, acetyl-CoA is carboxylated, producing malonyl-CoA, a key in- important in the biosynthesis of fatty acids termediate in fatty acid biosynthesis (Figure 21.14). This reaction is catalyzed by the acetyl-CoA carboxylase complex, which consists of three enzymes and re- quires Mn21, biotin, and ATP for activity. We have already seen that enzymes catalyzing reactions that take place in several steps frequently consist of several separate protein molecules, and this enzyme follows that pattern. In this case, acetyl-CoA carboxylase consists of the three proteins biotin carboxylase, the bio- tin carrier protein, and carboxyl transferase. Biotin carboxylase catalyzes the trans- fer of the carboxyl group to biotin. The “activated CO 2” (the carboxyl group derived from the bicarbonate ion HCO32) is covalently bound to biotin. Biotin (whether carboxylated or not) is bound to the biotin carrier protein by an amide linkage to the ´-amino group of a lysine side chain. The amide linkage to the side chain that bonds biotin to the carrier protein is long enough and flexible enough to move the carboxylated biotin into position to transfer the Cytosol Mitochondrial membrane Hexoses Glycolysis CoA-SH CoA-SH (in cytosol) Pyruvate Pyruvate Acetyl-CoA Citrate Citrate Acetyl-CoA Oxaloacetate Oxaloacetate Some Lipids amino acids Figure 21.13 The transport of acetyl groups from the mitochondrion to the cytosol. Mitochondrion Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-6 Fatty Acid Biosynthesis 649 O – Biotin H3C C S CoA + ATP + HCO3 Acetyl-CoA Mn2+ O – OOC CH2 C S CoA + ADP + P + H+ Figure 21.14 The formation of malonyl-CoA, Malonyl-CoA catalyzed by acetyl-CoA carboxylase. A O – CH3 C S CoA + ATP + HCO3 O O C CH2 C S CoA + ADP + P + H+ –O B Step 1 The carboxylation of biotin ADP O O P O O ATP + HCO3– –O C O P O– –O C N NH O– O S Lys HN NH Biotin S Lys Step 2 The transcarboxylation of biotin O H2C C S CoA Figure 21.15 The acetyl-CoA carboxylase – reaction. (A) The acetyl-CoA carboxylase reaction produces malonyl-CoA for fatty acid synthesis. (B) A –O mechanism for the acetyl-CoA carboxylase reaction. O O O Bicarbonate is activated for carboxylation reactions C by formation of N-carboxybiotin. ATP drives the O N NH H2C C S CoA + HN NH reaction forward, with transient formation of a COO– carbonyl-phosphate intermediate (Step 1). In a typical biotin-dependent reaction, nucleophilic S Lys S Lys attack by the acetyl-CoA carbanion on the carboxyl carbon of N-carboxybiotin—a transcarboxylation— yields the carboxylated product (Step 2). carboxyl group to acetyl-CoA in the reaction catalyzed by carboxyl transferase, producing malonyl-CoA (Figure 21.15). In addition to its role as a starting point in fatty acid synthesis, malonyl-CoA strongly inhibits the carnitine ac- yltransferase I on the outer face of the inner mitochondrial membrane. This avoids a futile cycle in which fatty acids are -oxidized in the mitochondria Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 650 CHAPTER 21 Lipid Metabolism to make acetyl-CoA just so they can be remade into fatty acids in the cytosol. BIOCHEMICAL CONNECTIONS 21B discusses other ways in which malonyl-CoA is a key player in lipid biosynthesis. 21B BIOCHEMICAL CONNECTIONS Nutrition Acetyl-CoA Carboxylase—A New Target in the Fight against Obesity? M alonyl-CoA has two very important functions in metabolism. First, it is the committed intermediate in fatty acid synthesis. Second, it strongly inhibits carnitine palmitoyltransferase I and there- of up to 50%. The mice showed no other abnormalities. They grew and reproduced normally and had normal life spans. The investigators concluded that reduced pools of malonyl-CoA due to fore fatty acid oxidation. The level of malonyl-CoA in the cytosol the lack of ACC2 results in increased b-oxidation via removal of can determine whether the cell will be oxidizing fats or storing fats. the block on carnitine palmitoyltransferase I, and a decrease in The enzyme that produces malonyl-CoA is acetyl-CoA carboxylase, fatty acid synthesis. They speculate that ACC2 would be a good or ACC. There are two isoforms of this enzyme encoded by separate target for drugs used to combat obesity. ◗ genes. ACC1 is found in the liver and adipose tissue, while ACC2 is found in cardiac and skeletal muscle. High glucose concentra- tions and high insulin concentrations lead to stimulation of ACC2. March 2001). Science 291 (5513) 2613. Used with Exercise has the opposite effect. During exercise, an AMP-dependent Reduced Fat Storage in Mice Lacking Acetyl-CoA Carboxylase 2 by Lufti Abu-Elheiga, et al., (30 protein kinase phosphorylates ACC2 and inactivates it. From Continuous Fatty Acid Oxidation and Some recent studies looked at the nature of weight gain and weight loss with respect to ACC2 (see papers by Ruderman and Flier and by Abu-Elheiga et al. cited in Further Reading). The investiga- tors created a strain of mice lacking the gene for ACC2. These mice Figure 21.16 The amount of white fat permission of AAAS. ate more than their wild-type counterparts but had significantly under the skin of the mouse on the left, lower stores of lipids (30% to 40% less in skeletal muscle and 10% which lacks the gene for ACC2, is less less in cardiac muscle) (Figure 21.16). Even the adipose tissue, than that for the mouse on the right, which still had ACC1, showed a reduction in stored triacylglycerols which has the gene. Two-Carbon Addition by Fatty Acid Synthase c What is the mode of action of fatty acid synthase? The biosynthesis of fatty acids involves the successive addition of two-carbon units to the growing chain. Two of the three carbon atoms of the malonyl group of malonyl-CoA are added to the growing fatty acid chain with each cycle of the biosynthetic reaction. This reaction, like the formation of the malonyl- CoA itself, requires a multienzyme complex located in the cytosol and not at- tached to any membrane. The complex, made up of the individual enzymes, is fatty acid synthase a giant enzyme complex that called fatty acid synthase. makes long-chain fatty acids from acetyl-CoA The usual product of fatty acid anabolism is palmitate, the 16-carbon satu- rated fatty acid. All 16 carbons come from the acetyl group of acetyl-CoA; we have already seen how malonyl-CoA, the immediate precursor, arises from acetyl-CoA. But first there is a priming step in which one molecule of acetyl-CoA is required for each molecule of palmitate produced. In this priming step, the ace- acyl carrier protein (ACP) a protein that tyl group from acetyl-CoA is transferred to an acyl carrier protein (ACP), which functions in fatty acid synthesis to carry activated is considered a part of the fatty acid synthase complex (Figure 21.17). The acetyl carbon groups group is bound to the protein as a thioester. The group on the protein to which the acetyl group is bonded is the 4-phosphopantetheine group, which in turn is bonded to a serine side chain; note in Figure 21.18 that this group is structurally similar to CoA-SH itself. The acetyl group is transferred from CoA-SH, to which it is bound by a thioester linkage, to the ACP; the acetyl group is bound to the ACP by a thioester linkage. Although the functional group of ACP is similar to that of Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203  21-6 Fatty Acid Biosynthesis 651 ACP-SH CoA-SH HS-KSase ACP-SH CH3 CH3 CH3 C O C O C O Acetyl transferase S CoA S ACP S KSase O O Acetyl-CoA CH3 C CH2 C S ACP CO2 Acetoacetyl-ACP COO– COO– NADPH + H+ -Ketoacyl-ACP ACP-SH CoA-SH reductase CH2 CH2 NADP+ C O C O OH O S CoA S ACP CH3 C CH2 C S ACP Malonyl-CoA H D- -Hydroxybutyryl-ACP -Hydroxyacyl- ACP dehydratase H2O Note that these H three steps are the reverse of those in CH3 C C C S ACP -oxidation H O Crotonyl-ACP NADPH + H+ 2,3-trans-Enoyl- ACP reductase NADP+ O CH3 CH2 CH2 C S ACP Butyryl-ACP Mal CoA + 4 H+ + 4 e– O CH3 CH2 CH2 CH2 CH2 C S ACP Mal CoA + 4 H+ + 4 e– O CH3 CH2 CH2 CH2 CH2 CH2 CH2 C S ACP Mal CoA + 4 H+ + 4 e– O CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 C S ACP Mal CoA + 4 H+ + 4 e– O CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 C S ACP Mal CoA + 4 H+ + 4 e– O CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 C S ACP Mal CoA + 4 H+ + 4 e– O CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 C S ACP H2O Figure 21.17 The pathway of palmitate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl building blocks are introduced as acyl carrier protein conjugates. In animals O ACP SH Decarboxylation drives the -ketoacyl-ACP synthase and results in the addition of two- carbon units to the growing chain. Concentrations of free fatty acids are extremely low in CH3 (CH2)14 C O– most cells, and newly synthesized fatty acids exist primarily as acyl-CoA esters. Palmitate Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-200-203 652 CHAPTER 21 Lipid Metabolism H H OH CH3 O HS CH2 CH2 N C CH2 CH2 N C C C CH2 O P O CH2 Ser ACP – O O H CH3 O Phosphopantetheine group of ACP H H OH CH3 O O HS CH2 CH2 N C CH2 CH2 N C C C CH2 O P O P O CH2 O Adenine O O – – H H