Fatty Acid Catabolism

CHAPTER 17 FATTY ACID CATABOLISM This advantage is compounded by the extreme insolubility of lipids in water; cellular triacylglycerols aggregate in lipid droplets, which do not raise the osmolarity of the cytosol, and they are unsolvated. (In storage polysaccharides, by contrast, water of solvation can account for two-thirds of the overall weight of the stored molecules.) And because of their relative chemical inertness, triacylglycerols can be stored in large quantity in cells without the risk of undesired chemical reactions with other cellular constituents. The properties that make triacylglycerols good storage compounds, however, present problems in their role as fuels. Because they are insoluble in water, ingested triacylglycerols must be emulsified before they can be digested by water-soluble enzymes in the intestine, and triacylglycerols absorbed in the intestine or mobilized from storage tissues must be carried in the blood bound to proteins that counteract their insolubility. Also, to overcome the relative stability of the C—C bonds in a fatty acid, the carboxyl group at C-1 is activated by attachment to coenzyme A, which allows stepwise oxidation of the fatty acyl group at the C- 3, or β , position — hence the name β oxidation. The principles we emphasize in this chapter are not new. They apply to the catabolic pathways of carbohydrates that we just studied. Metabolites of diverse origin funnel into a few central pathways. Fatty acid catabolism and glycolysis convert quite different starting materials into the same product (acetyl-CoA). The electrons from the oxidative reactions of these pathways and of the citric acid cycle are carried by common cofactors (NAD and FAD) to the mitochondrial respiratory chain leading to oxygen, providing the energy for ATP synthesis by oxidative phosphorylation. Evolution selects for chemical mechanisms that make useful reactions more energetically favorable, and those same mechanisms are used in different pathways. In the breakdown of fatty acids we see the activation of a carboxylic acid by its conversion to a thioester, as we saw with acetyl-CoA in the citric acid cycle. To break the C—C bonds in the long chain of relatively inert —CH2—CH2— groups in fatty acids, a carbonyl group is created adjacent to the —CH2— group, as we saw in the reactions of the citric acid cycle. Allosteric mechanisms and posttranslational regulation (protein phosphorylation) coordinate metabolic processes within a cell. Hormones and growth factors coordinate metabolic activities among tissues and organs. Reciprocal regulation of catabolic and anabolic pathways prevents the inefficiency of futile cycling. When a process lacks a critical component — an enzyme, a cofactor, or a regulatory agent — the resulting loss of homeostasis may cause disease across a spectrum of severity. Defects in fatty acid breakdown are no exception. The liver plays a unique role in whole-body metabolism. When glucose is unavailable, the liver makes glucose by gluconeogenesis and releases it to the blood for distribution to other tissues, including the brain. During starvation, the liver processes fatty acids into ketone bodies, which, unlike fatty acids, can cross the blood brain barrier and fuel the brain. 17.1 Digestion, Mobilization, and Transport of Fats Cells can obtain fatty acid fuels from four sources: fats consumed in the diet, fats stored in cells as lipid droplets, fats synthesized in one organ for export to another, and fats obtained by autophagy (which degrades the cell’s own organelles). Some species use all four sources under various circumstances; others use one or two. Vertebrates, for example, obtain fats in the diet; mobilize fats stored in specialized tissue (adipose tissue, consisting of cells called adipocytes); and, in the liver, convert excess dietary carbohydrates to fats for export to other tissues. During starvation, they can recycle lipids by autophagy. On average, 40% or more of the daily energy requirement of humans in highly industrialized countries is supplied by dietary triacylglycerols. Triacylglycerols provide more than half the energy requirements of some organs, particularly the liver, heart, and resting skeletal muscle. Stored triacylglycerols are virtually the sole source of energy in hibernating animals and migrating birds. Vascular plants mobilize fats stored in seeds during germination, but do not otherwise depend on fats for energy. Dietary Fats Are Absorbed in the Small Intestine In vertebrates, before ingested triacylglycerols can be absorbed through the intestinal wall they must be converted from insoluble macroscopic fat particles to finely dispersed microscopic micelles. This solubilization is carried out by bile salts, such as taurocholic acid (p. 352), which are synthesized from cholesterol in the liver, stored in the gallbladder, and released into the small intestine aer ingestion of a fatty meal. Bile salts are amphipathic compounds that act as biological detergents, emulsifying dietary fats into mixed micelles of bile salts and triacylglycerols (Fig. 17- 1, step ). Micelle formation enormously increases the fraction of lipid molecules accessible to the action of water-soluble lipases in the intestine, and lipase action converts triacylglycerols to monoacylglycerols (monoglycerides) and diacylglycerols (diglycerides) and free fatty acids (step ). These products of lipase action diffuse or are transported into the epithelial cells lining the intestinal surface (the intestinal mucosa) (step ), where they are reconverted to triacylglycerols and packaged with dietary cholesterol and specific apolipoproteins (from the Greek apo, meaning “detached” or “separate,” designating the protein in its lipid-free form) into lipoprotein aggregates called chylomicrons (step ). It is the protein moieties that target triacylglycerols, phospholipids, cholesterol, and cholesteryl esters for transport between organs. FIGURE 17-1 Processing of dietary lipids in vertebrates. Digestion and absorption of dietary lipids occur in the small intestine, and the fatty acids released from triacylglycerols are packaged and delivered to muscle and adipose tissues. The eight steps are discussed in the text. Apolipoproteins combine with lipids to form several classes of lipoprotein particles, spherical aggregates with hydrophobic lipids at the core and hydrophilic protein side chains and lipid head groups at the surface. Various combinations of lipid and protein produce particles of different densities, ranging from chylomicrons and very-low-density lipoproteins (VLDL) to very- high-density lipoproteins (VHDL). These particles can be separated by ultracentrifugation. The structures of these lipoprotein particles and their roles in lipid transport are detailed in Chapter 21 (Fig. 21-39). Apolipoprotein B-48 (apoB-48) is the primary protein component of chylomicrons. In lipid uptake from the intestine, chylomicrons move from the intestinal mucosa into the lymphatic system, and then enter the blood, where they can exchange apolipoproteins with other types of circulating lipoprotein. In the blood, chylomicrons pick up apolipoprotein C-II (apoC-II) from high- density lipoprotein (HDL) particles and are carried to muscle and adipose tissue (Fig. 17-1, step ). In the capillaries of these tissues, the extracellular enzyme lipoprotein lipase, activated by apoC-II, hydrolyzes triacylglycerols to free fatty acids and monoacylglycerols (step ), which are taken up by specific transporters in the plasma membranes of cells in the target tissues (step ). In muscle, the fatty acids are oxidized for energy; in adipose tissue, they are reesterified for storage as triacylglycerols (step ). The remnants of chylomicrons, depleted of most of their triacylglycerols but still containing cholesterol and apolipoproteins, travel in the blood to the liver, where they are taken up by endocytosis mediated by receptors for their apolipoproteins. Triacylglycerols that enter the liver by this route may be oxidized to provide energy or to provide precursors for the synthesis of ketone bodies, as described in Section 17.3. When the diet contains more fatty acids than are needed immediately for fuel or as precursors, the liver converts them to triacylglycerols, which are packaged with specific apolipoproteins into VLDLs. These VLDLs are secreted by hepatocytes and transported in the blood to adipose tissue, where the triacylglycerols are removed and stored in lipid droplets within adipocytes. Hormones Trigger Mobilization of Stored Triacylglycerols Neutral lipids (triacylglycerols, sterols, and steroyl esters) are stored in adipocytes (and in steroid-synthesizing cells of the adrenal cortex, ovary, and testis) in lipid droplets, which have a core of triacylglycerols and sterol esters surrounded by a monolayer of phospholipids. The surface of these droplets is coated with perilipins, a family of proteins that restrict access to lipid droplets, preventing untimely lipid mobilization. When hormones signal the need for metabolic energy, triacylglycerols stored in adipose tissue are mobilized (brought out of storage) and transported to tissues (skeletal muscle, heart, and renal cortex) in which fatty acids can be oxidized for energy production. The hormones epinephrine and glucagon, secreted in response to low blood glucose levels or a fight-or-flight situation, stimulate the enzyme adenylyl cyclase in the adipocyte plasma membrane (Fig. 17-2), which produces the intracellular second messenger cyclic AMP (cAMP; see Fig. 12-4). Cyclic AMP– dependent protein kinase (PKA) triggers changes that open the lipid droplet to the action of three cytosolic lipases, which act on tri-, di-, and monoacylglycerols, releasing fatty acids and glycerol. FIGURE 17-2 Mobilization of triacylglycerols stored in adipose tissue. When low levels of glucose in the blood trigger the release of glucagon, the hormone binds its receptor in the adipocyte membrane and thus stimulates adenylyl cyclase, via a G protein, to produce cAMP. This activates PKA, which phosphorylates the hormone-sensitive lipase (HSL) and perilipin molecules on the surface of the lipid droplet. Phosphorylation of perilipin causes dissociation of the protein CGI-58 from perilipin. CGI-58 (comparative gene identification-58), a protein closely associated with lipid droplets, then recruits adipose triacylglycerol lipase (ATGL) to the droplet surface and stimulates its lipase activity. Active ATGL converts triacylglycerols to diacylglycerols. The phosphorylated perilipin associates with phosphorylated HSL, allowing it access to the surface of the lipid droplet, where it converts diacylglycerols to monoacylglycerols. A third lipase, monoacylglycerol lipase (MGL), hydrolyzes monoacylglycerols. Fatty acids leave the adipocyte, and are transported in the blood bound to serum albumin. They are released from the albumin and enter a myocyte via a specific fatty acid transporter. In the myocyte, fatty acids are oxidized to CO2, and the energy of oxidation is conserved in ATP, which fuels muscle contraction and other energy-requiring metabolism in the myocyte. The fatty acids thus released (free fatty acids, FFAs) pass from the adipocyte into the blood, where they bind to the blood protein serum albumin (Fig. 17-3). This protein (Mr66,000), which makes up about half of the total serum protein, noncovalently binds as many as seven fatty acids. Bound to this soluble protein, the otherwise insoluble fatty acids are carried to tissues such as skeletal muscle, heart, and renal cortex. In these target tissues, fatty acids dissociate from albumin and are moved by plasma membrane transporters into cells to serve as fuel. FIGURE 17-3 Human serum albumin complexed with stearate. Each day the human liver releases 10–15 g of serum albumin into the bloodstream, where this workhorse protein transports many ligands, drugs, and particularly fatty acids through the blood. The structure has nooks and crannies that can carry up to seven fatty acids. Its passengers are both hydrophobic and hydrophilic, and include steroid hormones, the blood thinner warfarin, the antibiotic penicillin, the anti-inflammatory drug ibuprofen, and the anxiolytic diazepam. About 95% of the biologically available energy of triacylglycerols resides in their three long-chain fatty acids; only 5% is contributed by the glycerol moiety. The glycerol released by lipase action is phosphorylated by glycerol kinase (Fig. 17-4), and the resulting glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate. The glycolytic enzyme triose phosphate isomerase converts this compound to glyceraldehyde 3-phosphate, which is oxidized via glycolysis. FIGURE 17-4 Entry of glycerol into the glycolytic pathway. Fatty Acids Are Activated and Transported into Mitochondria The enzymes of fatty acid oxidation in animal cells are located in the mitochondrial matrix, as demonstrated in 1948 by Eugene P. Kennedy and Albert Lehninger. Short- and medium-chain fatty acids, those with chain lengths of 12 or fewer carbons, enter mitochondria without the help of membrane transporters. Long- chain fatty acids, those with 14 or more carbons, which constitute the majority of the FFAs obtained in the diet or released from adipose tissue, cannot pass directly through the mitochondrial membranes: they must be transported through the carnitine shuttle. First, the fatty acid must be activated by a fatty acyl–CoA synthetase isozyme specific for long-chain fatty acids. The isozymes are present in the outer mitochondrial membrane, where they promote the general reaction Fattyacid+ CoA + ATP ⇌  fattyacyl–CoA + AM P + PPi Fatty acyl–CoA synthetases catalyze the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl–CoA, coupled to the cleavage of ATP to AMP and PPi. (Recall the description of this reaction in Chapter 13, which illustrates how the free energy released by cleavage of phosphoanhydride bonds in ATP could be coupled to the formation of a high-energy compound; p. 485.) The reaction catalyzed by a fatty acyl–CoA synthetase occurs in two steps and involves a fatty acyl–adenylate intermediate (Fig. 17-5). MECHANISM FIGURE 17-5 Activation of a fatty acid by conversion to a fatty acyl–CoA. Formation of the fatty acyl–CoA derivative occurs in two steps, catalyzed by fatty acyl–CoA synthetase. Hydrolysis of the pyrophosphate created in the first step of that reaction is catalyzed by inorganic pyrophosphatase. The overall reaction is highly exergonic. Fatty acyl–CoAs, like acetyl-CoA, are high-energy compounds; their hydrolysis to FFAs and CoA has a large, negative standard free-energy change. The formation of a fatty acyl–CoA is made more favorable by the hydrolysis of two high-energy bonds in ATP; the pyrophosphate formed in the activation reaction is immediately hydrolyzed by inorganic pyrophosphatase (le side of Fig. 17-5), which pulls the preceding activation reaction in the direction of fatty acyl–CoA formation. The overall reaction (three steps) is Fattyacid+ CoA + ATP ⇌ fattyacyl–CoA + AM P + 2Pi ΔG′°=−34kJ /mol (17-1) Fatty acyl–CoA esters formed on the cytosolic side of the outer mitochondrial membrane can be transported into the mitochondrion and oxidized to produce ATP, or they can be used in the cytosol to synthesize membrane lipids. Fatty acyl–CoAs destined for mitochondrial oxidation must be attached to carnitine to be shuttled across the inner mitochondrial membrane. In a transesterification catalyzed by carnitine acyltransferase 1, CAT1 (also called carnitine palmitoyltransferase 1, CPT1) in the outer mitochondrial membrane, the fatty acyl–CoA is transiently attached to the hydroxyl group of carnitine to form fatty acyl– carnitine (Fig. 17-6). The fatty acyl–carnitine ester then diffuses across the intermembrane space and enters the matrix by passive transport through the acyl-carnitine/carnitine cotransporter of the inner mitochondrial membrane. This cotransporter moves one molecule of carnitine from the matrix to the intermembrane space as one molecule of fatty acyl–carnitine moves into the matrix. Once inside the matrix, the fatty acyl group is transferred from carnitine back to coenzyme A from the intramitochondrial pool by carnitine acyltransferase 2 (CAT2, or CPT2). This isozyme, located on the inner face of the inner mitochondrial membrane, regenerates fatty acyl–CoA and releases it, along with free carnitine, into the matrix. The carnitine is then available to be transferred back through the acyl-carnitine/carnitine cotransporter to be used to shuttle the next fatty acid across. Once inside the mitochondrion, the fatty acyl–CoA is acted upon by a set of enzymes in the matrix. FIGURE 17-6 Fatty acid entry into mitochondria via the acyl-carnitine/carnitine transporter. Fatty acyl–carnitine formed on the outer mitochondrial membrane moves into the matrix by passive cotransport through the inner membrane. In the matrix, the acyl group is transferred to mitochondrial coenzyme A, freeing carnitine to leave the matrix through the same transporter. This process for transferring fatty acids into the mitochondrion — esterification to CoA, transesterification to carnitine, translocation across the inner membrane, and transesterification back to CoA — links two separate pools of coenzyme A and of fatty acyl–CoA, one in the cytosol, the other in mitochondria. These pools have different functions. Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids, and some amino acids, whereas cytosolic coenzyme A is used in the biosynthesis of fatty acids (see Fig. 21-10). Fatty acyl– CoA in the cytosolic pool can be used there for membrane lipid synthesis or can be moved into the mitochondrial matrix for oxidation and ATP production. Conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate. The carnitine-mediated entry process is the rate-limiting step for oxidation of fatty acids in mitochondria and, as discussed later, is a control point. Carnitine acyltransferase 1 is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis (see Fig. 21-1). This inhibition prevents the simultaneous synthesis and degradation of fatty acids, a wasteful futile cycle. SUMMARY 17.1 Digestion, Mobilization, and Transport of Fats Dietary triacylglycerols are emulsified in the small intestine by bile salts, hydrolyzed by intestinal lipases, absorbed by intestinal epithelial cells, and reconverted into triacylglycerols. They are combined with specific apolipoproteins for passage through the blood and lymph to adipose tissue, where they are stored as lipid droplets. Triacylglycerols stored in adipose tissue are mobilized by a hormone-sensitive triacylglycerol lipase. The released fatty acids bind to serum albumin and are carried in the blood to the heart, skeletal muscle, and other tissues that use fatty acids for fuel. Once inside cells, fatty acids are activated at the outer mitochondrial membrane by conversion to fatty acyl–CoA thioesters. Fatty acyl–CoA that is to be oxidized enters mitochondria via the carnitine shuttle, which is a major control point. Malonyl-CoA, the first intermediate in fatty acid synthesis, inhibits carnitine acyltransferase 1, assuring that fatty acid oxidation and fatty acid synthesis do not occur simultaneously. 17.2 Oxidation of Fatty Acids As noted earlier, mitochondrial oxidation of fatty acids takes place in three stages (Fig. 17-7). In the first stage — β oxidation — fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acyl chain. For example, the 16-carbon palmitic acid (palmitate at pH 7) undergoes seven passes through the oxidative sequence, in each pass losing two carbons as acetyl-CoA. At the end of seven cycles, the last two carbons of palmitate (originally C-15 and C-16) remain as acetyl-CoA. The overall result is the conversion of the 16-carbon chain of palmitate to eight two- carbon acetyl groups of acetyl-CoA molecules. Formation of each acetyl-CoA requires removal of four hydrogen atoms (two pairs of electrons and four H+) from the fatty acyl moiety by dehydrogenases. FIGURE 17-7 Stages of fatty acid oxidation. Stage 1: A long-chain fatty acid is oxidized to yield acetyl residues in the form of acetyl-CoA. This process is called β oxidation. Stage 2: The acetyl groups are oxidized to CO2 via the citric acid cycle. Stage 3: Electrons derived from the oxidations of stages 1 and 2 pass to O2 via the mitochondrial respiratory chain, providing the energy for ATP synthesis by oxidative phosphorylation. In the second stage of fatty acid oxidation, the acetyl groups of acetyl-CoA are oxidized to CO2 in the citric acid cycle, which also takes place in the mitochondrial matrix. Acetyl-CoA derived from fatty acids thus enters a final common pathway of oxidation with the acetyl-CoA derived from glucose via glycolysis and pyruvate oxidation (see Fig. 16-1). The first two stages of fatty acid oxidation produce the reduced electron carriers NADH and FAD H2, which in the third stage donate electrons to the mitochondrial respiratory chain, through which the electrons pass to oxygen with the concomitant phosphorylation of ADP to ATP (Fig. 17-7). The energy released by fatty acid oxidation is thus conserved as ATP. We now take a closer look at the first stage of fatty acid oxidation, beginning with the simple case of a saturated fatty acyl chain with an even number of carbons, then turning to the slightly more complicated cases of unsaturated and odd-number chains. We also consider the regulation of fatty acid oxidation, the β - oxidative processes as they occur in organelles other than mitochondria, and, finally, a less-common mode of fatty acid catabolism — α oxidation. The β Oxidation of Saturated Fatty Acids Has Four Basic Steps Four enzyme-catalyzed reactions make up the first stage of fatty acid oxidation (Fig. 17-8a). First, dehydrogenation of fatty acyl– CoA produces a double bond between the α and β carbon atoms (C-2 and C-3), yielding a trans-Δ2-enoyl-CoA (the symbol Δ 2 designates the position of the double bond; you may want to review fatty acid nomenclature, p. 342). Note that the new double bond has the trans configuration, whereas the double bonds in naturally occurring unsaturated fatty acids are normally in the cis configuration. We consider the significance of this difference later.

FIGURE 17-8 The β -oxidation pathway. (a) In each pass through this four-step sequence, one acetyl residue (shaded in light red) is removed in the form of acetyl-CoA from the carboxyl end of the fatty acyl chain — in this example palmitate (C16), which enters as palmitoyl-CoA. Electrons from the first oxidation pass through electron transfer flavoprotein (ETF), and then through a second flavoprotein (ETF:ubiquinone oxidoreductase), into the respiratory chain. Electrons from the second oxidation enter the respiratory chain through NADH dehydrogenase. (b) Six more passes through the β - oxidation pathway yield seven more molecules of acetyl-CoA, the seventh arising from the last two carbon atoms of the 16-carbon chain. Eight molecules of acetyl-CoA are formed in all. The acetyl-CoA may be oxidized in the citric acid cycle, donating more electrons to the respiratory chain. This first step is catalyzed by three isozymes of acyl-CoA dehydrogenase, each specific for a range of fatty-acyl chain lengths: very-long-chain acyl-CoA dehydrogenase (VLCAD), acting on fatty acids of 12 to 18 carbons; medium-chain (MCAD), acting on fatty acids of 4 to 14 carbons; and short-chain (SCAD), acting on fatty acids of 4 to 8 carbons. VLCAD is in the inner mitochondrial membrane; MCAD and SCAD are in the matrix. All three isozymes are flavoproteins with tightly bound FAD (see Fig. 13-27) as a prosthetic group. The electrons removed from the fatty acyl–CoA are transferred to FAD, and the reduced form of the dehydrogenase immediately donates its electrons to an electron carrier, the electron transfer flavoprotein (ETF) (see Fig. 19-15). Electrons move from ETF to a second flavoprotein, ETF:ubiquinone oxidoreductase, and through ubiquinone into the mitochondrial respiratory chain. The oxidation catalyzed by an acyl-CoA dehydrogenase is analogous to succinate dehydrogenation in the citric acid cycle (p. 586); in both reactions the enzyme is bound to the inner membrane, a double bond is introduced into a carboxylic acid between the α and β carbons, FAD is the electron acceptor, and electrons from the reaction ultimately enter the respiratory chain and pass to O2, with the concomitant synthesis of about 1.5 ATP molecules per electron pair. In the second step of the β -oxidation cycle (Fig. 17-8a), water is added to the double bond of the trans-Δ 2-enoyl-CoA to form the L stereoisomer of β-hydroxyacyl-CoA (3-hydroxyacyl-CoA). This reaction, catalyzed by enoyl-CoA hydratase, is formally analogous to the fumarase reaction in the citric acid cycle, in which H2O adds across an α–β double bond (p. 587). In the third step, L-β -hydroxyacyl-CoA is dehydrogenated to form β-ketoacyl-CoA, by the action of β-hydroxyacyl-CoA dehydrogenase; NAD + is the electron acceptor. This enzyme is absolutely specific for the L stereoisomer of hydroxyacyl-CoA. The NADH formed in the reaction donates its electrons to NADH dehydrogenase (Complex I), an electron carrier of the respiratory chain (see Fig. 19-15), and ATP is formed from ADP as the electrons pass to O2. The reaction catalyzed by β - hydroxyacyl-CoA dehydrogenase is closely analogous to the malate dehydrogenase reaction of the citric acid cycle (p. 587). The fourth and last step of the β -oxidation cycle is catalyzed by acyl-CoA acetyltransferase, more commonly called thiolase, which promotes reaction of β -ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA. The other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms (Fig. 17-8a). This reaction is called thiolysis, by analogy with the process of hydrolysis, because the β -ketoacyl-CoA is cleaved by reaction with the thiol group of coenzyme A. The thiolase reaction is a reverse Claisen condensation (see Fig. 13-4). The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of α4β4 subunits. Each α subunit contains two activities, the enoyl-CoA hydratase and the β -hydroxyacyl-CoA dehydrogenase; the β subunits contain the thiolase activity. This tight association of three enzymes allows efficient substrate channeling from one active site to the next, without diffusion of the intermediates away from the enzyme surface. When TFP has shortened the fatty acyl chain to 12 or fewer carbons, further oxidations are catalyzed by a set of four soluble enzymes in the matrix. As noted earlier, the single bond between methylene (—CH2—) groups in fatty acids is relatively stable. The β - oxidation sequence is an elegant mechanism for destabilizing and breaking these bonds. The first three reactions of β oxidation create a much less stable C—C bond, in which the α carbon (C-2) is bonded to two carbonyl carbons (the β -ketoacyl-CoA intermediate). The ketone function on the β carbon (C-3) makes it a good target for nucleophilic attack by the — SH of coenzyme A, catalyzed by thiolase. The acidity of the α hydrogen and the resonance stabilization of the carbanion generated by the departure of this hydrogen make the terminal —CH2—CO—S-CoA a good leaving group, facilitating breakage of the α–β bond. We have already seen a reaction sequence nearly identical with these four steps of fatty acid oxidation, in the citric acid cycle reaction steps between succinate and oxaloacetate (see Fig. 16-7). A nearly identical reaction sequence occurs again in the pathways by which the branched-chain amino acids (isoleucine, leucine, and valine) are oxidized as fuels (see Fig. 18-28). Figure 17-9 shows the common features of these three sequences, almost certainly an example of the conservation of a mechanism by gene duplication and evolution of a new specificity in the enzyme products of the duplicated genes. FIGURE 17-9 A conserved reaction sequence to introduce a carbonyl function on the carbon β to a carboxyl group. The β -oxidation pathway for fatty acyl–CoAs, the pathway from succinate to oxaloacetate in the citric acid cycle, and the pathway by which the deaminated carbon skeletons from isoleucine, leucine, and valine are oxidized as fuels — all use the same reaction sequence. The Four β -Oxidation Steps Are Repeated to Yield Acetyl-CoA and ATP In one pass through the β -oxidation sequence, one molecule of acetyl-CoA, two pairs of electrons, and four protons (H+) are removed from the long-chain fatty acyl–CoA, shortening it by two carbon atoms. The equation for one pass, beginning with the coenzyme A ester of our example, palmitate, is Palmitoyl-CoA + CoA + FAD + NAD + + H2O → myristoyl-CoA + acetyl-CoA + FAD H2+ NAD H + H+ (17-2) Following removal of one acetyl-CoA unit from palmitoyl-CoA, the coenzyme A thioester of the shortened fatty acid (now the 14- carbon myristate) remains. The myristoyl-CoA can now go through another set of four β -oxidation reactions, exactly analogous to the first, to yield a second molecule of acetyl-CoA and lauroyl-CoA, the coenzyme A thioester of the 12-carbon laurate. Altogether, seven passes through the β -oxidation sequence are required to oxidize one molecule of palmitoyl-CoA to eight molecules of acetyl-CoA (Fig. 17-8b). The overall equation is Palmitoyl-CoA + 7CoA + 7FAD + 7NAD + + 7H2O → 8acetyl-CoA + 7FAD H2+ 7NAD H + 7H+ (17-3) Each molecule of FAD H2 formed during oxidation of the fatty acid donates a pair of electrons to ETF of the respiratory chain, and about 1.5 molecules of ATP are generated during the ensuing transfer of each electron pair to O2. Similarly, each molecule of NADH formed delivers a pair of electrons to the mitochondrial NADH dehydrogenase, and the subsequent transfer of each pair of electrons to O2 results in formation of about 2.5 molecules of ATP. Thus four molecules of ATP are formed for each two-carbon unit removed in one pass through the sequence. Note that water is also produced in this process. Each pair of electrons transferred from NADH or FAD H2 to O2 yields one H2O, referred to as “metabolic water.” Reduction of O2 by NADH also consumes one H+ per NADH molecule: NAD H + H+ + ½O2 → NAD + + H2O. In hibernating animals, fatty acid oxidation provides metabolic energy, heat, and water — all essential for survival of an animal that neither eats nor drinks for long periods (Box 17-1). Camels obtain water to supplement the meager supply available in their natural environment by oxidation of fats stored in their hump. BOX 17-1 A Long Winter’s Nap: Oxidizing Fats during Hibernation Many animals depend on fat stores for energy during hibernation, during migratory periods, and in other situations involving radical metabolic adjustments. One of the most pronounced adjustments of fat metabolism occurs in hibernating grizzly bears. These animals remain in a continuous state of dormancy for as long as seven months. Unlike most hibernating species, the bear maintains a body temperature of about 31 °C, close to its normal (nonhibernating) temperature near 40 °C. Although expending about 25,000 kJ/day (6,000 kcal/day) while hibernating, the bear does not eat, drink, urinate, or defecate for months at a time. Its heart rate drops from 90 to 8 beats per minute, and its respiration rate drops from 6 to 10 breaths to approximately 1 breath per minute. As we shall see in Chapter 19, mitochondrial electron transfer can be uncoupled from ATP production so that all of the energy of fuel oxidation is dissipated as heat, to maintain a body temperature near normal in the face of much lower ambient temperatures. One form of fat tissue, brown adipose tissue, is especially important in thermogenesis; we discuss it in more detail in Chapter 23.

A grizzly bear prepares its hibernation nest near the McNeil River in Canada. Experimental studies have shown that hibernating grizzly bears use body fat as their sole fuel. Fat oxidation yields sufficient energy to maintain body temperature, synthesize amino acids and proteins, and carry out other energy- requiring activities, such as membrane transport. Fat oxidation also releases large amounts of water, as described in the text, which replenishes water lost in breathing. The glycerol released by degradation of triacylglycerols is converted into blood glucose by gluconeogenesis. Urea formed during breakdown of amino acids is reabsorbed in the kidneys and recycled, with the amino groups reused to make new amino acids for maintaining body proteins. Bears store an enormous amount of body fat in preparation for their long sleep. An adult grizzly consumes about 38,000 kJ/day during the late spring and summer, but as winter approaches it feeds for 20 hours a day, consuming up to 84,000 kJ daily. This increase in feeding is a response to a seasonal change in hormone secretion. Large amounts of triacylglycerols are formed from the huge intake of carbohydrates during the fattening-up period. The bear will emerge from hibernation having lost 15% to 40% of its maximum body weight. The winter sleep of bears is sometimes called torpor, and it differs in important ways from the hibernation behavior of a group of smaller animals that undergo alternating periods of high and low body temperature. In these animals, body temperature approaches ambient temperature, close to 0 °C, for much of the time during hibernation, but rises to almost prehibernation level during brief periods of wakefulness. During these periods, the animals eat, drink, and defecate. In the Arctic ground squirrel (Urocitellus parryii), for example, body temperature (37 °C prehibernation) drops to 0 °C during hibernation, and respiration drops to less than 10% of its prehibernation rate. Studies of hibernation mechanisms may yield insight into several problems in human medicine; for example, slowing the metabolism of organs donated for transplantation might extend the period of their viability. And if humans are to make long trips into space, inducing a torporlike state might relieve the monotony of long missions and conserve onboard resources such as food and oxygen. The overall equation for the oxidation of palmitoyl-CoA to eight molecules of acetyl-CoA, including the electron transfers and oxidative phosphorylations, is Palmitoyl-CoA + 7CoA + 7O2+ 28Pi+ 28AD P → 8 acetyl-CoA + 28AT P + 7H2O (17-4) Acetyl-CoA Can Be Further Oxidized in the Citric Acid Cycle The acetyl-CoA produced from the oxidation of fatty acids can be oxidized to CO2 and H2O by the citric acid cycle. The following equation represents the balance sheet for the second stage in the oxidation of palmitoyl-CoA, together with the coupled phosphorylations of the third stage: 8Acetyl-CoA + 16O2+ 80Pi+ 80AD P → 8CoA + 80AT P + 16CO2+ 16H2O (17-5) Combining Equations 17-4 and 17-5, we obtain the overall equation for the complete oxidation of palmitoyl-CoA to carbon dioxide and water: Palmitoyl-CoA + 23O2+ 108Pi+ 108AD P → CoA + 108AT P + 16CO2+ 23H2O (17-6) Table 17-1 summarizes the yields of NADH, FAD H2, and ATP in the successive steps of palmitoyl-CoA oxidation. Note that because the activation of palmitate to palmitoyl-CoA breaks both phosphoanhydride bonds in ATP (Fig. 17-5), the energetic cost of activating a fatty acid is equivalent to two ATP, and the net gain per molecule of palmitate is 106 ATP. The standard free-energy change for the oxidation of palmitate to CO2 and H2O is about 9,800 kJ/mol. Under standard conditions, the energy recovered as the phosphate bond energy of ATP is 106× 30.5kJ /mol= 3,230kJ /mol, about 33% of the theoretical maximum. However, when the free-energy changes are calculated from actual concentrations of reactants and products under intracellular conditions, the free-energy recovery is more than 60%; the energy conservation is remarkably efficient (see Worked Example 13-2, p. 480). TABLE 17.1 Yield of ATP during Oxidation of One Molecule of Palmitoyl-CoA to CO2 and H2O Enzyme catalyzing the oxidation step Number of NADH or FADH2 formed Number of ATP ultimately formed β Oxidation Acyl-CoA dehydrogenase 7FAD H2     10.5 β -Hydroxyacyl-CoA 7 NADH     17.5 a dehydrogenase Citric acid cycle Isocitrate dehydrogenase 8 NADH    20 α -Ketoglutarate dehydrogenase 8 NADH    20 Succinyl-CoA synthetase       8 Succinate dehydrogenase 8FAD H2    12 Malate dehydrogenase 8 NADH    20     Total 108 These calculations assume that mitochondrial oxidative phosphorylation produces 1.5 ATP per FAD H2 oxidized and 2.5 ATP per NADH oxidized. GTP produced directly in this step yields ATP in the reaction catalyzed by nucleoside diphosphate kinase (p. 487). Oxidation of Unsaturated Fatty Acids Requires Two Additional Reactions The fatty acid oxidation sequence just described is typical when the incoming fatty acid is saturated (that is, has only single bonds in its carbon chain). However, most of the fatty acids in the triacylglycerols and phospholipids of animals and plants are unsaturated, having one or more double bonds. These bonds are in the cis configuration and cannot be acted upon by enoyl-CoA hydratase, the enzyme catalyzing the addition of H2O to the trans double bond of the Δ 2-enoyl-CoA generated during β oxidation. b a b Two auxiliary enzymes — an isomerase and a reductase — must act on the common unsaturated fatty acids to transform them into substrates for the β -oxidation pathway. We illustrate these auxiliary reactions with two examples. Oleate is an abundant 18-carbon monounsaturated fatty acid with a cis double bond between C-9 and C-10 (denoted Δ 9). In the first step of oxidation, oleate is converted to oleoyl-CoA and, like the saturated fatty acids, enters the mitochondrial matrix via the carnitine shuttle (Fig. 17-6). Oleoyl-CoA then undergoes three passes through the fatty acid oxidation cycle to yield three molecules of acetyl-CoA and the coenzyme A ester of a Δ 3, 12- carbon unsaturated fatty acid, cis-Δ 3-dodecenoyl-CoA (Fig. 17-10). This product cannot serve as a substrate for enoyl-CoA hydratase, which acts only on trans double bonds. The auxiliary enzyme Δ3,Δ2-enoyl-CoA isomerase isomerizes the cis-Δ 3-enoyl-CoA to the trans-Δ 2-enoyl-CoA, which is converted by enoyl-CoA hydratase into the corresponding L-β hydroxyacyl-CoA (trans-Δ 2- dodecenoyl-CoA). This intermediate is now acted upon by the remaining enzymes of β oxidation to yield acetyl-CoA and the coenzyme A ester of a 10-carbon saturated fatty acid, decanoyl- CoA. The latter undergoes four more passes through the β - oxidation pathway to yield five more molecules of acetyl-CoA. Altogether, nine acetyl-CoAs are produced from one molecule of the 18-carbon oleate. FIGURE 17-10 Oxidation of a monounsaturated fatty acid. Oleic acid, as oleoyl-CoA (Δ 9), is the example used here. Oxidation requires an additional enzyme, enoyl-CoA isomerase, to reposition the double bond, converting the cis isomer to a trans isomer, an intermediate in β oxidation. The other auxiliary enzyme (a reductase) is required for oxidation of polyunsaturated fatty acids — for example, the 18-carbon linoleate, which has a cis-Δ 9,cis-Δ 12 configuration (Fig. 17-11). Linoleoyl-CoA undergoes three passes through the β -oxidation sequence to yield three molecules of acetyl-CoA and the coenzyme A ester of a 12-carbon unsaturated fatty acid with a cis-Δ 3, cis-Δ 6 configuration. This intermediate cannot be used by the enzymes of the β -oxidation pathway: its double bonds are in the wrong position and have the wrong configuration (cis, not trans). However, the combined action of enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase, as shown in Figure 17-11, transforms this intermediate into one that can enter the β -oxidation pathway and be degraded to six acetyl-CoAs. The overall result is conversion of linoleate to nine molecules of acetyl-CoA.

FIGURE 17-11 Oxidation of a polyunsaturated fatty acid. The example here is linoleic acid, as linoleoyl-CoA (Δ 9,12). Oxidation requires a second auxiliary enzyme in addition to enoyl-CoA isomerase: NADPH-dependent 2,4-dienoyl-CoA reductase. The combined action of these two enzymes converts a trans-Δ 2, cis-Δ 4-dienoyl-CoA intermediate to the trans-Δ 2- enoyl-CoA substrate necessary for β oxidation. Complete Oxidation of Odd-Number Fatty Acids Requires Three Extra Reactions Although most naturally occurring lipids contain fatty acids with an even number of carbon atoms, fatty acids with an odd number of carbons are common in the lipids of many plants and some marine organisms. Cattle and other ruminant animals form large amounts of the three-carbon propionate (CH3—CH2—COO−) during fermentation of carbohydrates in the rumen. The propionate is absorbed into the blood and oxidized by the liver and other tissues. Long-chain odd-number fatty acids are oxidized in the same pathway as the even-number acids, beginning at the carboxyl end of the chain. However, the substrate for the last pass through the β -oxidation sequence is a fatty acyl–CoA with a five-carbon fatty acid. When this is oxidized and cleaved, the products are acetyl- CoA and propionyl-CoA. The acetyl-CoA can be oxidized in the citric acid cycle, of course, but propionyl-CoA enters a different pathway, having three enzymes. Propionyl-CoA is first carboxylated to form the D stereoisomer of methylmalonyl-CoA by propionyl-CoA carboxylase, which contains the cofactor biotin (Fig. 17-12). In this enzymatic reaction, as in the pyruvate carboxylase reaction (see Fig. 16-16), bicarbonate ion (HCO− 3) is activated by attachment to biotin before its transfer to the substrate, in this case the propionate moiety. Formation of the carboxybiotin intermediate requires energy, which is provided by ATP. The D-methylmalonyl-CoA thus formed is enzymatically epimerized to its L stereoisomer by methylmalonyl-CoA epimerase. The L-methylmalonyl-CoA then undergoes an intramolecular rearrangement to form succinyl- CoA, which can enter the citric acid cycle. This rearrangement is catalyzed by methylmalonyl-CoA mutase, which requires as its coenzyme 5′-deoxyadenosylcobalamin, or coenzyme B12, which is derived from vitamin B12 (cobalamin). Box 17-2 describes the role of coenzyme B12 in this remarkable exchange reaction. FIGURE 17-12 Oxidation of propionyl-CoA produced by β oxidation of odd-number fatty acids. The sequence involves the carboxylation of propionyl-CoA to -methylmalonyl-CoA and conversion of the latter to succinyl-CoA. This conversion requires epimerization of - to - methylmalonyl-CoA, followed by a remarkable reaction in which substituents on adjacent carbon atoms exchange positions (see Box 17-2). BOX 17-2 Coenzyme B12: A Radical Solution to a Perplexing Problem In the methylmalonyl-CoA mutase reaction (see Fig. 17-12), the group —CO—S-CoA at C-2 of the original propionate exchanges position with a hydrogen atom at C-3 of the original propionate (Fig. 1a). Coenzyme B12 is the cofactor for this general type of reaction (Fig. 1b). These coenzyme B12– dependent processes are among the very few enzymatic reactions in biology in which there is an exchange of an alkyl or substituted alkyl group (X) with a hydrogen atom on an adjacent carbon, with no mixing of the transferred hydrogen atom with the hydrogen of the solvent, H2O. How can the hydrogen atom move between two carbons without mixing with the enormous excess of hydrogen atoms in the solvent? FIGURE 1 Coenzyme B12 is the cofactor form of vitamin B12, which is unique among all the vitamins in that it contains an essential trace element, cobalt. The complex corrin ring system of vitamin B12 (colored blue in Fig. 2), to which cobalt (as Co3+) is coordinated, is chemically related to the porphyrin ring system of heme (see Fig. 5-1). A fi h coordination position of cobalt is filled by dimethylbenzimidazole ribonucleotide (shaded yellow), bound covalently by its 3′-phosphate group to a side chain of the corrin ring, through aminoisopropanol. The formation of this complex cofactor occurs in one of only two known reactions in which triphosphate is cleaved from ATP (Fig. 3); the other reaction is the formation of S-adenosylmethionine from ATP and methionine (see Fig. 18-18). FIGURE 2 FIGURE 3 Vitamin B12 is usually isolated as cyanocobalamin, because it contains a cyano group (picked up during purification) attached to cobalt in the sixth coordination position. In 5′-deoxyadenosylcobalamin, the cofactor for methylmalonyl-CoA mutase, the cyano group is replaced by the 5′- deoxyadenosyl group (red in Fig. 2), covalently bound through C-5′ to the cobalt. The three-dimensional structure of the cofactor was determined by Dorothy Crowfoot Hodgkin in 1956, using x-ray crystallography. Dorothy Crowfoot Hodgkin, 1910–1994 The key to understanding how coenzyme B12 catalyzes hydrogen exchange lies in the properties of the covalent bond between cobalt and C-5′ of the deoxyadenosyl group (Fig. 2). This is a relatively weak bond; merely illuminating the compound with visible light is enough to break this Co—C bond. (This extreme photolability probably accounts for the absence of vitamin B12 in plants.) Dissociation produces a 5′-deoxyadenosyl radical and the Co2+ form of the vitamin. By generating free radicals in this way, 5′- deoxyadenosylcobalamin initiates a series of transformations such as that illustrated in Figure 4 — a postulated mechanism for the reaction catalyzed by methylmalonyl-CoA mutase and several other coenzyme B12–dependent transformations. In this postulated mechanism, the migrating hydrogen atom never exists as a free species and is thus never free to exchange with the hydrogen of surrounding water molecules. MECHANISM FIGURE 4 Vitamin B12 is not made by plants or animals and can be synthesized only by a few species of microorganisms. It is required by healthy people in only minute amounts, about 3μg/day. Vitamin B12 deficiency results in pernicious anemia, caused by a failure to absorb vitamin B12 efficiently from the intestine, where it is synthesized by intestinal bacteria or obtained from digestion of meat. Individuals with this disease do not produce sufficient amounts of intrinsic factor, a glycoprotein essential to vitamin B12 absorption. The pathology in pernicious anemia includes reduced production of erythrocytes, reduced levels of hemoglobin, and severe, progressive impairment of the central nervous system. Administration of large doses of vitamin B12 alleviates these symptoms in at least some cases.

About 1 in 100,000 babies are born with a genetic deficiency in propionyl-CoA carboxylase, making them unable to metabolize the propionyl-CoA that results from catabolism of odd-number fatty acids, fatty acids with methyl branches, and the amino acids isoleucine, valine, threonine, and methionine. The absence of functional propionyl-CoA carboxylase leads to an accumulation of propionyl-CoA in mitochondria, depleting the available supply of coenzyme A for continuing β -oxidation and other metabolism. The propionyl-CoA is esterified to carnitine, transported out of the mitochondria through the carnitine shuttle, and eventually released to the blood as propionate, which severely acidifies the blood and urine, a condition called propionic acidemia. Symptoms generally manifest in the first few days of life, with vomiting, low blood glucose, seizures, and neurologic abnormalities. The condition is diagnosed by detection of propionate and its metabolites in the blood and urine. Treatments include severe restriction of dietary protein, supplying carnitine, and the use of antibiotics targeted at gut bacteria that produce odd-chain and branched-chain fatty acids. Some individuals who are defective in the incorporation of biotin into the enzyme benefit from treatment with high doses of biotin. Fatty Acid Oxidation Is Tightly Regulated Oxidation of fatty acids consumes a precious fuel, and it is regulated so as to occur only when the organism’s need for energy requires it. In the liver, fatty acyl–CoA formed in the cytosol has two major pathways open to it: (1) β oxidation by enzymes in mitochondria or (2) conversion into triacylglycerols and phospholipids by enzymes in the cytosol. The pathway taken depends on the rate of transfer of long-chain fatty acyl–CoA into mitochondria. The three-step carnitine shuttle by which fatty acyl groups are carried into the mitochondrial matrix as fatty acyl– carnitine (Fig. 17-6) is rate-limiting for fatty acid oxidation and is an important point of regulation. Once fatty acyl groups have entered the mitochondrion, they are committed to oxidation to acetyl-CoA. Figure 17-13 illustrates, with numbered steps described below, the coordinated down-regulation of β oxidation when carbohydrate levels are sufficient. Ingestion of a high- carbohydrate meal raises the blood glucose level and thus triggers the release of insulin. Insulin-dependent protein phosphatase dephosphorylates the enzyme acetyl-CoA carboxylase (ACC), activating it. ACC catalyzes the formation of malonyl-CoA (the first intermediate of cytosolic fatty acid synthesis; see Figs 21-1 and 21-2), and malonyl-CoA inhibits carnitine acyltransferase 1, thereby preventing fatty acid entry into the mitochondrial matrix through the carnitine shuttle. The inhibition of carnitine acyltransferase 1 by malonyl-CoA ensures that the oxidation of fatty acids is inhibited whenever the liver is amply supplied with glucose as fuel and is actively making triacylglycerols from excess glucose. Two of the enzymes of β oxidation ( ) are also regulated by metabolites that signal energy sufficiency. When the [NAD H]/[NAD +] ratio is high, β - hydroxyacyl-CoA dehydrogenase is inhibited; in addition, high concentrations of acetyl-CoA inhibit thiolase. FIGURE 17-13 Coordinated regulation of fatty acid synthesis and breakdown. The steps are explained in the text. Figure 17-13 also shows that conversely, when blood glucose levels drop between meals, glucagon release activates protein kinase A (PKA), which phosphorylates and inactivates ACC. Recall from Chapter 14 that in a low-energy state, [AMP] rises relative to [ATP], activating AMP kinase (AMPK). Activated AMPK also phosphorylates and inactivates ACC. The concentration of malonyl-CoA falls, and the inhibition of fatty acid entry into mitochondria is relieved. Fatty acids enter the mitochondrial matrix, allowing β oxidation to replenish the supply of ATP. Because glucagon also triggers the mobilization of fatty acids in adipose tissue, a supply of fatty acids begins arriving in the blood. Transcription Factors Turn on the Synthesis of Proteins for Lipid Catabolism In addition to the various short-term regulatory mechanisms that modulate the activity of existing enzymes, transcriptional regulation can change the number of molecules of the enzymes of fatty acid oxidation on a longer time scale — minutes to hours. The PPAR family of nuclear receptors are transcription factors that affect many metabolic processes in response to a variety of fatty acid–like ligands. (They were originally recognized as peroxisome proliferator-activated receptors, then were found to function more broadly.) PPARα acts in muscle, adipose tissue, and liver to turn on a set of genes essential for fatty acid oxidation, including the fatty acid transporter, carnitine acyltransferases 1 and 2; the fatty acyl–CoA dehydrogenases for short, medium, long, and very long acyl chains; and related enzymes. This response is triggered when a cell or an organism has an increased demand for energy from fat catabolism, such as during a fast between meals or under conditions of longer-term starvation. Glucagon, released in response to low blood glucose, can act through cAMP and the transcription factor CREB to turn on certain genes for lipid catabolism. Another situation that is accompanied by major changes in the expression of the enzymes of fatty acid oxidation is the transition from fetal to neonatal metabolism in the heart. In the fetus, the principal fuels in heart muscle are glucose and lactate, but in the neonatal heart, fatty acids are the main fuel. At the time of this transition, PPARα is activated and in turn activates the genes essential for fatty acid metabolism. As we will see in Chapter 23, two other transcription factors in the PPAR family also play crucial roles in determining the enzyme complements — and therefore the metabolic activities — of specific tissues at particular times (see Fig. 23-38). The major sites of fatty acid oxidation, at rest and during exercise, are skeletal and heart muscle. Endurance training increases PPARα expression in muscle, leading to increased levels of fatty acid–oxidizing enzymes and increased oxidative capacity of the muscle. Genetic Defects in Fatty Acyl–CoA Dehydrogenases Cause Serious Disease Stored triacylglycerols are typically the chief source of energy for muscle contraction, and an inability to oxidize fatty acids from triacylglycerols has serious consequences for health. The most common genetic defect in fatty acid catabolism in U.S. and northern European populations is due to a mutation in the gene encoding the medium-chain acyl-CoA dehydrogenase (MCAD), mentioned earlier in the chapter. Among northern Europeans, the frequency of carriers (individuals with this recessive mutation on one of the two homologous chromosomes) is about 1 in 40, and about 1 individual in 10,000 has the disease — that is, has two copies of the mutant MCAD allele and is unable to oxidize fatty acids of 6 to 12 carbons. The disease is characterized by recurring episodes of a syndrome that includes fat accumulation in the liver, high blood levels of octanoic acid (8:0), low blood glucose (hypoglycemia), sleepiness, vomiting, and coma. Although individuals may have no symptoms between episodes, the episodes are very serious; mortality due to this disease is 25% to 60% in early childhood. If the genetic defect is detected shortly aer birth, the infant can be started on a low-fat, high-carbohydrate diet. With early detection and careful management of the diet — including avoiding long intervals between meals, to prevent the body from turning to its fat reserves for energy — the prognosis for these individuals is good. More than 20 human genetic defects in fatty acid transport or oxidation have been documented, most much less common than the defect in MCAD. One of the most severe disorders results from loss of the long-chain β -hydroxyacyl-CoA dehydrogenase activity of the trifunctional protein, TFP. Other disorders include defects in the α or β subunits that affect all three activities of TFP and cause serious heart disease and abnormal skeletal muscle. Peroxisomes Also Carry Out β Oxidation The mitochondrial matrix is the major site of fatty acid oxidation in animal cells, but other compartments also contain enzymes capable of oxidizing fatty acids to acetyl-CoA, by a pathway similar but not identical to that in mitochondria. In plant cells, the major site of β oxidation is not mitochondria but peroxisomes. In peroxisomes, organelles also found in animal cells, the intermediates for β oxidation of fatty acids are coenzyme A derivatives, and the process consists of four steps, as in mitochondrial β oxidation (Fig. 17-14): (1) dehydrogenation, (2) addition of water to the resulting double bond, (3) oxidation of the β -hydroxyacyl-CoA to a ketone, and (4) thiolytic cleavage by coenzyme A. The identical reactions also occur in glyoxysomes, organelles found only in germinating seeds. FIGURE 17-14 Comparison of β oxidation in mitochondria and in peroxisomes and glyoxysomes. The peroxisomal/glyoxysomal system differs from the mitochondrial system in three respects: (1) the peroxisomal system prefers very-long-chain fatty acids; (2) in the first oxidative step, electrons pass directly to O2, generating H2O2; and (3) the NADH formed in the second oxidative step cannot be reoxidized in the peroxisome or glyoxysome, so reducing equivalents are exported to the cytosol, eventually entering mitochondria. The acetyl-CoA produced by peroxisomes and glyoxysomes is also exported; the acetate from glyoxysomes serves as a biosynthetic precursor. Acetyl-CoA produced in mitochondria is further oxidized in the citric acid cycle. One difference between the peroxisomal and mitochondrial pathways is in the chemistry of the first step (Fig. 17-14). In peroxisomes, the flavoprotein acyl-CoA oxidase that introduces the double bond passes electrons directly to O2, producing H2O2 (thus the name “peroxisomes”). This strong and potentially damaging oxidant is immediately cleaved to H2O and O2 by catalase. Recall that in mitochondria, the electrons removed in the first oxidation step pass through the respiratory chain to O2 to produce H2O, and this process is accompanied by ATP synthesis. In peroxisomes, the energy released in the first oxidative step of fatty acid breakdown is not conserved as ATP, but is dissipated as heat. A second important difference between mitochondrial and peroxisomal β oxidation in mammals is in the specificity for fatty acyl–CoAs; the peroxisomal system is much more active on very- long-chain fatty acids such as hexacosanoic acid (26:0) and on branched-chain fatty acids such as phytanic acid and pristanic acid (see Fig. 17-15). These less-common fatty acids are obtained from dietary intake of dairy products, the fat of ruminant animals, meat, and fish. Their catabolism in the peroxisome involves several auxiliary enzymes unique to this organelle. The inability to oxidize these compounds is responsible for several serious human genetic diseases. Individuals with Zellweger syndrome are unable to make peroxisomes and therefore lack all the metabolism unique to that organelle. In X- linked adrenoleukodystrophy (XALD), peroxisomes fail to oxidize very-long-chain fatty acids, apparently due to lack of a functional transporter for these fatty acids in the peroxisomal membrane. Both defects lead to accumulation in the blood of very-long-chain fatty acids, especially 26:0. XALD affects young boys before the age of 10 years, causing loss of vision, behavioral disturbances, and death within a few years. In mammals, high concentrations of fats in the diet result in increased synthesis of the enzymes of peroxisomal β oxidation in the liver. Liver peroxisomes do not contain the enzymes of the citric acid cycle and cannot catalyze the oxidation of acetyl-CoA to CO2. Instead, long-chain or branched fatty acids are catabolized in peroxisomes to shorter-chain products, such as hexanoyl-CoA, which are exported to mitochondria and completely oxidized there. As we will see in Chapter 20, the germinating seeds of plants can synthesize carbohydrates and many other metabolites from acetyl-CoA produced in peroxisomes, using a pathway (the glyoxylate cycle) not present in vertebrates. Phytanic Acid Undergoes α Oxidation in Peroxisomes Phytanic acid, a long-chain fatty acid with methyl branches, is derived from the phytol side chain of chlorophyll (see Fig. 20-5). The presence of a methyl group on the β carbon of this fatty acid prevents the formation of a β -keto intermediate, making its β oxidation impossible. Humans obtain phytanic acid in the diet, primarily from dairy products and from the fats of ruminant animals; microorganisms in the rumen of these animals produce phytanic acid as they digest plant chlorophyll. The typical western diet includes 50 to 100 mg of phytanic acid per day. Phytanic acid is metabolized in peroxisomes by α oxidation, in which a single carbon is removed from the carboxyl end of the fatty acid (Fig. 17-15). Phytanoyl-CoA is first hydroxylated on its α carbon in a reaction that involves molecular oxygen. The product is decarboxylated to form an aldehyde one carbon shorter, and then oxidized to the corresponding carboxylic acid, which now has no substituent on the β carbon. Further β oxidation produces acetyl-CoA and then propionyl-CoA in successive oxidation cycles. Refsum disease, resulting from a genetic defect in phytanoyl-CoA hydroxylase, leads to the accumulation of very high blood levels of phytanic acid, causing (by unknown mechanisms) severe neurological deficits, including blindness and deafness.

FIGURE 17-15 The α oxidation of a branched-chain fatty acid (phytanic acid) in peroxisomes. Phytanic acid has a methyl-substituted β carbon and therefore cannot undergo β oxidation. The combined action of the enzymes shown here removes the carboxyl carbon of phytanic acid to produce pristanic acid, in which the β carbon is unsubstituted, allowing β oxidation. Notice that β oxidation of pristanic acid releases propionyl-CoA, not acetyl-CoA. This is further catabolized as in Figure 17-12. (The details of the reaction that produces pristanal remain controversial.) SUMMARY 17.2 Oxidation of Fatty Acids In the first stage of β oxidation, four sequential reactions remove each acetyl-CoA unit, in turn, from the carboxyl end of a saturated fatty acyl–CoA: (1) dehydrogenation of the α and β carbons by acyl–CoA dehydrogenases; (2) hydration of the resulting trans-Δ 2 double bond by enoyl-CoA hydratase; (3) dehydrogenation of the resulting L-β -hydroxyacyl-CoA; and (4) cleavage of the resulting β -ketoacyl–CoA by thiolase, to form acetyl-CoA and a fatty acyl–CoA shortened by two carbons. The shortened fatty acyl–CoA then reenters the β oxidation sequence for removal of another, and then another, acetyl-CoA. In the second stage of fatty acid oxidation, the acetyl-CoA is oxidized to CO2 in the citric acid cycle. Much of the free energy from fatty acid oxidation is recovered as ATP by oxidative phosphorylation, the final stage of the oxidative pathway. Oxidation of unsaturated fatty acids requires two additional enzymes: enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. Odd-number fatty acids are oxidized by the β -oxidation pathway to yield acetyl-CoA and a molecule of propionyl-CoA. The propionyl-CoA is carboxylated to methylmalonyl-CoA, which is isomerized to succinyl-CoA in a reaction catalyzed by methylmalonyl-CoA mutase, an enzyme requiring coenzyme B12. To prevent futile cycling, entry of fatty acids into mitochondria is blocked by the first intermediate in fatty acid synthesis, malonyl-CoA. The transcription factor PPARα stimulates the synthesis of several enzymes required in β oxidation when glucose is not available as an energy source. Individuals who lack medium-chain acyl-CoA dehydrogenase experience fatty liver, high blood levels of octanoic acid, coma, and sometimes death. Peroxisomes, of plants and animals, and glyoxysomes, of plants, carry out β oxidation in four steps similar to those of the mitochondrial pathway. The first oxidation step, however, transfers electrons directly to O2, generating H2O2. Peroxisomes of animal tissues specialize in the oxidation of very-long-chain fatty acids and branched fatty acids. The reactions of α oxidation convert branched fatty acids such as phytanic acid to products that can undergo β oxidation, yielding acetyl-CoA and propionyl-CoA. 17.3 Ketone Bodies In humans and most other mammals, acetyl-CoA formed in the liver during oxidation of fatty acids can either enter the citric acid cycle (stage 2 of Fig. 17-7) or undergo conversion to the “ketone bodies,” acetone, acetoacetate, and D-β -hydroxybutyrate, for export to other tissues. (The term “bodies” is an historical artifact; the compounds are soluble in blood and urine, not particulate, and not all of them are ketones.) Acetone, produced in smaller quantities than the other ketone bodies, is exhaled. Acetoacetate and D-β -hydroxybutyrate are transported by the blood to tissues other than the liver (extrahepatic tissues), where they are converted to acetyl-CoA and oxidized in the citric acid cycle, providing much of the energy required by tissues such as skeletal and heart muscle and the renal cortex. The brain, which preferentially uses glucose as fuel, can adapt to the use of acetoacetate and D-β -hydroxybutyrate under starvation conditions, when glucose is unavailable. The brain cannot use fatty acids as fuel, because they do not cross the blood-brain barrier. The production and export of ketone bodies from the liver to extrahepatic tissues allows continued oxidation of fatty acids in the liver when acetyl-CoA is not being oxidized in the citric acid cycle. Ketone Bodies, Formed in the Liver, Are Exported to Other Organs as Fuel The first step in the formation of acetoacetate, occurring in the liver (Fig. 17-16), is the enzymatic condensation of two molecules of acetyl-CoA, catalyzed by thiolase; this is simply the reversal of the last step of β oxidation. The acetoacetyl-CoA then condenses with another molecule of acetyl-CoA to form β -hydroxy-β - methylglutaryl-CoA (HMG-CoA), which is cleaved to free acetoacetate and acetyl-CoA. The acetoacetate is reversibly reduced by D-β -hydroxybutyrate dehydrogenase, a mitochondrial enzyme, to D-β -hydroxybutyrate. This enzyme is specific for the D stereoisomer; it does not act on L-β -hydroxyacyl-CoAs and is distinct from L-β -hydroxyacyl-CoA dehydrogenase of the β - oxidation pathway. This difference in stereospecificity of the two enzymes that use β -hydroxyacyl-CoAs as substrates in fatty acid breakdown and fatty acid synthesis means that the cell can maintain separate pools of β -hydroxyacyl-CoAs, earmarked for either breakdown or synthesis.

FIGURE 17-16 Formation of ketone bodies from acetyl-CoA. Healthy, well-nourished individuals produce ketone bodies at a relatively low rate. When acetyl-CoA accumulates (as in starvation or untreated diabetes, for example), thiolase catalyzes the condensation of two acetyl-CoA molecules to acetoacetyl-CoA, the parent compound of the three ketone bodies. The reactions of ketone body formation occur in the matrix of liver mitochondria. The six-carbon compound β -hydroxy-β -methylglutaryl-CoA (HMG- CoA) is also an intermediate of sterol biosynthesis, but the enzyme that forms HMG-CoA in that pathway is cytosolic. HMG-CoA lyase is present only in the mitochondrial matrix. In extrahepatic tissues, D-β -hydroxybutyrate is oxidized to acetoacetate by D-β -hydroxybutyrate dehydrogenase (Fig. 17-17). The acetoacetate is activated to its coenzyme A ester by transfer of CoA from succinyl-CoA, an intermediate of the citric acid cycle (see Fig. 16-7), in a reaction catalyzed by β -ketoacyl-CoA transferase, also called thiophorase. The acetoacetyl-CoA is then cleaved by thiolase to yield two molecules of acetyl-CoA, which enter the citric acid cycle. Thus the ketone bodies are used as fuels in all tissues except liver, which lacks β -ketoacyl-CoA transferase. The liver is therefore a producer of ketone bodies for other tissues, but not a consumer. FIGURE 17-17 D-β -Hydroxybutyrate as a fuel. -β -Hydroxybutyrate, synthesized in the liver, passes into the blood and thus to other tissues, where it is converted in three steps to acetyl-CoA. It is first oxidized to acetoacetate, which is activated with coenzyme A donated from succinyl- CoA, then split by thiolase. The acetyl-CoA thus formed enters the citric acid cycle. The production and export of ketone bodies by the liver allows continued oxidation of fatty acids with only minimal oxidation of acetyl-CoA. When intermediates of the citric acid cycle are being siphoned off for glucose synthesis by gluconeogenesis, for example, oxidation of cycle intermediates slows — and so does acetyl-CoA oxidation. Moreover, the liver contains only a limited amount of coenzyme A, and when most of it is tied up in acetyl- CoA, β oxidation slows for want of the free coenzyme. The production and export of ketone bodies frees coenzyme A, allowing continued fatty acid oxidation. Ketone Bodies Are Overproduced in Diabetes and during Starvation Starvation (including very-low-calorie diets) and untreated diabetes mellitus lead to overproduction of ketone bodies in the liver, with several adverse effects on health. During starvation, gluconeogenesis depletes citric acid cycle intermediates, diverting acetyl-CoA from oxidation of mobilized stored fats to ketone body production (Fig. 17-18). In untreated diabetes, when the insulin level is insufficient, extrahepatic tissues cannot take up glucose efficiently from the blood, either for fuel or for conversion to fat. Under these conditions, levels of malonyl-CoA fall, inhibition of carnitine acyltransferase 1 is relieved, and fatty acids enter mitochondria to be degraded to acetyl-CoA (see Fig. 17-13). But this acetyl-CoA cannot pass through the citric acid cycle because cycle intermediates have been drawn off for use as substrates in gluconeogenesis.

The resulting accumulation of acetyl-CoA accelerates the formation of ketone bodies and their release into the blood beyond the capacity of extrahepatic tissues to oxidize them. The increased blood levels of acetoacetate and D-β -hydroxybutyrate lower the blood pH, causing the condition known as acidosis. Extreme acidosis can lead to coma and in some cases death. Ketone bodies in the blood and urine of individuals with untreated diabetes can reach extraordinary levels — a blood concentration of 90 mg/100 mL (compared with a normal level of <3mg/100mL) and urinary excretion of 5,000 mg/24 h (compared with a normal rate of ≤125mg/24h). This condition is called ketosis or, when combined with acidosis, ketoacidosis. FIGURE 17-18 Ketone body formation and export from the liver. Conditions that promote gluconeogenesis (untreated diabetes, severely reduced food intake) slow the citric acid cycle (by drawing off oxaloacetate) and enhance the conversion of acetyl-CoA to acetoacetate. The released coenzyme A allows continued β oxidation of fatty acids. In healthy people, acetone is formed in very small amounts from acetoacetate, which is easily decarboxylated, either spontaneously or by the action of acetoacetate decarboxylase (Fig. 17-16). Because individuals with untreated diabetes produce large quantities of acetoacetate, their blood contains significant amounts of acetone, which is toxic. Acetone is volatile and imparts a characteristic odor to the breath, which is sometimes useful in diagnosing diabetes. SUMMARY 17.3 Ketone Bodies The ketone bodies — acetone, acetoacetate, and D-β - hydroxybutyrate — are formed in the liver when fatty acids are the principal fuel supporting whole-body metabolism. Acetoacetate and β -hydroxybutyrate serve as fuel molecules in extrahepatic tissues, including the brain, through oxidation to acetyl-CoA and entry into the citric acid cycle. Overproduction of ketone bodies in uncontrolled diabetes or severely reduced calorie intake can lead to life-threatening ketoacidosis, characterized by high concentrations of ketones in blood and urine and lower blood pH. Chapter Review KEY TERMS Terms in bold are defined in the glossary. β oxidation apolipoprotein chylomicron lipoprotein perilipin free fatty acids serum albumin carnitine shuttle fatty acyl–CoA synthetase fatty acyl–CoA carnitine acyltransferase 1 (CAT1) acyl-carnitine/carnitine cotransporter carnitine acyltransferase 2 (CAT2) electron-transfer flavoprotein (ETF) NADH dehydrogenase (Complex I) trifunctional protein (TFP) methylmalonyl-CoA mutase coenzyme B12 malonyl-CoA pernicious anemia intrinsic factor PPAR (peroxisome proliferator-activated receptor) medium-chain acyl-CoA dehydrogenase (MCAD) peroxisome α oxidation ketone bodies acetone acetoacetate D-β -hydroxybutyrate acidosis ketosis ketoacidosis PROBLEMS 1. Energy in Triacylglycerols On a per-carbon basis, where does the largest amount of biologically available energy in triacylglycerols reside: in the fatty acid portions or in the glycerol portion? Indicate how knowledge of the chemical structure of triacylglycerols provides the answer. 2. Effect of PDE Inhibitor on Adipocytes How would the addition of a cAMP phosphodiesterase (PDE) inhibitor affect the response of an adipocyte to epinephrine? (Hint: See Fig. 12-4.) 3. Compartmentation in β Oxidation The activation of free palmitate to its coenzyme A derivative (palmitoyl-CoA) in the cytosol occurs before it can be oxidized in the mitochondrion. Aer adding palmitate and [14C]coenzyme A to a liver homogenate, you find palmitoyl-CoA isolated from the cytosolic fraction is radioactive, but that isolated from the mitochondrial fraction is not. Explain. 4. Mutant Carnitine Acyltransferase What changes in metabolic pattern would result from a mutation in the muscle carnitine acyltransferase 1 in which the mutant protein has lost its affinity for malonyl-CoA but not its catalytic activity? 5. Effect of Carnitine Deficiency An individual developed a condition characterized by progressive muscular weakness and aching muscle cramps. The symptoms were aggravated by fasting, exercise, and a high-fat diet. An homogenate of a skeletal muscle specimen from the patient oxidized added oleate more slowly than did control homogenates consisting of muscle specimens from healthy individuals. When the pathologist added carnitine to the patient’s muscle homogenate, the rate of oleate oxidation equaled that in the control homogenates. Based on these results, the attending physician diagnosed the patient as having a carnitine deficiency. a. Why did added carnitine increase the rate of oleate oxidation in the patient’s muscle homogenate? b. Why did fasting, exercise, and a high-fat diet aggravate the patient’s symptoms? c. Suggest two possible reasons for the deficiency of muscle carnitine in this individual. 6. Fuel Reserves in Adipose Tissue Triacylglycerols, with their hydrocarbon-like fatty acids, have the highest energy content of the major nutrients. a. If 15% of the body mass of a 70.0 kg adult consists of triacylglycerols, what is the total available fuel reserve, in both kilojoules and kilocalories, in the form of triacylglycerols? Recall that 1.00kcal=4.18kJ. b. If the basal energy requirement is approximately 8,400 kJ/day (2,000 kcal/day), how long could this person survive if the oxidation of fatty acids stored as triacylglycerols were the only source of energy? c. What would be the weight loss in pounds per day under such starvation conditions (1lb=0.454kg)? 7. Common Reaction Steps in the Fatty Acid Oxidation Cycle and Citric Acid Cycle Cells oen use the same enzyme reaction pattern for analogous metabolic conversions. For example, the steps in the oxidation of pyruvate to acetyl-CoA and of α -ketoglutarate to succinyl-CoA, although catalyzed by different enzymes, are very similar. The first stage of fatty acid oxidation follows a reaction sequence closely resembling a sequence in the citric acid cycle. Use equations to show the analogous reaction sequences in the two pathways. 8. β Oxidation: How Many Cycles? How many cycles of β oxidation are required for the complete oxidation of activated oleic acid, 18:1(Δ9)? 9. Chemistry of the Acyl-CoA Synthetase Reaction Fatty acids are converted to their coenzyme A esters in a reversible reaction catalyzed by acyl-CoA synthetase: a. The enzyme-bound intermediate in this reaction has been identified as the mixed anhydride of the fatty acid and adenosine monophosphate (AMP), acyl- AMP: Write two equations corresponding to the two steps of the reaction catalyzed by acyl-CoA synthetase. b. The acyl-CoA synthetase reaction is readily reversible, with an equilibrium constant near 1. How can this reaction be made to favor formation of fatty acyl–CoA? 10. Intermediates in Oleic Acid Oxidation What is the structure of the partially oxidized fatty acyl group that is formed when oleic acid, 18:1(Δ9), has undergone three cycles of β oxidation? What are the next two steps in the continued oxidation of this intermediate? 11. β Oxidation of an Odd-Number Fatty Acid What are the direct products of β oxidation of a fully saturated, straight-chain fatty acid of 11 carbons? 12. Oxidation of Tritiated Palmitate An investigator adds palmitate uniformly labeled with tritium (3H) to a specific activity of 2.48×108 counts per minute (cpm) per micromole of palmitate to a mitochondrial preparation that oxidizes it to acetyl-CoA. She then isolates the acetyl-CoA and hydrolyzes it to acetate. The specific activity of the isolated acetate is 1.00×107cpm/μmol. Is this result consistent with the β - oxidation pathway? Explain. What is the final fate of the removed tritium? (Note: Specific activity is a measure of the degree of labeling with a radioactive tracer expressed as radioactivity per unit mass. In a uniformly labeled compound, all atoms of a given type are labeled.) 13. Comparative Biochemistry: Energy-Generating Pathways in Birds One indication of the relative importance of various ATP-producing pathways is the Vmax of certain enzymes of these pathways. The values of Vmax of several enzymes from the pectoral muscles (chest muscles used for flying) of pigeon and pheasant are listed below. Vmax(μ mol substrate/min/g tissue) Enzyme Pigeon Pheasant Hexokinase     3.0     2.3 Glycogen phosphorylase   18.0 120.0 Phosphofructokinase-1   24.0  143.0 Citrate synthase 100.0     15.0 Triacylglycerol lipase         0.07           0.01 a. Discuss the relative importance of glycogen metabolism and fat metabolism in generating ATP in the pectoral muscles of these birds. b. Compare oxygen consumption in the two birds. c. Judging from the data in the table, which bird is the long-distance flyer? Justify your answer. d. Why were these particular enzymes selected for comparison? Would the activities of triose phosphate isomerase and malate dehydrogenase be equally good bases for comparison? Explain. 14. Fatty Acids as a Source of Water Contrary to legend, camels do not store water in their humps, which actually consist of large fat deposits. How can these fat deposits serve as a source of water? Calculate the amount of water (in liters) that a camel can produce from 1.0 kg of fat. Assume for simplicity that the fat consists entirely of tripalmitoylglycerol. 15. Metabolism of a Straight-Chain Phenylated Fatty Acid Investigators isolate a crystalline metabolite from the urine of a rabbit that has been fed a straight-chain fatty acid containing a terminal phenyl group: The addition of 22.2 mL of 0.100 M NaOH completely neutralized a 302 mg sample of the metabolite in aqueous solution. a. What is the probable molecular weight and structure of the metabolite? b. Did the straight-chain fatty acid contain an even or an odd number of methylene (—CH2—) groups (i.e., is n even or odd)? Explain. 16. Fatty Acid Oxidation in Uncontrolled Diabetes When the acetyl-CoA produced during β oxidation in the liver exceeds the capacity of the citric acid cycle, the excess acetyl-CoA forms ketone bodies — acetone, acetoacetate, and D-β - hydroxybutyrate. This occurs in people with severe, uncontrolled diabetes; because their tissues cannot use glucose, they oxidize large amounts of fatty acids instead. Although acetyl-CoA is not toxic, the mitochondrion must divert the acetyl-CoA to ketone bodies. What problem would arise if acetyl-CoA were not converted to ketone bodies? How does the diversion to ketone bodies solve the problem? 17. Consequences of a High-Fat Diet with No Carbohydrates Suppose you had to subsist on a diet of whale blubber and seal blubber, with little or no carbohydrate. a. What would be the effect of carbohydrate deprivation on the utilization of fats for energy? b. If your diet were totally devoid of carbohydrate, would it be better to consume odd- or even-number fatty acids? Explain. 18. Even- and Odd-Number Fatty Acids in the Diet In a laboratory experiment, investigators feed two groups of rats two different fatty acids as their sole source of carbon for a month. The first group gets heptanoic acid (7:0), and the second gets octanoic acid (8:0). Aer the experiment, those in the first group are healthy and have gained weight, whereas those in the second group are weak and have lost weight as a result of losing muscle mass. What is the biochemical basis for this difference? 19. Metabolic Consequences of Ingesting ω -Fluorooleate The shrub Dichapetalum toxicarium, native to Sierra Leone, produces ω -fluorooleate, which is highly toxic to warm-blooded animals. This substance has been used as an arrow poison, and powdered fruit from the plant is sometimes used as a rat poison (hence the plant’s common name, ratsbane). Why is this substance so toxic? (Hint: Review Chapter 16, Problem 21.) 20. Mutant Acetyl-CoA Carboxylase What would be the consequences for fat metabolism of a mutation in acetyl-CoA carboxylase that replaced the Ser residue normally phosphorylated by AMPK with an Ala residue? What might happen if the same Ser were replaced by Asp? (Hint: Compare the structures of phosphoserine, alanine, aspartate; see Fig. 17-13.) 21. Role of FAD as Electron Acceptor Acyl-CoA dehydrogenase uses enzyme-bound FAD as a prosthetic group to dehydrogenate the α and β carbons of fatty acyl–CoA. What is the advantage of using FAD as an electron acceptor rather than NAD+? Explain in terms of the standard reduction potentials for the Enz-FAD/FADH2(E′°=−0.219V) and NAD+/NADH(E′°=−0.320V) half- reactions. 22. β Oxidation of Arachidic Acid How many turns of the fatty acid oxidation cycle are required for complete oxidation of arachidic acid (20:0) to acetyl-CoA? 23. Fate of Labeled Propionate Adding [3−14C]propionate (14C in the methyl group) to a liver homogenate leads to the rapid production of 14C-labeled oxaloacetate. Draw a flowchart for the pathway by which propionate is transformed to oxaloacetate, and indicate the location of the 14C in oxaloacetate. 24. Phytanic Acid Metabolism A mouse fed phytanic acid uniformly labeled with 14C produces detectable levels of radioactive malate, a citric acid cycle intermediate, within minutes. Draw a metabolic pathway that could account for this. Which of the carbon atoms in malate would contain 14C label? 25. Sources of H2O Produced in β Oxidation The complete oxidation of palmitoyl- CoA to carbon dioxide and water is represented by the overall equation Palmitoyl-CoA+23O2+108Pi+108ADP → CoA+16CO2+108ATP+23H2O Water also forms in the reaction ADP+Pi →  ATP+H2O but is not included as a product in the overall equation. Why? 26. Biological Importance of Cobalt Cattle, deer, sheep, and other ruminant animals produce large amounts of propionate in the rumen through the bacterial fermentation of ingested plant matter. Propionate is the principal source of glucose for these animals, via the route propionate → oxaloacetate → glucose. In some areas of the world, notably Australia, ruminant animals sometimes show symptoms of anemia with concomitant loss of appetite and retarded growth, resulting from an inability to transform propionate to oxaloacetate. This condition is due to a cobalt deficiency caused by very low cobalt levels in the soil and thus in plant matter. Explain. 27. Fat Loss during Hibernation Bears expend about 25×106J/day during periods of hibernation, which may last as long as seven months. The energy required to sustain life is obtained from fatty acid oxidation. How much weight (in kilograms) do bears lose aer 7 months of hibernation? How could a bear’s body minimize ketosis during hibernation? (Assume the oxidation of fat yields 38 kJ/g.) DATA ANALYSIS PROBLEM 28. β Oxidation of Trans Fats Unsaturated fats with trans double bonds are commonly referred to as “trans fats.” In their investigations of the effects of trans fatty acid metabolism on health, Yu and colleagues (2004) showed that a model trans fatty acid was processed differently from its cis isomer. They used three related 18-carbon fatty acids to explore the difference in β oxidation between cis and trans isomers of the same-size fatty acid. The researchers incubated the coenzyme A derivative of each acid with rat liver mitochondria for 5 minutes, then separated the remaining CoA derivatives in each mixture by HPLC (high-performance liquid chromatography). The results are shown below, with separate panels for the three experiments. In the figure, IS indicates an internal standard (pentadecanoyl-CoA) added to the mixture aer the reaction as a molecular marker. The researchers abbreviated the CoA derivatives as follows: stearoyl-CoA, C18-CoA; cis-Δ5-tetradecenoyl-CoA, cΔ5C14-CoA; oleoyl-CoA, cΔ9C18-CoA; trans-Δ5-tetradecenoyl-CoA, tΔ5C14-CoA; and elaidoyl-CoA, tΔ9C18-CoA.

a. Why did Yu and colleagues need to use CoA derivatives rather than the free fatty acids in these experiments? b. Why were no lower molecular weight CoA derivatives found in the reaction with stearoyl-CoA? c. How many rounds of β oxidation would be required to convert the oleoyl-CoA and the elaidoyl-CoA to cis-Δ5-tetradecenoyl-CoA and trans-Δ5-tetradecenoyl- CoA, respectively? Yu and coworkers measured the kinetic parameters of two forms of the enzyme acyl-CoA dehydrogenase: long-chain acyl-CoA dehydrogenase (LCAD) and very-long-chain acyl-CoA dehydrogenase (VLCAD). They used the CoA derivatives of three fatty acids: tetradecanoyl-CoA (C14-CoA), cis-Δ5- tetradecenoyl-CoA (cΔ5C14-CoA), and trans-Δ5-tetradecenoyl-CoA (tΔ5C14-CoA). The results are shown below. (See Chapter 6 for definitions of the kinetic parameters.) LCAD VLCAD C14-CoA cΔ5C14-CoA tΔ5C14-CoA C14-CoA cΔ5C14-CoA tΔ5C14-CoA Vmax 3.3 3.0 2.9 1.4 0.32 0.88 Km   0.41   0.40 1.6   0.57 0.44 0.97 kcat 9.9 8.9 8.5 2.0 0.42 1.12 kcat/Km 24       22       5    4    1       1       d. For LCAD, the Km differs dramatically for the cis and trans substrates. Provide a plausible explanation for this observation in terms of the structures of the substrate molecules. (Hint: You may want to refer to Fig. 10-1.) e. The kinetic parameters of the two enzymes are relevant to the differential processing of these fatty acids only if the LCAD or VLCAD reaction (or both) is the rate-limiting step in the pathway. What evidence is there to support this assumption? f. How do these different kinetic parameters explain the different levels of the CoA derivatives found aer incubation of rat liver mitochondria with stearoyl-CoA, oleoyl-CoA, and elaidoyl-CoA (shown in the three-panel figure)? Yu and coworkers measured the substrate specificity of rat liver mitochondrial thioesterase, which hydrolyzes acyl-CoA to CoA and free fatty acid. This enzyme was approximately twice as active with C14-CoA thioesters as with C18-CoA thioesters. g. Other research has suggested that free fatty acids can pass through membranes. In their experiments, Yu and colleagues found trans-Δ5-tetradecenoic acid outside (i.e., in the medium surrounding) mitochondria that had been incubated with elaidoyl-CoA. Describe the pathway that led to this extramitochondrial trans-Δ5-tetradecenoic acid. Be sure to indicate where in the cell the various transformations take place, as well as the enzymes that catalyze the transformations. h. It is sometimes said in the popular press that “trans fats are not broken down by your cells and instead accumulate in your body.” In what sense is this statement correct and in what sense is it an oversimplification? Reference Yu, W., X. Liang, R. Ensenauer, J. Vockley, L. Sweetman, and H. Schultz. 2004. Leaky β - oxidation of a trans-fatty acid. J. Biol. Chem. 279:52,160–52,167.