Chapter 24

Fatty Acid Catabolism

The hummingbird’s tremendous capacity to store and use fatty
acids enables it to make migratory journeys of remarkable
distances. (Two Hummingbirds Lithograph; The Academy of Natural
Sciences of Philadelphia/Corbis Images)

Fatty acids represent the principal form of stored energy for many organisms. There are two important advantages to storing energy in the form of fatty acids. (1) The carbon in fatty acids (mostly -CH₂Ogroups) is almost completely reduced compared to the carbon in other simple biomolecules (sugars, amino acids). Therefore, oxidation of fatty acids will yield more energy (in the form of ATP) than any other form of carbon. (2) Fatty acids are not generally hydrated as mono- and polysaccharides are, and thus can pack more closely in storage tissues. This chapter will be devoted to several important aspects of fatty acid catabolism. Lipid biosynthetic processes will be considered in Chapter 25.

24.1 × Mobilization of Fats from Dietary Intake and Adipose Tissue

Modern Diets Are Often High in Fat

Fatty acids are acquired readily in the diet and can also be made from carbohydrates and the carbon skeletons of amino acids. Fatty acids provide 30% to 60% of the calories in the diets of most Americans. For our caveman and cavewoman ancestors, the figure was probably closer to 20%. Dairy products were apparently not part of their diet, and the meat they consumed (from fast-moving animals) was low in fat. In contrast, modern domesticated cows and pigs are actually bred for high fat content (and better taste). However, woolly mammoth burgers and saber-toothed tiger steaks are hard to find these days—even in the gourmet sections of grocery stores—and so, by default, we consume (and metabolize) large quantities of fatty acids.

Triacylglycerols Are a Major Form of Stored Energy in Animals

Although some of the fat in our diets is in the form of phospholipids, triacylglycerols are a major source of fatty acids. Triacylglycerols are also our principal stored energy reserve. As shown in Table 24.1, the energy available in stores of fat in the average person far exceeds the energy available from protein, glycogen, and glucose. Overall, fat accounts for approximately 83% of available energy, partly because more fat is stored than protein and carbohydrate, and partly because of the substantially higher energy yield per gram for fat compared with protein and carbohydrate. Complete combustion of fat yields about 37 kJ/g, compared with about 16 to 17 kJ/g for sugars, glycogen, and amino acids. In animals, fat is stored mainly as triacylglycerols in specialized cells called adipocytes or adipose cells. As shown in Figure 24.1, triacylglycerols, aggregated to form large globules, occupy most of the volume of adipose cells. Much smaller amounts of triacylglycerols are stored as small, aggregated globules in muscle tissue.

Figure 24.1 · Scanning electron micrograph of an adipose cell (fat cell). Globules of triacylglycerols occupy most of the volume of such cells. (Prof. P. Motta, Dept. of Anatomy, University “La Sapienza,” Rome/Science Photo Library/Photo Researchers, Inc.)

Hormones Signal the Release of Fatty Acids from Adipose Tissue

The pathways for liberation of fatty acids from triacylglycerols, either from adipose cells or from the diet, are shown in Figures 24.2 and 24.3. Fatty acids are mobilized from adipocytes in response to hormone messengers such as adrenaline, glucagon, and ACTH (adrenocorticotropic hormone). These signal mole-cules bind to receptors on the plasma membrane of adipose cells and lead to the activation of adenylyl cyclase, which forms cyclic AMP from ATP. (Second messengers and hormonal signaling are discussed in Chapter 34.) In adipose cells, cAMP activates protein kinase A, which phosphorylates and activates a riacylglycerol lipase (also termed hormone-sensitive lipase) that hydrolyzes a fatty acid from C-1 or C-3 of triacylglycerols. Subsequent actions of diacylglycerol lipase and monoacylglycerol lipase yield fatty acids and glycerol. The cell then releases the fatty acids into the blood, where they are carried (in complexes with serum albumin) to sites of utilization.

Figure 24.2 · Liberation of fatty acids from triacylglycerols in adipose tissue is hormone-dependent.

Figure 24.3 · (a) A duct at the junction of the pancreas and duodenum secretes pancreatic juice into the duodenum, the first portion of the small intestine. (b) Hydrolysis of triacylglycerols by pancreatic and intestinal lipases. Pancreatic lipases cleave fatty acids at the C-1 and C-3 positions. Resulting monoacylglycerols with fatty acids at C-2 are hydrolyzed by intestinal lipases. Fatty acids and monoacylglycerols are absorbed through the intestinal wall and assembled into lipoprotein aggregates termed chylomicrons (discussed in Chapter 25).

Degradation of Dietary Fatty Acids Occurs Primarily in the Duodenum

Dietary triacylglycerols are degraded to a small extent (via fatty acid release) by lipases in the low-pH environment of the stomach, but mostly pass untouched into the duodenum. Alkaline pancreatic juice secreted into the duodenum (Figure 24.3a) raises the pH of the digestive mixture, allowing hydrolysis of the triacylglycerols by pancreatic lipase and by nonspecific esterases, which hydrolyze the fatty acid ester linkages. Pancreatic lipase cleaves fatty acids from the C-1 and C-3 positions of triacylglycerols, and other lipases and esterases attack the C-2 position (Figure 24.3b). These processes depend upon the presence of bile salts, a family of carboxylic acid salts with steroid backbones (see also Chapter 25). These agents act as detergents to emulsify the triacylglycerols and facilitate the hydrolytic activity of the lipases and esterases. Short-chain fatty acids (10 carbons or less) released in this way are absorbed directly into the villi of the intestinal mucosa, whereas long-chain fatty acids, which are less soluble, form mixed micelles with bile salts, and are carried in this fashion to the surfaces of the epithelial cells that cover the villi (Figure 24.4). The fatty acids pass into the epithelial cells, where they are condensed with glycerol to form new triacylglycerols. These triacylglycerols aggregate with lipoproteins to form particles called chylomicrons, which are then transported into the lymphatic system and on to the bloodstream, where they circulate to the liver, lungs, heart, muscles, and other organs (Chapter 25). At these sites, the triacylglycerols are hydrolyzed to release fatty acids, which can then be oxidized in a highly exergonic metabolic pathway known as b-oxidation.

Figure 24.4 · In the small intestine, fatty acids combine with bile salts in mixed micelles, which deliver fatty acids to epithelial cells that cover the intestinal villi. Triacylglycerols are formed within the epithelial cells.

24.2 × b-Oxidation of Fatty Acids

Figure 24.5 · The oxidative breakdown of phenyl fatty acids observed by Franz Knoop. He observed that fatty acid analogs with even numbers of carbon atoms yielded phenyl acetate, whereas compounds with odd numbers of carbon atoms produced only benzoate.

Franz Knoop and the Discovery of b-Oxidation

The earliest clue to the secret of fatty acid oxidation and breakdown came in the early 1900s, when Franz Knoop carried out experiments in which he fed dogs fatty acids in which the terminal methyl group had been replaced with a phenyl ring (Figure 24.5). Knoop discovered that fatty acids containing an even number of carbon atoms were broken down to yield phenyl acetate as the final product, whereas fatty acids with an odd number of carbon atoms yielded benzoate as the final product (Figure 24.5). From these experiments, Knoop concluded that the fatty acids must be degraded by oxidation at the b-carbon (Figure 24.6), followed by cleavage of the C_a¾C_b bond. Repetition of this process yielded 2-carbon units, which Knoop assumed must be acetate. Much later, Albert Lehninger showed that this degradative process took place in the mitochondria, and F. Lynen and E. Reichart showed that the 2-carbon unit released is acetyl-CoA, not free acetate. Because the entire process begins with oxidation of the carbon that is “b” to the carboxyl carbon, the process has come to be known as b-oxidation.

Figure 24.6 · Fatty acids are degraded by repeated cycles of oxidation at the b-carbon and cleavage of the C_a¾C_b bond to yield acetate units.

Coenzyme A Activates Fatty Acids for Degradation

Figure 24.7 · The acyl-CoA synthetase reaction activates fatty acids for b-oxidation. The reaction is driven by hydrolysis of ATP to AMP and pyrophosphate and by the subsequent hydrolysis of pyrophosphate.

Figure 24.8 · The mechanism of the acyl-CoA synthetase reaction involves fatty acid carboxylate attack on ATP to form an acyl-adenylate intermediate. The fatty acyl CoA thioester product is formed by CoA attack on this intermediate.

The process of b-oxidation begins with the formation of a thiol ester bond between the fatty acid and the thiol group of coenzyme A. This reaction, shown in Figure 24.7, is catalyzed by acyl-CoA synthetase, which is also called acyl-CoA ligase or fatty acid thiokinase. This condensation with CoA activates the fatty acid for reaction in the b-oxidation pathway. For long-chain fatty acids, this reaction normally occurs at the outer mitochondrial membrane, prior to entry of the fatty acid into the mitochondrion, but it may also occur at the surface of the endoplasmic reticulum. Short- and medium-length fatty acids undergo this activating reaction in the mitochondria. In all cases, the reaction is accompanied by the hydrolysis of ATP to form AMP and pyrophosphate. As shown in Figure 24.7, the two combined reactions have a net DG°' of about -0.8 kJ/mol, so that the reaction is favorable but easily reversible. However, there is more to the story. As we have seen in several similar cases, the pyrophosphate produced in this reaction is rapidly hydrolyzed by inorganic pyrophosphatase to two molecules of phosphate, with a net DG°' of about -33.6 kJ/mol. Thus, pyrophosphate is maintained at a low concentration in the cell (usually less than 1 mM) and the synthetase reaction is strongly promoted. The mechanism of the acyl-CoA synthetase reaction is shown in Figure 24.8 and involves attack of the fatty acid carboxylate on ATP to form an acyladenylate intermediate, which is subsequently attacked by CoA, forming a fatty acyl-CoA thioester.

Carnitine Carries Fatty Acyl Groups Across the Inner Mitochondrial Membrane

All of the other enzymes of the b-oxidation pathway are located in the mitochondrial matrix. Short-chain fatty acids, as already mentioned, are transported into the matrix as free acids and form the acyl-CoA derivatives there. However, long-chain fatty acyl-CoA derivatives cannot be transported into the matrix directly. These long-chain derivatives must first be converted to acylcarnitine derivatives, as shown in Figure 24.9. Carnitine acyltransferase I, located on the outer side of the inner mitochondrial membrane, catalyzes the formation of the O-acylcarnitine, which is then transported across the inner membrane by a translocase. At this point, the acylcarnitine is passed to carnitine acyltransferase II on the matrix side of the inner membrane, which transfers the fatty acyl group back to CoA to re-form the fatty acyl-CoA, leaving free carnitine, which can return across the membrane via the translocase.
      Several additional points should be made. First, although oxygen esters usually have lower group-transfer potentials than thiol esters, the O¾acyl bonds in acylcarnitines have high group-transfer potentials, and the transesterification reactions mediated by the acyltransferases have equilibrium constants close to 1. Second, note that eukaryotic cells maintain separate pools of CoA in the mitochondria and in the cytosol. The cytosolic pool is utilized principally in fatty acid biosynthesis (Chapter 25), and the mitochondrial pool is important in the oxidation of fatty acids and pyruvate, as well as some amino acids.

Figure 24.9 · The formation of acylcarnitines and their transport across the inner mitochondrial membrane. The process involves the coordinated actions of carnitine acyltransferases on both sides of the membrane and of a translocase that shuttles O-acylcarnitines across the membrane.

b-Oxidation Involves a Repeated Sequence of Four Reactions

Figure 24.10 · The b-oxidation of saturated fatty acids involves a cycle of four enzyme-catalyzed reactions. Each cycle produces single molecules of FADH₂, NADH, and acetyl-CoA and yields a fatty acid shortened by two carbons. (The delta [D] symbol connotes a double bond, and its superscript indicates the lower-numbered carbon involved.)

For saturated fatty acids, the process of b-oxidation involves a recurring cycle of four steps, as shown in Figure 24.10. The overall strategy in the first three steps is to create a carbonyl group on the b-carbon by oxidizing the C_a¾C_b bond to form an olefin, with subsequent hydration and oxidation. In essence, this cycle is directly analogous to the sequence of reactions converting succinate to oxaloacetate in the TCA cycle. The fourth reaction of the cycle cleaves the b-keto ester in a reverse Claisen condensation, producing an acetate unit and leaving a fatty acid chain that is two carbons shorter than it began. (Recall from Chapter 20 that Claisen condensations involve attack by a nucleophilic agent on a carbonyl carbon to yield a b-keto acid.)

Acyl-CoA Dehydrogenase—The First Reaction of b-Oxidation

Figure 24.11 · The acyl-CoA dehydrogenase reaction. The two electrons removed in this oxidation reaction are delivered to the electron transport chain in the form of reduced coenzyme Q (UQH₂).

The first reaction, the oxidation of the C_a¾C_b bond, is catalyzed by acyl-CoA dehydrogenases, a family of three soluble matrix enzymes (with molecular weights of 170 to 180 kD), which differ in their specificity for either long-, medium-, or short-chain acyl-CoAs. They carry noncovalently (but tightly) bound FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure 24.11, FADH₂ transfers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is reoxidized by a specific oxidoreductase (an iron-sulfur protein), which in turn sends the electrons on to the electron transport chain at the level of coenzyme Q. As always, mitochondrial oxidation of FAD in this way eventually results in the net formation of about 1.5 ATP. Themechanism of the acyl-CoA dehydrogenase (Figure 24.12) involves deprotonation of the fattyacid chain at the a-carbon, followed by hydride transfer from the b-carbon to FAD. The structure of the medium-chain dehydrogenase from pig liver places an FAD molecule in an extended conformation between a bundle of a-helices and a distorted b-barrel (Figure 24.13).

Figure 24.12 · The mechanism of acyl-CoA dehydrogenase. Removal of a proton from the a-C is followed by hydride transfer from the b-carbon to FAD.

Figure 24.13 · The subunit structure of medium chain acyl-CoA dehydrogenase from pig liver mitochondria. Note the location of the bound FAD (red). (Adapted from Kim, J.-T., and Wu, J., 1988. Structure of the medium-chain acyl-CoA dehydrogenase from pig liver mitochondria at 3-Å resolution. Proceedings of the National Academy of Sciences, USA 85:6671-6681.)

A Deeper Look
The Akee Tree
The akee (also spelled ackee) tree is native to West Africa and was brought to the Caribbean by African slaves. It was introduced to science by William Bligh, captain of the infamous sailing ship the Bounty, and its botanical name is (appropriately) Blighia sapida (the latter name from the Latin sapidus meaning “tasty”). A popular dish in the Caribbean consists of akee and salt fish. “Akee, rice, salt fish are nice, And the rum is fine any time of year.” —From the song  Jamaica Farewell

A Metabolite of Hypoglycin from Akee Fruit Inhibits Acyl-CoA Dehydrogenase

The unripened fruit of the akee tree contains hypoglycin, a rare amino acid (Figure 24.14). Metabolism of hypoglycin yields methylenecyclopropylacetyl-CoA (MCPA-CoA). Acyl-CoA dehydrogenase will accept MCPA-CoA as a substrate, removing a proton from the a-carbon to yield an intermediate that irreversibly inactivates acyl-CoA dehydrogenase by reacting covalently with FAD on the enzyme. For this reason, consumption of unripened akee fruit can lead to vomiting and, in severe cases, convulsions, coma, and death. The condition is most severe in individuals with low levels of acyl-CoA dehydrogenase.

Figure 24.14 · The conversion of hypoglycin from akee fruit to a form that inhibits acyl-CoA dehydrogenase. 

Enoyl-CoA Hydratase Adds Water Across the Double Bond

Figure 24.15 · The conversion of trans- and cis-enoyl CoA derivatives to L- and D-b-hydroxyacyl CoA, respectively. These reactions are catalyzed by enoyl-CoA hydratases (also called crotonases), enzymes that vary in their acyl-chain length specificity. A recently discovered enzyme converts trans-enoyl-CoA directly to D-b-hydroxyacyl-CoA.

The next step in b-oxidation is the addition of the elements of H₂O across the new double bond in a stereospecific manner, yielding the corresponding hydroxyacyl-CoA (Figure 24.15). The reaction is catalyzed by enoyl-CoA hydratase. At least three different enoyl-CoA hydratase activities have been detected in various tissues. Also called crotonases, these enzymes specifically convert trans-enoyl-CoA derivatives to L-b-hydroxyacyl-CoA. As shown in Figure 24.15, these enzymes will also metabolize cis-enoyl-CoA (at slower rates) to give specifically D-b-hydroxyacyl-CoA. Recently, a novel enoyl-CoA hydratase was discovered, which converts trans-enoyl-CoA to D-b-hydroxyacyl-CoA, as shown in Figure 24.15.

L-Hydroxyacyl-CoA Dehydrogenase Oxidizes the b-Hydroxyl Group

The third reaction of this cycle is the oxidation of the hydroxyl group at the b-position to produce a b-ketoacyl-CoA derivative. This second oxidation reaction is catalyzed by L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD⁺ as a coenzyme. NADH produced in this reaction represents metabolic energy. Each NADH produced in mitochondria by this reaction drives the synthesis of 2.5 molecules of ATP in the electron transport pathway. L-Hydroxyacyl-CoA dehydrogenase shows absolute specificity for the L-hydroxyacyl isomer of the substrate (Figure 24.16). (D-Hydroxyacyl isomers, which arise mainly from oxidation of unsaturated fatty acids, are handled differently.)

Figure 24.16 · The L-b-hydroxyacyl-CoA dehydrogenase reaction.

b-Ketoacyl-CoA Intermediates Are Cleaved in the Thiolase Reaction

The final step in the b-oxidation cycle is the cleavage of the b-ketoacyl-CoA. This reaction, catalyzed by thiolase (also known as b-ketothiolase), involves the attack of a cysteine thiolate from the enzyme on the b-carbonyl carbon, followed by cleavage to give the enolate of acetyl-CoA and an enzyme-thioester intermediate (Figure 24.17). Subsequent attack by the thiol group of a second CoA and departure of the cysteine thiolate yields a new (shorter) acyl-CoA. If the reaction in Figure 24.17 is read in reverse, it is easy to see that it is a Claisen condensation—an attack of the enolate anion of acetyl-CoA on a thioester. Despite the formation of a second thioester, this reaction has a very favorable K_eq, and it drives the three previous reactions of b-oxidation.

Figure 24.17 · The mechanism of the thiolase reaction. Attack by an enzyme cysteine thiolate group at the b-carbonyl carbon produces a tetrahedral intermediate, which decomposes with departure of acetyl-CoA, leaving an enzyme thioester intermediate. Attack by the thiol group of a second CoA yields a new (shortened) acyl-CoA.

Repetition of the b-Oxidation Cycle Yields a Succession of Acetate Units

In essence, this series of four reactions has yielded a fatty acid (as a CoA ester) that has been shortened by two carbons, and one molecule of acetyl-CoA. The shortened fatty acyl-CoA can now go through another b-oxidation cycle, as shown in Figure 24.10. Repetition of this cycle with a fatty acid with an even number of carbons eventually yields two molecules of acetyl-CoA in the final step. As noted in the first reaction in Table 24.2, complete b-oxidation of palmitic acid yields eight molecules of acetyl-CoA as well as seven molecules of FADH₂ and seven molecules of NADH. The acetyl-CoA can be further metabolized in the TCA cycle (as we have already seen). Alternatively, acetyl-CoA can also be used as a substrate in amino acid biosynthesis (Chapter 26). As noted in Chapter 23, however, acetyl-CoA cannot be used as a substrate for gluconeogenesis.

Complete b-Oxidation of One Palmitic Acid Yields 106 Molecules of ATP

If the acetyl-CoA is directed entirely to the TCA cycle in mitochondria, it can eventually generate approximately 10 high-energy phosphate bonds—that is, 10 molecules of ATP synthesized from ADP (Table 24.2). Including the ATP formed from FADH₂ and NADH, complete b-oxidation of a molecule of palmitoyl-CoA in mitochondria yields 108 molecules of ATP. Subtracting the two high-energy bonds needed to form palmitoyl-CoA, the substrate for b-oxidation, one concludes that b-oxidation of a molecule of palmitic acid yields 106 molecules of ATP. The DG°' for complete combustion of palmitate to CO₂ is -9790 kJ/mol. The hydrolytic energy embodied in 106 ATPs is 106 x 30.5 kJ/mol = 3233 kJ/mol, so the overall efficiency of b-oxidation under standard-state conditions is approximately 33%. The large energy yield from fatty acid oxidation is a reflection of the highly reduced state of the carbon in fatty acids. Sugars, in which the carbon is already partially oxidized, produce much less energy, carbon for carbon, than do fatty acids. The breakdown of fatty acids is regulated by a variety of metabolites and hormones. Details of this regulation are described in Chapter 25, following a discussion of fatty acid synthesis.

Migratory Birds Travel Long Distances on Energy from Fatty Acid Oxidation

Because they represent the most highly concentrated form of stored biological energy, fatty acids are the metabolic fuel of choice for sustaining the incredibly long flights of many migratory birds. Although some birds migrate over land masses and dine frequently, other species fly long distances without stopping to eat. The American golden plover flies directly from Alaska to Hawaii, a 3300-kilometer flight requiring 35 hours (at an average speed of nearly 60 miles/hr) and more than 250,000 wing beats! The ruby-throated hummingbird, which winters in Central America and nests in southern Canada, often flies nonstop across the Gulf of Mexico. These and similar birds accomplish these prodigious feats by storing large amounts of fatty acids (as triacylglycerols) in the days before their migratory flights. The percentage of dry-weight body fat in these birds may be as high as 70% when migration begins (compared with values of 30% and less for nonmigratory birds).

Fatty Acid Oxidation Is an Important Source of Metabolic Water for Some Animals

Large amounts of metabolic water are generated by b-oxidation (130 H₂O per palmitoyl-CoA). For certain animals—including desert animals, such as gerbils, and killer whales (which do not drink seawater)—the oxidation of fatty acids can be a significant source of dietary water. A striking example is the camel (Figure 24.18), whose hump is essentially a large deposit of fat. Metabolism of fatty acids from this store provides needed water (as well as metabolic energy) during periods when drinking water is not available. It might well be said that “the ship of the desert” sails on its own metabolic water!

Figure 24.18 · Animals whose existence is strongly dependent on fatty acid oxidation: (a) gerbil, (b) ruby-throated hummingbird, (c) golden plover, (d) orca (killer whale), and (e) camels. (a, Photo Researchers, Inc.; b, Tom J. Ulrich/Visuals Unlimited; c, S. J. Krasemann/Photo Researchers, Inc.; d, (c) Francois Gohier/Photo Researchers, Inc.; e, (c) George Holton/Photo Researchers, Inc.)

b-Oxidation of Odd-Carbon Fatty Acids Yields Propionyl-CoA

Figure 24.19 · The conversion of propionyl-CoA (formed from b-oxidation of odd-carbon fatty acids) to succinyl-CoA is carried out by a trio of enzymes as shown. Succinyl-CoA can enter the TCA cycle or be converted to acetyl-CoA.

Fatty acids with odd numbers of carbon atoms are rare in mammals, but fairly common in plants and marine organisms. Humans and animals whose diets include these food sources metabolize odd-carbon fatty acids via the b-oxidation pathway. The final product of b-oxidation in this case is the 3-carbon propionyl-CoA instead of acetyl-CoA. Three specialized enzymes then carry out the reactions that convert propionyl-CoA to succinyl-CoA, a TCA cycle intermediate. (Because propionyl-CoA is a degradation product of methionine, valine, and isoleucine, this sequence of reactions is also important in amino acid catabolism, as we shall see in Chapter 26.) The pathway involves an initial carboxylation at the a-carbon of propionyl-CoA to produce D-methylmalonyl-CoA (Figure 24.19). The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxylation of biotin at N₁, followed by nucleophilic attack by the a-carbanion of propionyl-CoA in a stereo-specific manner.
      D-Methylmalonyl-CoA, the product of this reaction, is converted to the L-isomer by methylmalonyl-CoA epimerase (Figure 24.19). (This enzyme has often and incorrectly been called “methylmalonyl-CoA racemase.” It is not a racemase because the CoA moiety contains five other asymmetric centers.) The epimerase reaction also appears to involve a carbanion at the a-position (Figure 24.20). The reaction is readily reversible and involves a reversible dissociation of the acidic a-proton. The L-isomer is the substrate for methylmalonyl-CoA mutase. Methylmalonyl-CoA epimerase is an impressive catalyst. The pK_a for the proton that must dissociate to initiate this reaction is approximately 21! If binding of a proton to the a-anion is diffusion-limited, with k_on=10⁹ M^-1 sec^-1, then the initial proton dissociation must be rate-limiting, and the rate constant must be

           k_off = K_a× k_on = (10^-21M) × (10⁹ M^-1sec^-1) = 10^-12 sec^-1

The turnover number of methylmalonyl-CoA epimerase is 100 sec^-1, and thus the enzyme enhances the reaction rate by a factor of 10¹⁴.

Figure 24.20 · The methylmalonyl-CoA epimerase mechanism involves a resonance-stabilized carbanion at the a-position.

A B₁₂-Catalyzed Rearrangement Yields Succinyl-CoAfrom L-Methylmalonyl-CoA

Figure 24.21 · A mechanism for the methylmalonyl-CoA mutase reaction. In the first step, Co³⁺ is reduced to Co²⁺ due to homolytic cleavage of the Co³⁺¾C bond in cobala-min. Hydrogen atom transfer from methylmalonyl-CoA yields a methylmalonyl-CoA radical that can undergo rearrangement to form a succinyl-CoA radical. Transfer of an H atom regenerates the coenzyme and yields succinyl-CoA.

he third reaction, catalyzed by methylmalonyl-CoA mutase, is quite unusual because it involves a migration of the carbonyl-CoA group from one carbon to its neighbor (Figure 24.21). The mutase reaction is vitamin B₁₂-dependent and begins with homolytic cleavage of the Co³⁺¾C bond in cobalamin, reducing the cobalt to Co²⁺. Transfer of a hydrogen atom from the substrate to the deoxyadenosyl group produces a methylmalonyl-CoA radical, which then can undergo a classic B₁₂-catalyzed rearrangement to yield a succinyl-CoA radical. Hydrogen transfer from the deoxyadenosyl group yields succinyl-CoA and regenerates the B₁₂ coenzyme.

A Deeper Look
The Activation of Vitamin B12
Conversion of inactive vitamin B12 to active 5'-deoxyadenosylcobalamin is thought to involve three steps (see figure). Two flavoprotein reductases sequentially convert Co3+ in cyanocobalamin to the Co2+ state and then to the Co+ state. Co+ is an extremely powerful nucleophile. It attacks the C-5' carbon of ATP as shown, expelling the triphosphate anion to form 59-deoxyadenosylcobalamin.	Because two electrons from Co+ are donated to the Co-carbon bond, the oxidation state of cobalt reverts to Co3+ in the active coenzyme. This is one of only two known adenosyl transfers (that is, nucleophilic attack on the ribose 5'-carbon of ATP) in biological systems. (The other is the formation of S-adenosylmethionine—see Chapter 26.)
Formation of the active coenzyme 5'-deoxyadenosylcobalamin from inactive vitamin B12 is initiated by the action of flavoprotein reductases. The resulting Co1 species, dubbed a supernucleophile, attacks the 5'-carbon of ATP in an unusual adenosyl transfer.

Net Oxidation of Succinyl-CoA Requires Conversion to Acetyl-CoA

Succinyl-CoA derived from propionyl-CoA can enter the TCA cycle. Oxidation of succinate to oxaloacetate provides a substrate for glucose synthesis. Thus, although the acetate units produced in b-oxidation cannot be utilized in gluconeogenesis by animals, the occasional propionate produced from oxidation of odd-carbon fatty acids can be used for sugar synthesis. Alternatively, succinate introduced to the TCA cycle from odd-carbon fatty acid oxidation may be oxidized to CO₂. However, all of the 4-carbon intermediates in the TCA cycle are regenerated in the cycle and thus should be viewed as catalytic species. Net consumption of succinyl-CoA thus does not occur directly in the TCA cycle. Rather, the succinyl-CoA generated from b-oxidation of odd-carbon fatty acids must be converted to pyruvate and then to acetyl-CoA (which is completely oxidized in the TCA cycle). To follow this latter route, succinyl-CoA entering the TCA cycle must be first converted to malate in the usual way, and then transported from the mitochondrial matrix to the cytosol, where it is oxidatively decarboxylated to pyruvate and CO₂ by malic enzyme, as shown in Figure 24.22. Pyruvate can then be transported back to the mitochondrial matrix, where it enters the TCA cycle via pyruvate dehydrogenase. Note that malic enzyme plays an important role in fatty acid synthesis (see Figure 25.1).

Figure 24.22 · The malic enzyme reaction proceeds by oxidation of malate to oxaloace-tate, followed by decarboxylation to yield pyruvate.

24.4 ×

An Isomerase and a Reductase Facilitate the b-Oxidation of Unsaturated Fatty Acids

Figure 24.23 · b-Oxidation of unsaturated fatty acids. In the case of oleoyl-CoA, three b-oxidation cycles produce three molecules of acetyl-CoA and leave cis-D³-dodecenoyl-CoA. Rearrangement of enoyl-CoA isomerase gives the trans-D² species, which then proceeds normally through the b-oxidation pathway.

Unsaturated fatty acids are also catabolized by b-oxidation, but two additional mitochondrial enzymes—an isomerase and a novel reductase—are required to handle the cis-double bonds of naturally occurring fatty acids. As an example, consider the breakdown of oleic acid, an 18-carbon chain with a double bond at the 9,10-position. The reactions of b-oxidation proceed normally through three cycles, producing three molecules of acetyl-CoA and leaving the degradation product cis-D³-dodecenoyl-CoA, shown in Figure 4.23. 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 b) position. As shown in Figure 24.23, this problem is solved by enoyl-CoA isomerase, an enzyme that rearranges this cis-D³ double bond to a trans-D² double bond. This latter species can proceed through the normal route of b-oxidation.

Degradation of Polyunsaturated Fatty Acids Requires 2,4-Dienoyl-CoA Reductase

Figure 24.24 · The oxidation pathway for polyunsaturated fatty acids, illustrated for linoleic acid. Three cycles of b-oxidation on linoleoyl-CoA yield the cis-D³, cis-D⁶ intermediate, which is converted to a trans-D², cis-D⁶ intermediate. An additional round of b-oxidation gives cis-D⁴ enoyl-CoA, which is oxidized to the trans-D², cis-D⁴ species by acyl-CoA dehydrogenase. The subsequent action of 2,4-dienoyl-CoA reductase yields the trans-D³ product, which is converted by enoyl-CoA isomerase to the trans-D² form. Normal b-oxidation then produces five molecules of acetyl-CoA.

Polyunsaturated fatty acids pose a slightly more complicated situation for the cell. Consider, for example, the case of linoleic acid shown in Figure 24.24. As with oleic acid, b-oxidation proceeds through three cycles, and enoyl-CoA isomerase converts the cis-D³ double bond to a trans-D² double bond to permit one more round of b-oxidation. What results this time, however, is a cis-D⁴ enoyl-CoA, which is converted normally by acyl-CoA dehydrogenase to a trans-D², cis-D⁴ 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-D³ enoyl product, as shown in Figure 24.24, which can be converted by an enoyl-CoA isomerase to the trans-D² enoyl-CoA, which can then proceed normally through the b-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-D² enoyl-CoA product in a single step.

Peroxisomal b-Oxidation Requires FAD-Dependent Acyl-CoA Oxidase

Although b-oxidation in mitochondria¹ is the principal pathway of fatty acid catabolism, several other minor pathways play important roles in fat catabolism. For example, organelles other than mitochondria carry out b-oxidation processes, including eroxisomes and glyoxysomes. Peroxisomes are so named because they carry out a variety of flavin-dependent oxidation reactions, regenerating oxidized flavins by reaction with oxygen to produce hydrogen peroxide, H₂O₂. Peroxisomal b-oxidation is similar to mitochondrial b-oxidation, except that the initial double bond formation is catalyzed by an FAD-dependent acyl-CoA oxidase (Figure 24.25). The action of this enzyme in the peroxisomes transfers the liberated electrons directly to oxygen instead of the electron transport chain. As a result, each 2-carbon unit oxidized in peroxisomes produces fewer ATPs. The enzymes responsible for fatty acid oxidation in peroxisomes are inactive with carbon chains of eight or fewer. Such short-chain products must be transferred to the mitochondria for further breakdown. Similar b-oxidation enzymes are also found in glyoxysomes—peroxisomes in plants that also carry out the reactions of the glyoxylate pathway.

Figure 24.25 · The acyl-CoA oxidase reaction in peroxisomes.

Branched-Chain Fatty Acids and a-Oxidation

Figure 24.26 · Branched-chain fatty acids are oxidized by a-oxidation, as shown for phytanic acid. The product of the phytanic acid oxidase, pristanic acid, is a suitable substrate for normal b-oxidation. Isobutyryl-CoA and propionyl-CoA can both be converted to succinyl-CoA, which can enter the TCA cycle.

Although b-oxidation is universally important, there are some instances in which it cannot operate effectively. For example, branched-chain fatty acids with alkyl branches at odd-numbered carbons are not effective substrates for b-oxidation. For such species, a-oxidation is a useful alternative. Consider phytol, a breakdown product 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 in dairy products is thus an important dietary consideration for humans. The methyl group at C-3 will block b-oxidation, but, as shown in Figure 24.26, phytanic acid a-hydroxylase places an -OH group at the a-carbon, and phytanic acid a-oxidase decarboxylates it to yield pristanic acid. The CoA ester of this metabolite can undergo b-oxidation in the normal manner. The terminal product, isobutyryl-CoA, can be sent into the TCA cycle by conversion to succinyl-CoA.

Refsum’s Disease Is a Result of Defects in a-Oxidation

The a-oxidation pathway is defective in Refsum’s disease, an inherited 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.

v-Oxidation of Fatty Acids Yields Small Amounts of Dicarboxylic Acids

Figure 24.27 · Dicarboxylic acids can be formed by oxidation of the methyl group of fatty acids in a cytochrome P-450-dependent reaction.

In the endoplasmic reticulum of eukaryotic cells, the oxidation of the terminal carbon of a normal fatty acid—a process termed v-oxidation—can lead to the synthesis of small amounts of dicarboxylic acids (Figure 24.27). Cytochrome P-450, a monooxygenase enzyme that requires NADPH as a coenzyme and uses O₂ as a substrate, places a hydroxyl group at the terminal carbon. Subsequent oxidation to a carboxyl group produces a dicarboxylic acid. Either end can form an ester linkage to CoA and be subjected to b-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 metabolism of toxic hydrocarbon agents.)

24.6 × Ketone Bodies

Ketone Bodies Are a Significant Source of Fuel and Energy for Certain Tissues

Figure 24.28 · The formation of ketone bodies, synthesized primarily in liver mitochondria.

Most of the acetyl-CoA produced by the oxidation of fatty acids in liver mitochondria undergoes further oxidation in the TCA cycle, as stated earlier. However, some of this acetyl-CoA is converted to three important metabolites: acetone, acetoacetate, and b-hydroxybutyrate. The process is known as ketogenesis, and these three metabolites are traditionally known as ketone bodies, in spite of the fact that b-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 peripheral tissues, including brain, heart, and skeletal muscle. The brain, for example, normally uses glucose as its source of metabolic energy. However, during periods of starvation, ketone bodies may be the major energy source for the brain. Acetoacetate and 3-hydroxybutyrate are the preferred and normal substrates for kidney cortex and for heart muscle.
      Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first 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 b-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give b-hydroxy-b-methylglutaryl-CoA, commonly abbreviated 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 25. 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, b-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to b-hydroxybutyrate.
      Acetoacetate and b-hydroxybutyrate are transported through the blood from liver to target organs and tissues, where they are converted to acetyl-CoA (Figure 24.29). Ketone bodies are easily transportable forms of fatty acids that move through the circulatory system without the need for complexation with serum albumin and other fatty acid-binding proteins.

Figure 24.29 · Reconversion of ketone bodies to acetyl-CoA in the mitochondria of many tissues (other than liver) provides significant metabolic energy.

Ketone Bodies and Diabetes Mellitus

Diabetes mellitus is the most common endocrine disease and the third leading cause of death in the United States, with approximately 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 I diabetes (representing 10% or fewer of all cases), elevated blood glucose results from inadequate secretion of insulin by the islets of Langerhans in the pancreas. Type II diabetes (at least 90% of all cases) results from an insensitivity to insulin. Type II diabetics produce normal or even elevated levels of insulin, but owing to a shortage of insulin receptors (Chapter 34), their cells are not responsive to insulin. 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 catabolism of fat and protein. In type I diabetes, increased gluconeogenesis 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 normally 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 I diabetics, an indication of high plasma levels of ketone bodies.