23.2 How Are Fatty Acids Broken Down? 703 chain derivatives must first be converted to acylcarnitine derivatives, as shown in Figure 23.8. Carnitine acyltransferase I, associated with the outer mitochondrial membrane, catalyzes the formation of the O-acylcarnitine, which is then trans- ported across the inner membrane by a translocase. At this point, the acylcarni- tine is passed to carnitine acyltransferase II on the matrix side of the inner mem- brane, 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 OOacyl bonds in acyl- carnitines 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 (see Chapter 24), and the mitochondrial pool is important in the oxidation of fatty acids and pyruvate, as well as some amino acids. N + CH 3 CH 3 CH 3 CO O CH CH 2 CH 2 CH 2 O-acylcarnitine CO O CH CH 2 CH 2 CH 2 COO – COO – COO – S C O SH N + H 3 CCH 3 CH 3 N + H 3 CCH 3 CH 3 HHO C CoA CoA H HO C C OO – S C O SH CH 2 CH 2 N + CH 3 CH 3 CH 3 CoA CoA Intermembrane space Inner mitochondrial membrane Outer mitochondrial membrane Mitochondrial matrix Carnitine: acylcarnitine translocase O-acylcarnitine L-Carnitine Carnitine acyltrans- ferase II Cytosol L-Carnitine Intermembrane space Carnitine acyltrans- ferase I FIGURE 23.8 The formation of acylcarnitines and their transport across the inner mitochondrial membrane. The process involves the coordinated actions of carni- tine acyltransferases on both the inner and outer mito- chondrial membranes and of a translocase that shuttles O-acylcarnitines across the inner membrane. 704 Chapter 23 Fatty Acid Catabolism ␤-Oxidation Involves a Repeated Sequence of Four Reactions For saturated fatty acids, the process of ␤-oxidation involves a recurring cycle of four steps, as shown in Figure 23.9. The overall strategy in the first three steps is to create a carbonyl group on the ␤-carbon by oxidizing the C ␣ OC ␤ 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 ␤-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 19 that Claisen con- densations involve attack by a nucleophilic agent on a carbonyl carbon to yield a ␤-keto acid.) A Family of Acyl-CoA Dehydrogenases Carry Out the First Reaction of ␤-Oxidation The enzymes of mitochondrial ␤-oxidation are organized in two functional systems: a membrane-bound complex that is specific for long-chain fatty acids (14 carbons and longer) and a family of soluble enzymes in the matrix that is specific for short- and medium-chain fatty acids (Figure 23.10). As a fatty acyl chain is shortened in successive cycles of ␤-oxidation, it moves from the membrane-bound complex to the family of soluble matrix enzymes. The first reaction of the ␤-oxidation cycle is catalyzed by one of four acyl-CoA dehydrogenases. These include the very long- OH C CH 2 H 2 O RCC S H HH O C SCH 2 R O C CH 2 RCC S H H O O C CH 2 RCCS H H O HO CCH 2 RCC S H H H ␣ ␣ ␤ ␤ ␣ ␤ O C C S H H H ␣ ␤ ␣ ␤ 1 2 3 4 CoA CoA CoA CoA CoA CoA Fatty acyl-CoA Fatty acyl-CoA shortened by two carbons ␤-Ketoacyl-CoA trans-Δ 2 -Enoyl-CoA L-␤-H y drox y ac y l-CoA FAD FADH 2 Acyl-CoA dehydrogenase Enoyl-CoA hydratase HydrationOxidation + L-Hydroxyacyl-CoA dehydrogenase Thiolase Acetyl-CoA Cleavage Oxidation Successive cycles H + CoASH NAD + NADH FIGURE 23.9 The ␤-oxidation of saturated fatty acids involves a cycle of four enzyme-catalyzed reactions. Each cycle produces single molecules of FADH 2 , NADH, and acetyl-CoA, consumes a water, and yields a fatty acid shortened by two carbons. (The delta [⌬] symbol connotes a double bond, and its superscript indicates the lower-numbered carbon involved.) 23.2 How Are Fatty Acids Broken Down? 705 chain acyl-CoA dehydrogenase (VLCAD), as well as acyl-CoA dehydrogenases spe- cific for long-chain (LCAD), medium-chain (MCAD), and short-chain (SCAD) sub- strates. VLCAD is a membrane-bound homodimer of 67-kD subunits (Figure 23.11), whereas the soluble LCAD, MCAD, and SCAD are homotetramers of 40- to 45-kD subunits. All acyl-CoA dehydrogenases carry noncovalently (but tightly) bound FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure 23.12, FADH 2 transfers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is re- oxidized 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. Recall from Chapter 20 that mitochondrial oxidation of FAD in this way eventually results in the net formation of about 1.5 ATPs. The mechanism of the acyl-CoA dehydroge- nase (Figure 23.13) involves deprotonation of the fatty acid chain at the ␣-carbon, fol- lowed by hydride transfer from the ␤-carbon to FAD. LCAD MCAD SCAD ECHKT ECH KTHAD HAD VLCAD SCoAR O SCoA CoA R O SCoAR O SCoAH 3 C O FIGURE 23.10 Very long-chain fatty acids proceed through several cycles of ␤-oxidation (left) via membrane- bound enzymes in mitochondria. A membrane-bound multifunctional complex includes the enoyl-CoA hydra- tase (ECH), hydroxyacyl-CoA dehydrogenase (HAD), and ketoacyl thiolase (KT) activities. As chains shorten pro- gressively, they become substrates for the separate, soluble enzymes of ␤-oxidation (right). (a) (b) FIGURE 23.11 (a) The VLCAD of mammalian mitochondria is a 67-kD membrane-bound homodimer with bound FAD (red) and myristoyl-CoA (blue) (pdb id ϭ 3B96).The tertiary structure of the N-terminal 400 residues of VLCAD is similar to that of (b) the soluble MCAD (pdb id ϭ 3MDE), shown with bound FAD (red) and octanoyl-CoA (blue).These similar structures each include an N-terminal ␣-helical domain (yellow), fol- lowed by a ␤-sheet domain (blue) and another ␣-helical domain (green).The acyl-CoA substrate lies in a long cleft between these three domains.The VLCAD also has a C-terminal ␣-helical domain (purple). 706 Chapter 23 Fatty Acid Catabolism Enoyl-CoA Hydratase Adds Water Across the Double Bond The next step in ␤-oxidation is the addition of the elements of H 2 O across the new double bond in a stereospecific manner, yielding the corresponding hydroxyacyl-CoA. The reaction is catalyzed by enoyl-CoA hydratase (Figure 23.14). A number of dif- ferent enoyl-CoA hydratase activities have been detected in various tissues. Also called crotonases, these enzymes specifically convert trans-enoyl-CoA derivatives to L-␤-hydroxyacyl-CoA. Enoyl-CoA hydratases will also metabolize cis-enoyl-CoA (at slower rates) to give specifically D-␤-hydroxyacyl-CoA. In addition, there is a novel enoyl-CoA hydratase that converts trans-enoyl-CoA to D-␤-hydroxyacyl-CoA. L-Hydroxyacyl-CoA Dehydrogenase Oxidizes the ␤-Hydroxyl Group The third reaction of this cycle is the oxidation of the hydroxyl group at the ␤-position to produce a ␤-ketoacyl-CoA derivative. This second oxidation reaction is catalyzed by L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD ϩ as a coen- zyme (see Figure 23.9). CH 2 SCoA R CH 2 R CC H CH H OH C CH 2 C O O SCoA H 2 O trans-Enoyl-CoA Crotonase L-␤-Hydroxyacyl-CoA R CH 2 H C β C α C SCoA H HHO UQH 2 UQ R CH 2 H CCC SCoA HO 1.5 ADP 1.5 P i 1.5 + ATP O 2 H 2 O Fatty acyl-CoA Acyl-CoA dehydrogenase FAD FADH 2 ETF red ETF ox ETF: UQ oxidoreductase ox ETF: UQ oxidoreductase red Mitochondrial electron- transport chain 1 2 trans-Δ 2 -Enoyl-CoA FIGURE 23.12 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 (UQH 2 ). CCH 2 H R H C H H CCH 2 H R H C HO C SCoA O C SCoA FAD CH 2 R CC H H B O C SCoA E FIGURE 23.13 The mechanism of acyl-CoA dehydrogenase. Removal of a proton from the ␣-C is followed by hydride transfer from the ␤-carbon to FAD. (a) (b) (c) FIGURE 23.14 Structures of mitochondrial (a) enoyl- CoA hydratase trimer (pdb id ϭ 2DUB), (b) hydroxyacyl- CoA dehydrogenase dimer (pdb id ϭ 1F0Y), and (c) ketoacyl thiolase dimer (pdb id ϭ 1AFW). (a) (b) (c) 23.2 How Are Fatty Acids Broken Down? 707 NADH produced in this reaction represents metabolic energy. Each NADH pro- duced in mitochondria by this reaction drives the synthesis of 2.5 molecules of ATP in the electron-transport pathway. L-Hydroxyacyl-CoA dehydrogenase shows ab- solute specificity for the L-hydroxyacyl isomer of the substrate. (D-Hydroxyacyl isomers, which arise mainly from the metabolism of unsaturated fatty acids, are handled differently.) ␤-Ketoacyl-CoA Intermediates Are Cleaved in the Thiolase Reaction The final step in the ␤-oxidation cycle is the cleavage of the ␤-ketoacyl-CoA. This reaction, catalyzed by ketoacyl thiolase (also known as ␤-ketothiolase, Figure 23.9), involves the attack of a cysteine thiolate from the enzyme on the ␤-carbonyl carbon, fol- lowed by cleavage to give the enolate of acetyl-CoA and an enzyme-thioester intermediate (Figure 23.15). 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 23.15 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 ␤-oxidation. Repetition of the ␤-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 ␤-oxidation cycle, as shown in Figure 23.9. 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. Complete ␤-oxidation of palmitic CCH 2 H R OH C O CCH 2 RCH 2 C O SCoACH 2 O SCoA L-␤-Hydroxyacyl-CoA ␤-Ketoacyl-CoA H + + NAD + NADH O – C C O CH 2 O R + S – BH E R C S BH + + H O S S CoA R C B BH O S R C C CCH 2 SCoA SCoA C R S E S – CH 2 C H 3 C SCoA C O R SCoA SCoA CoA + O – C SCoA CH 2 O – O E E O O E ␤-Ketoacyl-CoA Acyl-CoA FIGURE 23.15 The mechanism of the thiolase reaction. Attack by an enzyme cysteine thiolate group at the ␤-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. Go to CengageNOW and click CengageInteractive to discover the main functions of coenzyme A. 708 Chapter 23 Fatty Acid Catabolism acid yields eight molecules of acetyl-CoA as well as seven molecules of FADH 2 and seven molecules of NADH (Figure 23.16 and Table 23.2). 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 (see Chapter 25). As noted in Chapter 22, however, acetyl-CoA cannot be used as a substrate for gluconeogenesis. Complete ␤-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 eventu- ally generate approximately ten high-energy phosphate bonds—that is, ten molecules of ATP synthesized from ADP (Table 23.2). Including the ATP formed from FADH 2 and NADH, complete ␤-oxidation of a molecule of palmitoyl-CoA in mitochondria HUMAN BIOCHEMISTRY Exercise Can Reverse the Consequences of Metabolic Syndrome Metabolic syndrome is a combination of disorders that increase the risk of diabetes and cardiovascular disease. The hallmarks of meta- bolic syndrome include high blood pressure, elevated serum triglycerides, reduced serum high-density lipoprotein (HDL) cho- lesterol, insulin resistance, and obesity. The prevalence of these conditions is increasing in the United States. By most estimates, more than 30% of Americans are obese, and rising obesity has con- tributed to an epidemic of type 2 diabetes. Insights into how the body deals with high fat and high sugar diets are emerging from a variety of studies, and evidence points clearly to the benefits of ex- ercise and dietary restriction. Endurance training (such as distance running) and resistance training (with weights) are both benefi- cial. Endurance training increases the mass of slow-twitch muscle fibers, resistance training builds fast-twitch muscle fibers, and both types of exercise reduce body fat, but in quite different ways. Slow-twitch muscles depend on fatty acid oxidation and TCA cycle activity to support long periods of exercise and are termed oxidative. Effects of endurance training include increased expres- sion of (1) peroxisome proliferator-activated receptor ␦, a tran- scription factor that builds slow-twitch muscle fiber, and (2) in- sulin receptors and glucose transporters. Fast-twitch muscles are adapted for short bursts of energy, which can be supplied by gly- colysis and thus are termed glycolytic. Effects of resistance training include activation of metabolic processes by the serine/threonine kinase Akt1. Induction of the Akt1 pathway results in growth of fast-twitch skeletal muscle fibers and subsequent effects on several other organs. These include increased fat uptake and oxidation by the liver and heart, reduction of adipose (fat cell) mass, and re- duced blood glucose and insulin levels. Local effects in muscle: Increased fatty acid oxidation Increased expression of insulin receptors and glucose transporters Adipose tissue: Reduced cell size Circulation: Reduced glucose, insulin, and le p tin levels Muscle: Increased glucose uptake and glycolysis Liver: Increased fat uptake and oxidation Heart: Increased fat uptake and oxidation Endurance training Circulating factors Weight training Fast-twitch muscle growth Slow-twitch muscle growth 23.2 How Are Fatty Acids Broken Down? 709 yields 108 molecules of ATP. Subtracting the two high-energy bonds needed to form palmitoyl-CoA, the substrate for ␤-oxidation, one concludes that ␤-oxidation of a mol- ecule of palmitic acid yields 106 molecules of ATP. The ⌬G°Ј for complete combustion of palmitate to CO 2 is Ϫ9790 kJ/mol. The hydrolytic energy embodied in 106 ATPs is 106 ϫ 30.5 kJ/mol ϭ 3233 kJ/mol, so the overall efficiency of ␤-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 less energy, carbon for car- bon, 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 24, fol- lowing 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 landmasses and eat frequently, other species fly long distances without stopping to eat. The American golden plover (Figure 23.17) flies directly from Alaska to Hawaii, a 3300-km 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 C SCoA O 7 CoA, 7 [ FAD ] , 7 NAD + 8 Acetyl-CoA ␤-Oxidation Electron transport Oxidative phosphorylation 24 NADH, 8 [ FADH 2 ] 16 CO 2 8 ATP TCA cycle 7 [ FADH 2 ] , 7 NADH + 7 H + 100 ADP + 100 P i 100 ATP FIGURE 23.16 Reduced coenzymes produced by ␤-oxidation and TCA cycle activity provide electrons that drive the synthesis of ATP in oxidative phosphorylation. Complete oxidation of palmitoyl-CoA yields a total of 108 ATP.Subtracting the 2 ATP equivalents consumed in forming the original CoA thioester, oxidation of palmi- tate produces 106 ATP. ATP Free Energy Equation Yield Yield (kJ/mol) CH 3 (CH 2 ) 14 CO-CoA ϩ 7 [FAD] ϩ 7 H 2 O ϩ 7 NAD ϩ ϩ 7 CoA 88n 8 CH 3 CO-CoA ϩ 7 [FADH 2 ] ϩ 7 NADH ϩ 7 H ϩ 7 [FADH 2 ] ϩ 10.5 P i ϩ 10.5 ADP ϩ 3.5 O 2 88n 7 [FAD] ϩ 17.5 H 2 O ϩ 10.5 ATP 10.5 320 7 NADH ϩ 7 H ϩ ϩ 17.5 P i ϩ 17.5 ADP ϩ 3.5 O 2 88n 7 NAD ϩ ϩ 24.5 H 2 O ϩ 17.5 ATP 17.5 534 8 Acetyl-CoA ϩ 16 O 2 ϩ 80 ADP ϩ 80 P i 88n 8 CoA ϩ 88 H 2 O ϩ 16 CO 2 ϩ 80 ATP 80 2440 CH 3 O(CH 2 ) 14 CO-CoA ϩ 108 P i ϩ 108 ADP ϩ 23 O 2 88n 108 ATP ϩ 16 CO 2 ϩ 123 H 2 O ϩ CoA 108 3294 Energetic “cost” of forming palmitoyl-CoA from palmitate and CoA Ϫ2 Ϫ61 Total 106 3233 TABLE 23.2 Equations for the Complete Oxidation of Palmitoyl-CoA to CO 2 and H 2 O 710 Chapter 23 Fatty Acid Catabolism 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 mi- gration 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 ␤-oxidation (123 H 2 O per palmitoyl-CoA, see Table 23.2). 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 23.17), 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! 23.3 How Are Odd-Carbon Fatty Acids Oxidized? ␤-Oxidation of Odd-Carbon Fatty Acids Yields Propionyl-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 ␤-oxidation pathway. The final prod- (a) Gerbil (b) Ruby-throated hummingbird (c) Golden p lover (d) Orca (e) Camels FIGURE 23.17 Animals whose existence is strongly dependent on fatty acid oxidation: (a) gerbil, (b) ruby-throated humming- bird, (c) golden plover, (d) orca (killer whale), and (e) camels. Photo Researchers, Inc. © Millard H. Sharp/Photo Researchers, Inc. © Eric and David Hosking/CORBIS © Francois Gohier/Photo Researchers, Inc. © George Holton/Photo Researchers, Inc. 23.3 How Are Odd-Carbon Fatty Acids Oxidized? 711 uct of ␤-oxidation in this case is the three-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 degra- dation product of methionine, valine, and isoleucine, this sequence of reactions is also important in amino acid catabolism, as we shall see in Chapter 25.) The pathway involves an initial carboxylation at the ␣-carbon of propionyl-CoA to produce D-methylmalonyl-CoA (Figure 23.18). The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxyla- tion of biotin at N 1 , followed by nucleophilic attack by the ␣-carbanion of propionyl- CoA in a stereospecific manner. D-Methylmalonyl-CoA, the product of this reaction, is converted to the L-isomer by methylmalonyl-CoA epimerase (Figure 23.18). (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 involves a carbanion at the ␣-position formed via a reversible dissociation of the acidic ␣-proton (Figure 23.19). The L-isomer is the substrate for methyl- malonyl-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 ␣-anion is diffusion limited, with k on ϭ 10 9 M Ϫ1 sec Ϫ1 , then the initial proton dissociation must be rate limiting and the rate constant must be k off ϭ K a и k on ϭ (10 Ϫ21 M) и (10 9 M Ϫ1 sec Ϫ1 ) ϭ 10 Ϫ12 sec Ϫ1 The turnover number of methylmalonyl-CoA epimerase is 100 sec Ϫ1 , and thus the enzyme enhances the reaction rate by a factor of 10 14 . A B 12 -Catalyzed Rearrangement Yields Succinyl-CoA from L-Methylmalonyl-CoA The third reaction, catalyzed by methylmalonyl-CoA mutase, is quite unusual be- cause it involves a migration of the carbonyl-CoA group from one carbon to its neighbor (Figure 23.18). The mutase reaction is vitamin B 12 –dependent and begins with homolytic cleavage of the Co 3ϩ OC bond in 5Ј-deoxyadenosylcobalamin, re- ducing the cobalt to Co 2ϩ (see A Deeper Look, page 712). Transfer of a hydrogen atom from the substrate to the deoxyadenosyl group produces a methylmalonyl- CoA radical, which then can undergo a classic B 12 -catalyzed rearrangement to yield SCoA O +CH 3 CH 2 C ++ H 2 O ATP CH 2 CH 2 C C H 3 C H 3 C O _ OOC _ OOC _ OOC C SCoA O C H H C SCoA O SCoA CO 2 Propionyl-CoA Methylmalonyl-Co A mutase Succinyl-CoA L-Methylmalonyl-CoA Methylmalonyl-Co A epimerase D-Methylmalonyl-CoA Propionyl-CoA carboxylase FIGURE 23.18 The conversion of propionyl-CoA (formed from ␤-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. O C – O 2 C C CH 3 H B O C – O 2 C C H H 3 C B O – C – O 2 C C CH 3 CH 3 HO – C – O 2 CCSCoA B H B SCoA SCoA SCoA E E E E (S)-Methylmalonyl-CoA (R)-Methylmalonyl-CoA Resonance-stabilized carbanion intermediate FIGURE 23.19 The methylmalonyl-CoA epimerase mechanism involves a resonance-stabilized carbanion at the ␣-position. 712 Chapter 23 Fatty Acid Catabolism a succinyl-CoA radical. Hydrogen transfer from the deoxyadenosyl group yields succinyl-CoA and regenerates the B 12 coenzyme (see problem 16 at the end of the chapter). 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, al- though the acetate units produced in ␤-oxidation cannot be utilized in gluco- neogenesis by animals, the occasional propionate produced from oxidation of odd-carbon fatty acids can be used for sugar synthesis. Alternatively, succinate in- troduced to the TCA cycle from odd-carbon fatty acid oxidation may be oxidized to CO 2 . However, all of the four-carbon intermediates in the TCA cycle are re- generated in the cycle and thus should be viewed as catalytic species. Net con- sumption of succinyl-CoA thus does not occur directly in the TCA cycle. Rather, the succinyl-CoA generated from ␤-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 mi- tochondrial matrix to the cytosol, where it is oxidatively decarboxylated to pyru- vate and CO 2 by malic enzyme, as shown in Figure 23.20. Pyruvate can then be transported back to the mitochondrial matrix, where it enters the TCA cycle via pyruvate dehydrogenase. Note that malic enzyme plays a role in fatty acid synthe- sis (see Figure 24.1). A DEEPER LOOK The Activation of Vitamin B 12 Conversion of inactive vitamin B 12 to active 5Ј-deoxyadenosylcobalamin involves three steps (see accompanying figure). Two flavoprotein reductases sequentially convert Co 3ϩ in cyanocobalamin to the Co 2ϩ state and then to the Co ϩ state. Co ϩ is an extremely power- ful nucleophile. It attacks the C-5Ј carbon of ATP as shown, expel- ling the triphosphate anion to form 5Ј-deoxyadenosylcobalamin. Because two electrons from Co ϩ are donated to the CoOcarbon bond, the oxidation state of cobalt reverts to Co 3ϩ in the active coenzyme. This is one of only two known adenosyl transfers (that is, nucleophilic attack on the ribose 5Ј-carbon of ATP) in biologi- cal systems. (The other is the formation of S-adenosylmethionine; see Chapter 25.) ATP CH 2 12 3 N N N N N N CH 3 CH 3 Inactive Vitamin B 12 (Co 3+ ) (cyanocobalamin) Flavoprotein reductases Supernucleophile (Co + ) O O P – OO O P O P O – O – O – OCH 2 O OH OH N N N N NH 2 CN N N N N N N CH 3 CH 3 DMBz Co + Co 3+ N N N N CH 2 O OH OH Reactive free radical O OH OH N N N N AA Co 3+ Co 2+ 5؅-Deoxyadenosyl-cobalamin (one of several B 12 coenzyme forms) ᮡ Formation of the active coenzyme 5Ј-deoxyadenosylcobalamin from inactive vitamin B 12 is initiated by the action of flavoprotein reductases. The resulting Co ϩ species, dubbed a supernucleophile, attacks the 5Ј-carbon of ATP in an unusual adenosyl transfer. Homolytic cleavage of the Co 3+ –C bond produces a reactive free radical that facilitates rearrangements such as that in the methylmalonyl-CoA mutase reaction.