23.4 How Are Unsaturated Fatty Acids Oxidized? 713 23.4 How Are Unsaturated Fatty Acids Oxidized? An Isomerase and a Reductase Facilitate the -Oxidation of Unsaturated Fatty Acids Unsaturated fatty acids are also catabolized by -oxidation, but two additional mito- chondrial enzymes—an isomerase and a novel reductase—are required to handle the cis double bonds of naturally occurring fatty acids. As an example, consider NADP + NADPH + C HO CH 2 CO 2 O O – C H COO – COO – COO – CH 2 C O CH 3 CO H + H + C O O – Malate Pyruvate Oxaloacetate (enzyme-bound) FIGURE 23.20 The malic enzyme reaction proceeds by oxidation of malate to oxaloacetate, followed by decar- boxylation to yield pyruvate. Go to CengageNOW and click CengageInteractive to interact with a mechanism for the methylmalonyl mutase reaction. A DEEPER LOOK Can Natural Antioxidants in Certain Foods Improve Fat Metabolism? Numerous beneficial effects have been attributed in recent years to the polyphenolic compounds in foods such as chocolate, red wine, and (black or green) tea. Principal among these compounds are the catechins, a class of antioxidants that have demonstrated protective effects against certain cancers, obesity, and heart disease, as well as other beneficial health effects. Is it possible to demonstrate specific effects of catechins on cellular metabolism? Takatoshi Murase and his colleagues have shown that green tea extract, consisting almost entirely of six catechins (shown in the figure), added to the diets of rats increased muscle glycogen content, decreased muscle fatty acid synthesis, and increased fatty acid oxidation by rat skeletal muscle by 36%. In these same rats, exercise endurance capacity for swimming and running increased significantly, suggesting that fatty acids can be an effective source of energy in exercising muscle. Measure- ments of the catechin content of a range of teas, wines, and other beverages indicate that the broadest range and the highest levels of these compounds are found in tea, but that red wine has significant levels of catechins. (Green tea and black tea are made from the same shrub, Camellia sinensis.) OH OH OH OH R HO O (a) R ϭ H (+)-catechin, R ϭ OH (+)-gallocatechin (GC) OH OH OH OH R HO O (b) R ϭ H (–)-epicatechin, R ϭ OH (–)-epigallocatechin (EGC) OH O O OH OH R HO OH O OH OH (c) R ϭ H (–)-epicatechin gallate (ECg), R ϭ OH (–)-epigallocatechin gallate (EGCg) © Michel de Nijs/iStockphoto.com ᮡ Tea plants 714 Chapter 23 Fatty Acid Catabolism the breakdown of oleic acid, an 18-carbon chain with a double bond at the 9,10-position. The reactions of -oxidation proceed normally through three cycles, producing three molecules of acetyl-CoA and leaving the degradation product cis- ⌬ 3 -dodecenoyl-CoA, shown in Figure 23.21. This intermediate is not a substrate for acyl-CoA dehydrogenase. With a double bond at the 3,4-position, it is not possible to form another double bond at the 2,3- (or -) position. As shown in Figure 23.21, this problem is solved by enoyl-CoA isomerase, an enzyme that rearranges this cis-⌬ 3 double bond to a trans-⌬ 2 double bond. This latter species can proceed through the normal route of -oxidation. Degradation of Polyunsaturated Fatty Acids Requires 2,4-Dienoyl-CoA Reductase Polyunsaturated fatty acids pose a slightly more complicated situation for the cell. Consider, for example, the case of linoleic acid shown in Figure 23.22. As with oleic acid, -oxidation proceeds throug h three cycles, and enoyl-CoA iso- merase converts the cis-⌬ 3 double bond to a trans-⌬ 2 double bond to permit one more round of -oxidation. What results this time, however, is a cis-⌬ 4 enoyl-CoA, which is converted normally by acyl-CoA dehydrogenase to a trans-⌬ 2 , cis-⌬ 4 species. This, however, is a poor substrate for the enoyl-CoA hydratase. This problem is solved by 2,4-dienoyl-CoA reductase, the product of which depends on the organism. The mammalian form of this enzyme produces a trans-⌬ 3 enoyl product, as shown in Figure 23.22; this enoyl product can be converted by an enoyl-CoA isomerase to the trans-⌬ 2 enoyl-CoA, which can then proceed nor- mally through the -oxidation pathway. Escherichia coli possesses a 2,4-dienoyl- CoA reductase that reduces the double bond at the 4,5-position to yield the trans-⌬ 2 enoyl-CoA product in a single step. 23.5 Are There Other Ways to Oxidize Fatty Acids? Peroxisomal -Oxidation Requires FAD-Dependent Acyl-CoA Oxidase Although -oxidation in mitochondria 1 is the principal pathway of fatty acid ca- tabolism, several other minor pathways play important roles in fat catabolism. For example, organelles other than mitochondria, including peroxisomes and gly- oxysomes, carry out -oxidation processes. Peroxisomes are so named because they carry out a variety of flavin-dependent oxidation reactions, regenerating ox- idized flavins by reaction with oxygen to produce hydrogen peroxide, H 2 O 2 . Per- oxisomal -oxidation is similar to mitochondrial -oxidation, except that the ini- tial double-bond formation is catalyzed by an FAD-dependent acyl-CoA oxidase (Figure 23.23). The action of this enzyme in the peroxisomes transfers the liber- ated electrons directly to oxygen instead of the electron-transport chain. As a re- sult, each two-carbon unit oxidized in peroxisomes produces fewer ATPs. (Com- pare Figure 23.23 with Figure 23.12.) The enzymes responsible for fatty acid oxidation in peroxisomes are inactive with acyl chains of eight carbons or fewer. Such short-chain products must be transferred to the mitochondria for further breakdown. Similar -oxidation enzymes are also found in glyoxysomes— peroxisomes in plants that also carry out the reactions of the glyoxylate pathway. Branched-Chain Fatty Acids Are Degraded Via ␣-Oxidation Although -oxidation is universally important, there are some instances in which it cannot operate effectively. For example, branched-chain fatty acids with alkyl CH 3 (CH 2 ) 7 C H C H CH 2 (CH 2 ) 6 C O SCoA C O SCoA3 CH 3 CH 3 (CH 2 ) 7 C H C H CH 2 O C CH 3 (CH 2 ) 7 CH 2 C H C O C H CH 3 (CH 2 ) 7 CH 2 CCH 2 O C H OH O 6 CH 3 C SCoA SCoA SCoA SCoA H 2 O Oleoyl-CoA -Oxidation (three cycles) cis-Δ 3 -Dodecenoyl-CoA Enoyl-CoA isomerase Enoyl-CoA hydratase Continuation of -oxidation trans-Δ 2 -Dodecenoyl-CoA FIGURE 23.21 -Oxidation of unsaturated fatty acids. In the case of oleoyl-CoA, three -oxidation cycles pro- duce three molecules of acetyl-CoA and leave cis-⌬ 3 - dodecenoyl-CoA. Rearrangement of enoyl-CoA iso- merase gives the trans-⌬ 2 species, which then proceeds normally through the -oxidation pathway. 1 -Oxidation does not occur significantly in plant mitochondria. Most -oxidation in plants occurs in peroxisomes. Structure of acyl-CoA oxidase from rat liver (pdb id ϭ 1IS2).The enzyme is a homodimer, each monomer consist- ing of an N-terminal ␣-helical domain (blue), a -sheet do- main (orange), and a C-terminal ␣-helical domain (violet). 23.5 Are There Other Ways to Oxidize Fatty Acids? 715 CH 3 (CH 2 ) 4 H C cis-Δ 9 , H C CH 2 O H C H C CH 2 (CH 2 ) 6 C cis-Δ 12 CH 3 (CH 2 ) 4 H C cis-Δ 3 , H C CH 2 O H C H C CH 2 SCoA cis-Δ 6 C + O 3 CH 3 C CH 3 (CH 2 ) 4 H C trans-Δ 2 , H C CH 2 cis-Δ 6 CH 2 H C C H O C CH 3 (CH 2 ) 4 H C H C CH 2 O CH 2 CoA cis-Δ 4 C + O CH 3 C CH 3 (CH 2 ) 4 H C H C H C C H O C CH 3 (CH 2 ) 4 CH 2 H C C H O CH 2 C trans-Δ 3 CH 3 (CH 2 ) 4 CH 2 CH 2 H C O C trans-Δ 2 O 5 CH 3 C H + C H trans-Δ 2 , cis-Δ 4 CoA SCoA SCoA SCoA SCoA SCoA SCoA SCoA -Oxidation (three cycles) Enoyl-CoA isomerase One cycle of -oxidation Acyl-CoA dehydrogenase 2,4-Dienoyl-CoA reductase Enoyl-CoA isomerase -Oxidation (four cycles) Acetyl-CoA + NADPH NADP + FIGURE 23.22 The oxidation pathway for polyunsaturated fatty acids in mammals, illustrated for linoleic acid. Three cycles of -oxidation on linoleoyl-CoA yield the cis-⌬ 3 , cis-⌬ 6 intermediate, which is converted to a trans-⌬ 2 , cis-⌬ 6 intermediate. An additional round of -oxidation gives cis-⌬ 4 enoyl-CoA, which is oxidized to the trans-⌬ 2 , cis-⌬ 4 species by acyl-CoA dehydrogenase.The subsequent action of 2,4-dienoyl-CoA reduc- tase yields the trans-⌬ 3 product, which is converted by enoyl-CoA isomerase to the trans-⌬ 2 form. Normal -oxidation then produces five molecules of acetyl-CoA. 716 Chapter 23 Fatty Acid Catabolism branches at odd-numbered carbons are not effective substrates for -oxidation. For such species, ␣-oxidation is a useful alternative. Consider phytol, a breakdown prod- uct of chlorophyll that occurs in the fat of ruminant animals such as sheep and cows and also in dairy products. Ruminants oxidize phytol to phytanic acid, and digestion of phytanic acid from dairy products is thus an important dietary consideration for humans. The methyl group at C-3 will block -oxidation, but, as shown in Figure 23.24, phytanic acid ␣-hydroxylase places an OOH group at the ␣-carbon, and phytanic acid ␣-oxidase decarboxylates it to yield pristanic acid. The CoA ester of this metabolite can undergo -oxidation in the normal manner. The terminal product, isobutyryl-CoA, can be sent into the TCA cycle by conversion to succinyl-CoA. -Oxidation of Fatty Acids Yields Small Amounts of Dicarboxylic Acids In the endoplasmic reticulum of eukaryotic cells, the oxidation of the terminal car- bon of a normal fatty acid—a process termed omega oxidation (-oxidation)—can O C  C ␣ CRCH 2 SCoA H H HH O CCCRCH 2 SCoA H H 2 O 2 O 2 H H 2 O + 1 O 2 2 Fatty acyl-CoA [ FAD ] Acyl-CoA Oxidase [ FADH 2 ] trans-Δ 2 -Enoyl-CoA FIGURE 23.23 The acyl-CoA oxidase reaction in peroxi- somes. Electrons captured as FADH 2 are used to pro- duce the hydrogen peroxide required for degradative processes in peroxisomes and thus are not available for eventual generation of ATP. (Compare this reaction with the acyl-coA dehydrogenase of mitochondria shown in Figure 23.12.) CH 3 O – CH 3 (CH CH 2 CH 2 CH 2 ) 3 CCHCH 2 OH CH 3 CH 3 CH 3 (CH CH 2 CH 2 CH 2 ) 3 CH CH 2 C CH 3 O O – CH 3 CH 3 (CH CH 2 CH 2 CH 2 ) 3 CH CH C CH 3 O OH O – CH 3 CH 3 (CH CH 2 CH 2 CH C CH 3 O CH 3 CH 3 CH 2 CH 2 ) 3 CH 2 ) 3 CH C O SCoA CH 3 CH C O SCoACH 3 + C O SCoA CH 3 + C O SCoA CH 3 CH 2 P P + + (CH CH 2 CH 3 CoA ATP AMP CO 2 H 2 O Phytol Phytanic acid Phytanic acid ␣-hydroxylase Phytanic acid ␣-oxidase Pristanic acid Acyl-CoA synthase Six cycles of -oxidation Isobutyryl-CoA 3 Acetyl-CoA 3 Propionyl-CoA FIGURE 23.24 Branched-chain fatty acids are oxidized by ␣-oxidation, as shown for phytanic acid.The product of the phytanic acid oxidase, pristanic acid, is a suitable substrate for normal -oxidation.Isobutyryl-CoA and propionyl-CoA can both be converted to succinyl-CoA, which can enter the TCA cycle. 23.6 What Are Ketone Bodies, and What Role Do They Play in Metabolism? 717 lead to the synthesis of small amounts of dicarboxylic acids (Figure 23.25). Cytochrome P-450, a monooxygenase enzyme that requires NADPH as a coenzyme and uses O 2 as a substrate, places a hydroxyl group at the terminal carbon. Subse- quent oxidation to a carboxyl group produces a dicarboxylic acid. Either end can form an ester linkage to CoA and be subjected to -oxidation, producing a variety of smaller dicarboxylic acids. (Cytochrome P-450–dependent monooxygenases also play an important role as agents of detoxication, the degradation and metab- olism of toxic hydrocarbon agents.) 23.6 What Are Ketone Bodies, and What Role Do They Play in Metabolism? Ketone Bodies Are a Significant Source of Fuel and Energy for Certain Tissues Most of the acetyl-CoA produced by the oxidation of fatty acids in liver mito- chondria undergoes further oxidation in the TCA cycle, as stated earlier. How- ever, some of this acetyl-CoA is converted to three important metabolites: ace- tone, acetoacetate, and -hydroxybutyrate. The process is known as ketogenesis, and these three metabolites are traditionally known as ketone bodies, despite the fact that -hydroxybutyrate does not contain a ketone function. These three metabolites are synthesized primarily in the liver but are important sources of fuel and energy for many tissues, including brain, heart, and skeletal muscle. The brain, for example, normally uses glucose as its source of metabolic energy. How- ever, during periods of starvation, ketone bodies may be the major energy source for the brain. Acetoacetate and 3-hydroxybutyrate are normal substrates for kid- ney cortex and for heart muscle. Ketone body synthesis occurs only in the mitochondrial matrix. The reactions re- sponsible for the formation of ketone bodies are shown in Figure 23.26. The first HUMAN BIOCHEMISTRY Refsum’s Disease Is a Result of Defects in ␣-Oxidation The ␣-oxidation pathway is defective in Refsum’s disease, an in- herited metabolic disorder that results in defective night vision, tremors, and other neurologic abnormalities. These symptoms are caused by accumulation of phytanic acid in the body. Treatment of Refsum’s disease requires a diet free of chlorophyll, the precursor of phytanic acid. This regimen is difficult to implement because all green vegetables and even meat from plant-eating animals, such as cows, pigs, and poultry, must be excluded from the diet. CO (CH 2 ) 10 (CH 2 ) 10 CH 3 O – COO – COO – -oxidation FIGURE 23.25 Dicarboxylic acids can be formed by oxi- dation of the omega carbon of fatty acids in a cyto- chrome P-450–dependent reaction. HUMAN BIOCHEMISTRY Large Amounts of Ketone Bodies Are Produced in Diabetes Mellitus Diabetes mellitus is the most common endocrine disease and the third leading cause of death in the United States, with approxi- mately 6 million diagnosed cases and an estimated 4 million more borderline but undiagnosed cases. Diabetes is characterized by an abnormally high level of glucose in the blood. In type 1 diabetes (representing 10% or less of all cases), elevated blood glucose re- sults from inadequate secretion of insulin by the islets of Langer- hans in the pancreas. Type 2 diabetes (at least 90% of all cases) results from an insensitivity to insulin. Type 2 diabetics produce normal or even elevated levels of insulin, but their cells are not responsive to insulin, often due to a shortage of insulin receptors (see Chapter 32). In both cases, transport of glucose into muscle, liver, and adipose tissue is significantly reduced, and despite abundant glucose in the blood, the cells are metabolically starved. They respond by turning to increased gluconeogenesis and ca- tabolism of fat and protein. In type 1 diabetes, increased gluco- neogenesis consumes most of the available oxaloacetate, but breakdown of fat (and, to a lesser extent, protein) produces large amounts of acetyl-CoA. This increased acetyl-CoA would nor- mally be directed into the TCA cycle, but with oxaloacetate in short supply, it is used instead for production of unusually large amounts of ketone bodies. Acetone can often be detected on the breath of type 1 diabetics, an indication of high plasma levels of ketone bodies. 718 Chapter 23 Fatty Acid Catabolism reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl- CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in -oxidation, but here it runs in reverse. The second reaction adds an- other molecule of acetyl-CoA to give 3-hydroxy-3-methylglutaryl-CoA, commonly ab- breviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 24. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, -hydroxybutyrate dehydrogenase, then can re- duce acetoacetate to -hydroxybutyrate. Acetoacetate and -hydroxybutyrate are transported through the blood from liver to target organs and tissues, where they are converted to acetyl-CoA (Figure 23.27). Ketone bodies are easily transportable forms of fatty acids that move through the cir- culatory system without the need for complexation with serum albumin and other fatty acid- binding proteins. CH 3 C O SCoA CH 3 C O CH 2 C O SCoA CH 3 C O CH 2 C CH 2 CH 3 COO – COO – H OH Succinate Succinyl-CoA H + NAD + NADH + CoA CoA Thiolase Acetoacetyl-CoA Acetoacetate -Hydroxybutyrate -Hydroxybutyrate dehydrogenase -Ketoacyl-CoA transferase 2 Acet yl-CoA FIGURE 23.27 Reconversion of ketone bodies to acetyl- CoA in the mitochondria of many tissues (other than liver) provides significant metabolic energy. OH CH 3 O – + CH 3 C O CH 3 C O CH 2 C O CH 3 C O C O – OCH 2 C CH 2 C O CH 3 C O CH 2 C O CH 3 C O C O CH 3 CH 3 Acetone C CH 2 CH 3 C O O – H OH H + 2 H 2 O CoA SCoA CoA SCoA CoA CoA SCoA SCoA NAD + NADH CO 2 + Thiolase Acetoacetyl-CoA HMG -CoA synthase 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) Acetoacetate -Hydroxybutyrate dehydrogenase -H y drox y but y rate HMG-CoA lyase FIGURE 23.26 The formation of ketone bodies, synthesized primarily in liver mitochondria. SUMMARY 23.1 How Are Fats Mobilized from Dietary Intake and Adipose Tissue? Triacylglycerols are a major source of fatty acids in the diet, and they are also our principal stored energy reserve in adipose tissue. Hormone messengers such as adrenaline, glucagon, and ACTH bind to receptors on the plasma membrane of adipose cells and lead to the activation of a triacylglycerol lipase that hydrolyzes a fatty acid from C-1 or C-3 of tri- acylglycerols. Subsequent actions of diacylglycerol lipase and monoacyl- glycerol lipase yield fatty acids and glycerol. The cell then releases the fatty acids into the blood, where they are carried to sites of utilization. Dietary triacylglycerols are degraded by lipases and esterases in the stomach and duodenum. Pancreatic lipase cleaves fatty acids from the C-1 and C-3 positions of triacylglycerols, and other lipases and es- terases attack the C-2 position. Bile salts act as detergents to emulsify the triacylglycerols and facilitate the hydrolytic activity of the lipases and esterases. 23.2 How Are Fatty Acids Broken Down? The process of -oxidation begins with the formation of a thiol ester bond between the fatty acid and the thiol group of coenzyme A, catalyzed by acyl-CoA synthetase. The en- zymes of the -oxidation pathway are located in the mitochondrial ma- trix. Short-chain fatty acids are transported into the matrix as free acids and form the acyl-CoA derivatives there. However, long-chain fatty acyl- CoA derivatives must first be converted to acylcarnitine derivatives, which are transported across the inner membrane by a translocase. On the ma- trix side of the inner membrane, a second acyl carnitine transferase re- forms the fatty acyl-CoA. The process of -oxidation involves a recurring cycle of four steps. A double bond is formed, water is added across the double bond, and the resulting alcohol is oxidized to a carbonyl group. The fourth reaction of the cycle cleaves the resulting -keto ester, pro- ducing an acetate unit and leaving a fatty acid chain that is two carbons shorter. 23.3 How Are Odd-Carbon Fatty Acids Oxidized? Humans and animals metabolize odd-carbon fatty acids via the -oxidation pathway, with the fi- nal product being propionyl-CoA. Three specialized enzymes then con- vert propionyl-CoA to succinyl-CoA, a TCA cycle intermediate. The path- way involves an initial ATP-dependent carboxylation (by propionyl-CoA carboxylase) at the ␣-carbon of propionyl-CoA to produce D-methyl- malonyl-CoA, which is converted to the L-isomer by methylmalonyl-CoA epimerase. The L-isomer is the substrate for methylmalonyl-CoA mutase, which catalyzes a migration of a carbonyl-CoA group from one carbon to its neighbor, yielding succinyl-CoA. 23.4 How Are Unsaturated Fatty Acids Oxidized? Two additional mito- chondrial enzymes—an isomerase and a novel reductase—are required to handle the cis-double bonds of naturally occurring fatty acids. Con- sider the breakdown of oleic acid. The reactions of -oxidation proceed normally through three cycles, producing three molecules of acetyl-CoA and leaving the product cis-⌬ 3 -dodecenoyl-CoA. This intermediate is not a substrate for acyl-CoA dehydrogenase. Instead, enoyl-CoA isomerase re- arranges the cis-⌬ 3 double bond to a trans-⌬ 2 double bond, which can pro- ceed through the normal route of -oxidation. 23.5 Are There Other Ways to Oxidize Fatty Acids? Organelles other than mitochondria, including peroxisomes and glyoxysomes, also carry out -oxidation processes. Peroxisomal -oxidation is similar to mito- chondrial -oxidation, except that the initial double-bond formation is catalyzed by an FAD-dependent acyl-CoA oxidase, which transfers the liberated electrons directly to oxygen instead of the electron-transport chain. Short-chain products must be transferred to the mitochondria for further breakdown. Similar -oxidation enzymes are also found in glyoxysomes. Branched-chain fatty acids with alkyl branches at odd-numbered carbons are not effective substrates for -oxidation. For such species, ␣-oxidation is a useful alternative. Ruminants oxidize phytol to phy- tanic acid. The methyl group at C-3 will block -oxidation, but phytanic acid ␣-hydroxylase places an OOH group at the ␣-carbon, and phy- tanic acid ␣-oxidase decarboxylates it to yield pristanic acid. The CoA ester of this metabolite can undergo -oxidation in the normal man- ner. The terminal product, isobutyryl-CoA, can be sent into the TCA cy- cle by conversion to succinyl-CoA. 23.6 What Are Ketone Bodies, and What Role Do They Play in Metabo- lism? Acetone, acetoacetate, and -hydroxybutyrate are known as ke- tone bodies. These three metabolites are synthesized primarily in the liver but are important sources of fuel and energy for many tissues, in- cluding brain, heart, and skeletal muscle. During periods of starvation, ketone bodies may be the major energy source for the brain. Aceto- acetate and 3-hydroxybutyrate are normal substrates for kidney cortex and for heart muscle. Problems 719 PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. Calculate the volume of metabolic water available to a camel through fatty acid oxidation if it carries 30 pounds of triacylglycerol in its hump. 2. Calculate the approximate number of ATP molecules that can be obtained from the oxidation of cis-11-heptadecenoic acid to CO 2 and water. 3. Phytanic acid, the product of chlorophyll that causes problems for individuals with Refsum’s disease, is 3,7,11,15-tetramethyl hexadec- anoic acid. Suggest a route for its oxidation that is consistent with what you have learned in this chapter. (Hint: The methyl group at C-3 effectively blocks hydroxylation and normal -oxidation. You may wish to initiate breakdown in some other way.) 4. Even though acetate units, such as those obtained from fatty acid oxidation, cannot be used for net synthesis of carbohydrate in animals, labeled carbon from 14 C-labeled acetate can be found in newly synthesized glucose (for example, in liver glycog en) in ani- mal tracer studies. Explain how this can be. Which carbons of glu- cose would you expect to be the first to be labeled by 14 C-labeled acetate? 5. Human serum albumin (66.4 kD) is a soluble protein present in blood at 0.75 mM or so. Among other functions, albumin acts as the major transport vehicle for fatty acids in the circulation, carrying fatty acids from storage sites in adipose tissue to their sites of oxi- dation in liver and muscle. The albumin molecule has up to 11 dis- tinct binding sites. Consult the biochemical literature to learn about the fatty acid–binding sites of albumin. Where are they lo- cated on the protein? What are their relative affinities? (Two suit- able references with which to begin your study are Bhattacharya, A. A., Grüne, T., and Curry, S., 2000. Crystallographic analysis reveals common modes of binding of medium- and long-chain fatty acids in human serum albumin. Journal of Molecular Biology 303:721–732; and Simard, J. R., Zunszain, P. A., Ha, C-E., et al., 2005. Locating high-affinity fatty acid-binding sites on albumin by X-ray crystallog- raphy and NMR spectroscopy. Proceedings of the National Academy of Sciences U.S.A. 102:17958–17963.) 6. Overweight individuals who diet to lose weight often view fat in neg- ative ways because adipose tissue is the repository of excess caloric intake. However, the “weighty” consequences might be even worse if excess calories were stored in other forms. Consider a person who is 10 pounds “overweight,” and estimate how much more he or she 720 Chapter 23 Fatty Acid Catabolism would weigh if excess energy were stored in the form of carbohy- drate instead of fat. 7. What would be the consequences of a deficiency in vitamin B 12 for fatty acid oxidation? What metabolic intermediates might accumulate? 8. Write properly balanced chemical equations for the oxidation to CO 2 and water of (a) myristic acid, (b) stearic acid, (c) ␣-linolenic acid, and (d) arachidonic acid. 9. How many tritium atoms are incorporated into acetate if a molecule of palmitic acid is oxidized in 100% tritiated water? 10. What would be the consequences of a carnitine deficiency for fatty acid oxidation? 11. The ruby-throated hummingbird flies 500 miles nonstop across the Gulf of Mexico. The flight takes 10 hours at 50 mph. The hum- mingbird weighs about 4 grams at the start of the flight and about 2.7 grams at the end. Assuming that all the lost weight is fat burned for the flight, calculate the total energy required by the humming- bird in this prodigious flight. Does anything about the results of this calculation strike you as unusual? 12. Energy production in animals is related to oxygen consumption. The ruby-throated hummingbird consumes about 250 mL of oxygen per hour during its migration across the Gulf of Mexico. Use this number and the data in problem 11 to determine a conversion fac- tor for energy expended per liter of oxygen consumed. If a human being consumes 12.7 kcal/min while running 8-minute miles, how long could a human run on the energy that the hummingbird con- sumes in its trans-Gulf flight? How many 8-minute miles would a per- son have to run to lose 1 pound of body fat? 13. Write a reasonable mechanism for the HMG-CoA synthase reaction shown in Figure 23.26. 14. The methylmalonyl-CoA mutase reaction (see Figure 23.18) in- volves vitamin B 12 as a coenzyme. Write a reasonable mechanism for this reaction. (Hint: The reaction begins with abstraction of a hy- drogen atom—that is, a proton plus an electron—from the sub- strate by vitamin B 12 . Consider the chemistry shown in A Deeper Look: The Activation of Vitamin B 12 as you write your mechanism.) 15. Discuss the changes of the oxidation state of cobalt in the course of the methylmalonyl-CoA mutase reaction. Why do they occur as you suggested in your answer to problem 14? 16. Based on the mechanism for the methylmalonyl-CoA mutase (see problem 14), write reasonable mechanisms for the following reac- tions shown. 17. A popular dish in the Caribbean islands consists of “akee and salt fish.” Unripened akee fruit (from the akee tree, native to West Africa but brought to the Caribbean by African slaves) is quite poisonous. The unripened fruit contains hypoglycin (structure shown below), a metabolite that serves as a substrate for acyl-CoA dehydrogenase. However, the product of this reaction irreversibly inhibits the acyl- CoA dehydrogenase by reacting covalently with FAD on the enzyme. Consumption of unripened akee fruit can lead to vomiting and, in severe cases, convulsions, coma, and death. Write a reaction scheme to show the product of the acyl-CoA dehydrogenase reaction that reacts with FAD. H Ϫ OOC COO Ϫ C CH 2 CH H NH 3 ϩ H Ϫ OOC COO Ϫ C CH 3 CH NH 3 ϩ H C OHCH CH 3 H OH C H CH 3 H C H ϩ O H 2 O H C HCH CH 2 OH OHOH CH 2 OH C H H C H ϩ O H 2 O H C H CH 2 OHNH 3 ϩ C H CH 3 ϩ O NH 4 ϩ The akee tree. Mark W. Skinner © USDA-NRCS PLANTS Database CH 2 C COO – H NH 3 + CHCH 2 C CH 2 Hypoglycin A 18. In mammalian mitochondria, three of the enzymes in the membrane- bound -oxidation system are components of a multifunctional enzyme (MFE). Similarly, glyoxysomes (in plants), peroxisomes (in most species), and Gram-negative bacteria carry out several reactions of -oxidation via MFEs. In all these cases, the components of an MFE must cooperate to carry or “channel” the acyl-CoA substrate from one active site to the next in a cyclic fashion. The structure of the com- plete MFE from Pseudomonas fragi provides insights into how this channeling might occur. Consult the journal article that describes this structure (Ishikawa, M., Tsuchiya, D., et al., 2004. EMBO Journal 23:2745–2754), and explain in your own words how this substrate channeling occurs. Preparing for the MCAT Exam 19. Study Figure 23.12 and comment on why nature uses FAD/FADH 2 as a cofactor in the acyl-CoA dehydrogenase reaction rather than NAD ϩ /NADH. 20. Study Figure 23.9. Where else in metabolism have you seen the chemical strategy and logic of the -oxidation pathway? Why is it that these two pathways are carrying out the same chemistry? FURTHER READING General Arts, I., van de Putte, B., et al., 2000. Catechin contents of foods com- monly consumed in the Netherlands: 2. Tea, wine, fruit juices, and chocolate milk. Journal of Agriculture and Food Chemistry 48:1752–1757. Bieber, L. L., 1988. Carnitine. Annual Review of Biochemistry 88:261–283. Ferry, G., 1998. Dorothy Hodgkin: A Life. New York: Cold Spring Harbor Laboratory. Gurr, M. I., Harwood, J. L., et al., 2002. Lipid Biochemistry: An Introduc- tion. Oxford: Wiley-Blackwell. Murase, T., Haramisu, S., et al., 2005. Green tea extract improves en- durance capacity and increases muscle lipid oxidation in mice. American Journal of Physiology—Regulatory, Integrative and Comparative Physiology 288:R708–R715. Murase, K., Haramisu, S., et al., 2006. Green tea extract improves run- ning endurance in mice by stimulating lipid utilization during ex- ercise. American Journal of Physiology—Regulatory, Integrative and Com- parative Physiology 290:R1550–R1556. Schrauwen, P., Hoeks, J., et al., 2006. Putative function and physiologi- cal relevance of the mitochondrial uncoupling protein-3: Involve- ment in fatty acid metabolism? Progress in Lipid Research 45:17–41. Scriver, C. R., Beaudet, A. L., et al., 1995. The Metabolic and Molecular Bases of Inherited Disease, 7th ed. New York: McGraw-Hill. Vance, D. E., and Vance, J. E., 2008. Biochemistry of Lipids, Lipoproteins and Membranes, 5th ed. Amsterdam: Elsevier. -Oxidation Bartlett, K., and Eaton, S., 2004. Mitochondrial beta-oxidation. European Journal of Biochemistry 271:462–469. Kim, J J., and Battaile, K. P., 2002. Burning fat: The structural basis of fatty acid beta-oxidation. Current Opinion in Structural Biology 12: 721–728. Liang, X., Zhang, D., et al., 2001. Impact of the intramitochondrial en- zyme organization on fatty acid oxidation. Biochemical Society Trans- actions 29:279–282. McGarry, J. D., and Brown, N. F., 1997. The mitochondrial carnitine palmitoyltransferase system. European Journal of Biochemistry 244:1–14. Zechner, R., Strauss, J., et al., 2005. Lipolysis: Pathway under construc- tion. Current Opinion in Lipidology 16:333–340. Acyl-CoA Dehydrogenases Battaile, K. P., Molin-Case, J., et al., 2002. Crystal structure of rat short chain acyl-CoA dehydrogenase complexed with acetoacetyl-CoA. Journal of Biological Chemistry 277: 12200–12207. Ghisla, S., and Thorpe, C., 2004. Acyl-CoA dehydrogenases: A mecha- nistic overview. European Journal of Biochemistry 271:494–508. Kim, J J., and Miura, R., 2004. Acyl-CoA dehydrogenases and acyl-CoA oxidases. European Journal of Biochemistry 271:483–493. McAndrew, R. P., Wang, Y., et al., 2008. Structural basis for substrate fatty-acyl chain specificity: Crystal structure of human very-long- chain acyl-CoA dehydrogenase. Journal of Biological Chemistry 283: 9435–9443. Nakajima, Y., Miyahara, I., et al., 2002. Three-dimensional structure of the flavoenyzme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase. Journal of Biochemistry (Tokyo) 131:365–374. Rao, K. S., Albro, M., et al., 2006. Kinetic mechanism of glutaryl-CoA de- hydrogenase. Biochemistry 45:15853–15861. Regulation and Defects of Fatty Acid Oxidation Gregersen, N., Bross, P ., et al., 2004. Genetic defects in fatty acid beta- oxidation and acyl-CoA dehydrogenases. European Journal of Bio- chemistry 271:470–482. Macfarlane, S., and Macfarlane, G. T., 2003. Regulation of short-chain fatty acid production. Proceedings of the Nutrition Society 62:67–72. Oey, N. A., Den Boer, M. E. J., et al., 2005. Long-chain fatty acid oxida- tion during early human development. Pediatric Research 57:755–759. Shekhawat, P., Bennett, M. J., et al., 2003. Human placenta metabolizes fatty acids: Iimplications for fetal fatty acid oxidation disorders and maternal liver diseases. American Journal of Endocrinology and Metabo- lism 284:E1098–E1105. Branched-Chain and Unsaturated Fatty Acid Oxidation Graham, I. A., and Eastmond, P. J., 2002. Pathways of straight and branched chain fatty acid catabolism in higher plants. Progress in Lipid Research 41:156–181. Peroxisomes and Phytanic Acid Oxidation Grimaldi, P. A., 2007. Peroxisome proliferator-activated receptors as sen- sors of fatty acids and derivatives. Cell and Molecular Life Science 64: 2459–2464. Schrader, M., and Fahimi, H. D., 2008. The peroxisome: Still a mysteri- ous organelle. Histochemistry and Cell Biology 129:421–440. Wanders, R. J. A., 2004. Peroxisomes, lipid metabolism, and peroxiso- mal disorders. Molecular Genetics and Metabolism 83:16–27. Wanders, R. J. A., and Waterham, H. R., 2006. Biochemistry of mam- malian peroxisomes revisited. Annual Review of Biochemistry 75: 295–332. Wanders, R. J. A., Jansen, G. A., et al., 2001. Refsum disease, peroxi- somes and phytanic acid oxidation. Journal of Neuropathology and Ex- perimental Neurology 60:1021–1031. Ylianttila, M. S., Pursiainen, N. V., et al., 2006. Crystal structure of yeast peroxisomal multifunctional enzyme: Structural basis for substrate specificity of (3R)-hydroxyacyl-CoA dehydrogenase units. Journal of Molecular Biology 358:1286–1295. Metabolic Syndrome Forest, C., Corvol, P., et al., 2005. New developments in metabolic syn- drome. Biochemie 87:1–3. Medina-Gomez, G., and Vidal-Puig, A., 2005. Gateway to the metabolic syndrome. Nature Medicine 11:602–603. Phinney, S., 2005. Fatty acids, inflammation, and the metabolic syn- drome. American Journal of Clinical Nutrition 82:1151–1152. Multifunctional Enzyme Complexes Ishikawa, M., Tsuchiya, D., et al., 2004. Structural basis for channeling mechanism of a fatty acid ß-oxidation multienzyme complex. EMBO Journal 23:2745–2754. Further Reading 721 © Alissa Crandall/CORBIS 24 Lipid Biosynthesis We have already seen several cases in which the synthesis of a class of biomolecules is conducted differently from degradation (glycolysis versus gluconeogenesis and glycogen or starch breakdown versus polysaccharide synthesis, for example). Like- wise, the synthesis of fatty acids and other lipid components is different from their degradation. Fatty acid synthesis involves a set of reactions that follow a strategy dif- ferent in several ways from the corresponding degradative process: 1. Intermediates in fatty acid synthesis are linked covalently to the sulfhydryl groups of special proteins, the acyl carrier proteins (ACPs). In contrast, fatty acid break- down intermediates are bound to the OSH group of coenzyme A. 2. Fatty acid synthesis occurs in the cytosol, whereas fatty acid degradation takes place in mitochondria. 3. In animals, the enzymes of fatty acid synthesis are components of one long polypeptide chain, the fatty acid synthase, whereas no similar association exists for the degradative enzymes. (Plants and bacteria employ separate enzymes to carry out the biosynthetic reactions.) 4. The coenzyme for the oxidation–reduction reactions of fatty acid synthesis is NADP ϩ /NADPH, whereas degradation involves the NAD ϩ /NADH couple. 24.1 How Are Fatty Acids Synthesized? Formation of Malonyl-CoA Activates Acetate Units for Fatty Acid Synthesis The design strategy for fatty acid synthesis is this: 1. Fatty acid chains are constructed by the addition of two-carbon units derived from acetyl-CoA. 2. The acetate units are activated by formation of malonyl-CoA (at the expense of ATP). 3. The addition of two-carbon units to the growing chain is driven by decarboxyla- tion of malonyl-CoA. 4. The elongation reactions are repeated until the growing chain reaches 16 carbons in length (palmitic acid). 5. Other enzymes then add double bonds and additional carbon units to the chain. Fatty Acid Biosynthesis Depends on the Reductive Power of NADPH The net reaction for the formation of palmitate from acetyl-CoA is Acetyl-CoA ϩ 7 malonyl-CoA Ϫ ϩ 14 NADPH ϩ 13 H ϩ ϩ H 2 O⎯⎯→ palmitate Ϫ ϩ 7 HCO 3 Ϫ ϩ 8 CoASH ϩ 14 NADP ϩ Walruses basking on the beach. To everything there is a season, and a time for every purpose under heaven…A time to break down and a time to build up. Ecclesiastes 3:1–3 KEY QUESTIONS 24.1 How Are Fatty Acids Synthesized? 24.2 How Are Complex Lipids Synthesized? 24.3 How Are Eicosanoids Synthesized, and What Are Their Functions? 24.4 How Is Cholesterol Synthesized? 24.5 How Are Lipids Transported Throughout the Body? 24.6 How Are Bile Acids Biosynthesized? 24.7 How Are Steroid Hormones Synthesized and Utilized? ESSENTIAL QUESTION We turn now to the biosynthesis of lipid structures.We begin with a discussion of the biosynthesis of fatty acids, stressing the basic pathways, additional means of elongation, mechanisms for the introduction of double bonds, and regulation of fatty acid synthesis. Sections then follow on the biosynthesis of glycerophospho- lipids, sphingolipids, eicosanoids, and cholesterol.The transport of lipids through the body in lipoprotein complexes is described, and the chapter closes with discussions of the biosynthesis of bile salts and steroid hormones. What are the pathways of lipid synthesis in biological systems? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login.