Conversion of Acetyl-CoA to Malonyl-CoA

The next reaction in the synthesis of fatty acids is catalyzed by acetyl-CoA carboxylase and involves an ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This is the first committed step in fatty acid synthesis and is highly regulated. Like pyruvate carboxylase, acetyl-CoA carboxylase utilizes bicarbonate as a source of carbon dioxide, has a two-step reaction mechanism, and requires a covalently bound biotin.

HCO3−+ATP+Biotin-enzyme→Carboxybiotin-enzyme+ADP+Pi

Carboxybiotin-enzyme+Acetyl-CoA→Malonyl-CoA+Biotin-enzyme

NET: Acetyl-CoA+HCO3−+ATP→Malonyl-CoA+ADP+Pi

In the first step, the biotin, which is bound to a specific lysine residue of the enzyme, is carboxylated with the energy furnished by the hydrolysis of ATP (biotin carboxylation). In the second step (transcarboxylation), the activated, biotin-bound carboxyl group is transferred to acetyl-CoA, regenerating enzyme-bound biotin and synthesizing malonyl-CoA. Both of these reactions are catalyzed by a single polypeptide chain that also contains a domain for the covalently linked biotin. In biotin deficiency, the acetyl-CoA carboxylase reaction is impaired, and thus fatty acid synthesis from acetyl-CoA is decreased. (See Chapter 26 for further discussion of biotin-dependent carboxylation.)

Acetyl-CoA carboxylase is regulated by an array of control mechanisms, thereby permitting the rate of fatty acid synthesis to fluctuate in response to physiological and developmental conditions (Sul and Smith, 2008; Wakil and Abu-Elheiga, 2009). At the same time, malonyl-CoA, the product of acetyl-CoA carboxylase, is a potent inhibitor of carnitine palmitoyltransferase 1 (CPT1), an enzyme that controls transport of long-chain fatty acids into mitochondria for oxidation (as discussed in subsequent text). Therefore acetyl-CoA carboxylase reciprocally regulates fatty acid synthesis and oxidation. There are two isoforms of acetyl-CoA carboxylase: ACC1 is for acetyl-CoA synthesis in lipogenic tissues (i.e., liver and adipose tissue), whereas ACC2 present in muscle and other tissues is for formation of malonyl-CoA for regulating fatty acid oxidation. The activity of acetyl-CoA carboxylase is stimulated by citrate, an allosteric activator, and inhibited by long-chain acyl-CoA, an allosteric inhibitor. In the fed state, production of cytosolic citrate is increased and the concentration of long-chain acyl-CoA is low, resulting in activation of acetyl-CoA carboxylase. Conversely, during starvation, the long-chain acyl-CoA level increases and the cytosolic citrate level decreases, resulting in inhibition of fatty acid synthesis. Activation of lipolysis in the adipose tissue gives rise to the increase in long-chain acyl-CoA concentration, and the lack of excess anaphlerotic substrate (e.g., pyruvate, oxaloacetate) for citrate formation in the mitochondrial citric acid cycle gives rise to the lack of cytosolic citrate. These allosteric mechanisms thus represent examples of feed-forward (citrate) and feedback (long-chain acyl-CoA) regulation of acetyl-CoA carboxylase as shown in Figure 16-3.

Acetyl-CoA carboxylase also is regulated by covalent modification (Brownsey et al., 2006; Saggerson, 2008), as shown in Figure 16-3. Up to seven serine residues in acetyl-CoA carboxylase can be phosphorylated. The phosphorylated enzyme is less active, less sensitive to the stimulatory effects of citrate, and more sensitive to the inhibitory action of long-chain acyl-CoA. A number of different protein kinases catalyze phosphorylation of acetyl-CoA carboxylase and do so at different specific serine residues on the enzyme. 5′-AMP–activated protein kinase (AMPK) is the physiologically important kinase that phosphorylates acetyl-CoA carboxylase. When the cellular energy state is low, with increased intracellular AMP levels, AMPK is allosterically activated by AMP. AMPK is also activated by phosphorylation by upstream kinases such as LKB1, and phosphorylation of AMPK is promoted by AMP (Steinberg and Kemp, 2009). Activated AMPK in turn phosphorylates acetyl-CoA carboxylase resulting in a decrease in its activity.

In liver, glucagon is an important effector increasing production of 3′-5′-cyclic adenosine monophosphate (cAMP). Catecholamines can also increase production of cAMP. An increase in cAMP in turn activates protein kinase A (see Figure 16-3). There may be a potential physiological role for protein kinase A in the hormone-mediated inactivation of acetyl-CoA carboxylase. However, regulation of acetyl-CoA carboxylase activity by protein kinase A–mediated phosphorylation is somewhat smaller in magnitude than that by AMPK-mediated phosphorylation. It is also possible that protein kinase A regulation of acetyl-CoA carboxylase may not occur via direct phosphorylation of this enzyme.

In addition to regulation by allosteric and phosphorylation–dephosphorylation mechanisms, acetyl-CoA carboxylase also is regulated by changes in the number of molecules present in the cell. The concentration of the enzyme is low in the liver of starved animals and high in the liver of fed animals, especially if the diet is high in carbohydrate. The acetyl-CoA carboxylase protein concentration in cells is controlled primarily by changes in the rate of transcription of the acetyl-CoA carboxylase gene. In this regard, in addition to acetyl-CoA carboxylase, transcription of many of the enzymes involved in fatty acid synthesis are coordinately regulated. These transcriptionally regulated enzymes include ATP-citrate lyase, which hydrolyzes citrate to provide the cytosolic acetyl-CoA that is used as a substrate for acetyl-CoA carboxylase and fatty acid synthase, and malic enzyme and some of the enzymes in the pentose phosphate pathway that generate the NADPH required for fatty acid synthase. Transcription of these enzymes is high in the fed state (especially if the diet is high in carbohydrate) and low in the fasting state. Fat, especially polyunsaturated fatty acids in the diet, decreases transcription. Changes in circulating insulin and glucagon, as well as changes in glucose levels, participate in the transcriptional regulation of lipogenic enzymes, including acetyl-CoA carboxylase. Insulin and glucose increase, whereas glucagon and catecholamines (via cAMP) decrease, the rate of transcription of acetyl-CoA carboxylase. When glucose levels are elevated in the fed state, insulin signaling via the insulin/phosphatidylinositol 3-kinase/Akt pathway results in activation of sterol regulatory element binding protein (SREBP) 1c, a basic helix-loop-helix transcription factor that resides at the ER but, upon cleavage, becomes a mature transcription factor and translocates to the nucleus to activate lipogenic gene transcription in the fed state. SREBP1c is expressed predominantly in the lipogenic tissues and SREBP1c gene transcription is itself suppressed in the fasted state but induced by high carbohydrate feeding. Liver X receptor (LXR), a ligand activated nuclear hormone receptor, activates transcription of SREBP1c as well as lipogenic enzymes in the fed state. LXR also increases transcription of another basic helix-loop-helix transcription factor, carbohydrate response element binding protein (ChREBP), which mediates transcriptional activation of lipogenic enzymes by glucose (Wong et al., 2009, Wong and Sul, 2010). The function of SREBP is discussed in more detail in Chapter 17.

Synthesis of Palmitate from Malonyl-CoA, Acetyl-CoA, and NADPH by Fatty Acid Synthase

The second and final committed step in fatty acid synthesis is catalyzed by a multifunctional polypeptide, fatty acid synthase, which catalyzes all the reactions shown in Figure 16-4 and which contains an acyl carrier protein (ACP) domain with a 4′-phosphopantetheine prosthetic group. The substrates for the synthesis of one palmitate molecule by fatty acid synthase are 7 molecules of malonyl-CoA, 1 of acetyl-CoA, 14 of NADPH, and 14 of H⁺. The products are 1 molecule of palmitate, 8 of CoA, 7 of CO₂, 14 of NADP, and 6 of H₂O. The reaction is complex in two ways. First, the fatty acid is built up by the serial addition of two-carbon fragments to a growing chain. Second, after each addition, the added carbons are reduced, dehydrated, and reduced again. The process then repeats itself—condensation, reduction, dehydration, and reduction—until a 16-carbon fatty acid (palmitate) has been formed and is released from the enzyme.

The first step in this complicated process involves an acyl transferase activity that transfers the acetyl moiety of

acetyl-CoA to a serine residue in the acyltransferase domain of the enzyme. The acetyl group is then transferred from the serine residue in the acyltransferase domain to the 4′-phosphopantetheine if the sulfhydryl of the covalently linked 4′-phosphopantetheine group in the acyl carrier protein domain of the enzyme is free. The 4′-phosphopantetheine group of the ACP domain of fatty acid synthase is described in Chapter 26. The β-ketoacyl synthase (the “condensing enzyme”) activity of fatty acid synthase then catalyzes transfer of the acetyl group to a cysteine residue at the active site of the condensation reaction. The serine residue in the acyl transferase domain is now free to accept a malonyl group. The malonyl group is then transferred to the sulfhydryl group of the phosphopantetheine side arm (see Figure 16-4), which has been freed of its acetyl group, and the enzyme is poised to carry out the first condensation reaction.

During the condensation reaction, the methylene carbon of the malonyl-phosphopantetheine form of the enzyme attacks the carbonyl group of the acetyl moiety in the active site of the β-ketoacyl transferase, forming the acetoacetyl-phosphopantetheine form of the enzyme and releasing the carboxyl group at C2 of the malonyl moiety as CO₂. The energy for this reaction is provided by coupling condensation of the acetyl and malonyl groups with decarboxylation. A condensation that started with two acetyl-CoAs would be energetically unfavorable. As a result, the energy for this reaction really comes from the hydrolysis of ATP during the carboxylation of acetyl-CoA to form malonyl-CoA; malonyl-CoA can thus be viewed as an activated form of acetyl-CoA. The carboxyl group added by acetyl-CoA carboxylase is the same one that is removed during the condensation reaction. Thus even though bicarbonate is a required substrate for the fatty acid synthesis pathway, incorporation of bicarbonate into the fatty acid does not occur.

The phosphopantetheine side arm in the ACP domain of fatty acid synthase is long and flexible, so that its attached acetoacetyl group can interact sequentially with the active sites of the β-ketoacyl reductase, β-hydroxyacyl dehydratase, and enoyl reductase domains to form a 4-carbon saturated fatty acyl group. At the end of this reaction sequence, the butyryl moiety remains attached to the phosphopantetheinyl moiety, but it is then transferred to the cysteine residue in the active site of the β-ketoacyl synthase domain. This leaves the sulfhydryl of the phosphopantetheinyl moiety free to receive a new malonyl-CoA moiety from the serine residue in the acyltransferase domain. The next cycle of condensation, reduction, dehydration, and reduction then forms a 6-carbon saturated fatty-acyl group (hexanoyl) attached to the phosphopantetheine side arm. The hexanoyl group is transferred to the active site cysteine residue of the β-ketoacyl synthase domain, and another malonyl group is condensed, reduced, dehydrated, and reduced. After seven cycles, the growing acyl chain reaches 16 carbons, and a thioesterase activity in the thioesterase domain of fatty acid synthase cleaves the free fatty acid from the enzyme (see Figure 16-4).

As mentioned previously, all of the reactions required for fatty acid synthesis from acetyl-CoA and malonyl-CoA are catalyzed by a single polypeptide (i.e., fatty acid synthase) that contains all activities in separate domains. The multifunctional fatty acid synthase can provide a greater efficiency by channeling intermediates from one active site to the next rather than relying on diffusion of intermediates between separate enzymes. In addition, the amounts of each enzyme can be regulated simultaneously by controlling expression of a single gene. Mammalian fatty acid synthase is synthesized as an inactive monomer of about 270 kDa (Figure 16-5). The active enzyme is an intertwined head-to-head homodimer of two fatty acid synthase monomers that has an X shape, with each of the two lateral clefts of the X defining a reaction chamber (Maier et al., 2008). One complete set of domains for progressive elongation of the fatty acid chain is present in each of the lateral clefts.

Similar to acetyl CoA carboxylase, fatty acid synthase activity is low in the fasted state but increases drastically in the fed state, especially when the diet is rich in carbohydrate. However, neither allosteric nor phosphorylation–dephosphorylation mechanisms appear to contribute significantly to these changes in fatty acid synthase activity. The major regulation is at the level of protein concentration as a consequence of changes in gene transcription by the previously described mechanism involving several transcription factors, including SREBP-1c (Wong and Sul, 2010).

Elongation of Fatty Acids

Fatty acids can be elongated in two-carbon steps. Elongation of fatty acids occurs in the endoplasmic reticulum (ER), mitochondria, and peroxisomes. In general, the enzyme systems for elongation are not well understood, but the ER elongation system is the most active and most studied. The ER elongation system uses both saturated and unsaturated fatty acyl-CoAs as substrates. The individual reactions are analogous to those catalyzed by fatty acid synthase, except that long-chain acyl-CoAs are used as primers rather than acetyl-CoA, and different gene products catalyze the individual reactions. The sequence of reactions is condensation, reduction, dehydration, and reduction. In the ER, malonyl-CoA is the elongating group, and NADPH is the electron donor. The initial rate-controlling step of a condensation reaction is referred to as elongation of very-long-chain fatty acids (ELOVL), and there are at least seven ELOVL enzymes that prefer either saturated and monounsaturated fatty acids or polyunsaturated fatty acids (Guillou et al., 2010). The mitochondrial elongation system appears to use acetyl-CoA as the elongating unit and NADH or NADH plus NADPH as electron donors. The mitochondrial elongation system is not a simple reversal of fatty acid oxidation involving two-carbon units (β-oxidation) because flavoenzymes are not involved in catalysis for either the first or the second reduction in each cycle. Fatty acid elongation in peroxisomes is even less understood but may produce very-long-chain fatty acids of 24 to 36 carbons in length.

Desaturation of Fatty Acids

Monounsaturated fatty acids are formed by direct oxidative desaturation of preformed long-chain saturated fatty acids in reactions catalyzed by a complex of enzymes located in the ER. The first double bond introduced into a saturated acyl chain is generally in the Δ9 position. The Δ9 desaturase complex (also called stearoyl-CoA desaturase, SCD) uses saturated fatty acids with 14 to 18 carbons; stearate is the most active. This complex, which is sometimes called a mixed function oxidase, uses two electrons and two protons donated by NADH (+ H⁺), two electrons and two protons from the fatty acid, and oxygen (as an electron acceptor) to generate the unsaturated fatty acid and two water molecules, as shown in Figure 16-6. The substrates and products are the acyl-CoA derivatives of the fatty acid. Electrons donated by NADH are passed via FADH₂ to the heme iron of cytochrome b₅, reducing it to the ferrous form, in a reaction catalyzed by NADH:cytochrome b₅ reductase. Cytochrome b₅ then donates two electrons to the nonheme iron of the desaturase component, reducing it to the ferrous form. This form then interacts with molecular oxygen and the fatty acyl-CoA to form water and the unsaturated fatty acyl-CoA. The desaturase activity regulates the overall reaction. Multiple forms of Δ9 desaturase are present with differing tissue distributions and regulation. The activity of SCD1, the isozyme present mostly in liver and adipose tissue, is controlled by diet and hormones in much the same manner as are the activities of the lipogenic enzymes, mainly by regulation of enzyme concentration.

Polyunsaturated fatty acids, usually containing double bonds interrupted by methylene groups, are produced in mammalian tissues. By using the enzymatic mechanism just described for desaturation at the Δ9 position by Δ9 desaturase, mammalian cells can further introduce double bonds into long-chain fatty acids at the Δ5 and Δ6 positions by Δ5 and Δ6 desaturases. However, due to the lack of Δ12 and Δ15 desaturases, double bonds cannot be introduced beyond the Δ9 position (i.e., at the third or sixth carbon from the methyl end, which is equivalent to a double bond at the twelfth or fifteenth carbon from the carboxyl end of an 18-carbon fatty acid). As a consequence, linoleate (18:2, Δ9, Δ12), an n-6 fatty acid, or α-linolenate (18:3, Δ9, Δ12, Δ15), an n-3 fatty acid, cannot be synthesized in humans, and they or longer chain members of the n-3 or n-6 classes must be provided in the diet. These fatty acids are essential for the synthesis of polyunsaturated fatty acids such as arachidonate (20:4, Δ5, Δ8, Δ11, Δ14) and eicosapentaenoate (20:5, Δ5, Δ8, Δ11, Δ14, Δ17). An alternating desaturation and elongation produces arachidonate and eicosapentaenoate from linoleate and α-linolenate, respectively.

18:2,Δ9,Δ12 (Linoleate)→18:3,Δ6,Δ9,Δ12→20:3Δ8,Δ11,Δ14→20:4,Δ5,Δ8,Δ11,Δ14(Arachidonate)

18:3,Δ9,Δ12,Δ15 (α-Linolenate)→18:4,Δ6,Δ9,Δ12,Δ15→20:4,Δ8,Δ11,Δ14,Δ17→20:5,Δ5,Δ8,Δ11,Δ14,Δ17 (Eicosapentaenoate)

Multiple desaturation and elongation reactions, plus a retroconversion step by peroxisomal β-oxidation, are involved in synthesis of docosahexaenoate (22:6, Δ4, Δ7, Δ10, Δ13, Δ16, Δ19) from α-linolenate. (See Chapters 6 and 18 for

Synthesis of Medium-Chain Fatty Acids

Medium-chain fatty acids are synthesized in mammary gland and are present as triacylglycerol in milk. Mammary gland has the same fatty acid synthase as does liver. Therefore fatty acid synthase purified from either tissue can synthesize palmitate because the thioesterase activity of fatty acid synthase is specific for 16-carbon acyl groups. However, secretory cells of the mammary gland also contain a second thioesterase that is specific for medium-chain fatty acids. This enzyme interacts with growing acyl chains on the fatty acid synthase and cleaves them from fatty acid synthase when they are 8 to 12 carbons in length.

Production of Simple Branched-Chain Fatty Acids

Sebaceous glands and certain related glands, such as the meibomian glands in the eyelids, synthesize fatty acids with one-carbon side chains. The “normal” fatty acid synthase carries out these reactions using unusual substrates. For example, propionyl-CoA can be used as a primer for fatty acid synthase rather than acetyl-CoA, and the resulting fatty acid will have a methyl branch at the C2 (i.e., the αC) position. Methyl groups also can be inserted at other positions along the fatty acid chain. Although it has a lower affinity for the condensing enzyme than does malonyl-CoA, methylmalonyl-CoA can be used as an elongating group if the concentration of malonyl-CoA is lower than that of methylmalonyl-CoA. In sebaceous glands, propionyl-CoA is converted to methylmalonyl-CoA in a reaction catalyzed by acetyl-CoA carboxylase. Acetyl-CoA carboxylase uses both acetyl-CoA and propionyl-CoA equally efficiently so the relative concentrations of malonyl-CoA and methylmalonyl-CoA depend on the relative concentrations of acetyl-CoA and propionyl-CoA. In sebaceous glands, a soluble malonyl-CoA decarboxylase keeps malonyl-CoA levels low. The mechanism for generating high levels of propionyl-CoA in sebaceous glands is not known.

Sources of Glycerol 3-Phosphate for Triacylglycerol Synthesis

Two reactions catalyze formation of glycerol 3-phosphate. First, dihydroxyacetone phosphate, a glycolytic (and gluconeogenic) intermediate, can be reduced to glycerol 3-phosphate. NADH is the electron donor in the reaction catalyzed by glycerol 3-phosphate dehydrogenase. This reaction is freely reversible, also allowing glycerol 3-phosphate to enter the glycolytic or gluconeogenic pathways under certain conditions.

Dihydroxyacetone phosphate+NADH+H+↔Glycerol 3-phosphate+NAD+

Second, in some tissues such as liver, but not appreciably in adipose or muscle tissues, glycerol can be phosphorylated to glycerol 3-phosphate in a reaction catalyzed by glycerol kinase.

Glycerol+ATP→Glycerol 3-phosphate+ADP

Sources of Fatty Acids for Triacylglycerol Synthesis

Fatty acids to be used for synthesis of triacylglycerol comprise fatty acids from de novo synthesis and those from hydrolysis of triacylglycerols in the cell, as well as fatty acids taken up from the circulation. The fatty acids taken up from the circulation may be free fatty acids (mainly released by lipolysis and associated with plasma albumin) or fatty acids generated locally from circulating lipoproteins (by the action of lipoprotein lipases localized on the endothelium of the capillaries within the tissue). These fatty acids from the plasma are taken up by the cell either by passive diffusion or by plasma membrane fatty acid transporters, such as fatty acid translocase (FAT/CD36), fatty acid transport proteins (FATPs), or plasma membrane fatty acid binding protein (FABPpm) (Glatz et al., 2010; Su and Abumrad, 2008). Because of their hydrophobic properties, once inside the cell, long-chain fatty acids are bound to fatty acid binding proteins (FABPs) to be transferred from membrane to membrane within the cell (Storch and Corsico, 2008).

Activation of Fatty Acids by Coenzyme a Thioester Formation

Before esterification to glycerol or transport to the mitochondria for oxidation, fatty acids must be activated to their CoA derivatives. This reaction is catalyzed by fatty acyl-CoA synthetase (also called fatty acyl-CoA ligase or fatty acid thiokinase). Different isoforms of fatty acyl-CoA synthetase are present in the membranes of mitochondria, ER, and peroxisomes, where fatty acids are used for either esterification or oxidation (Ellis et al., 2010). One of the proposed fatty acid transporters FATP also has intrinsic fatty acyl-CoA synthetase activity and may function to trap fatty acids in the cell and thereby facilitate fatty acid transport.

The ATP-dependent fatty acyl-CoA synthetase reaction has three steps. In the first step, fatty acid reacts with ATP to form a fatty acyl-AMP intermediate plus pyrophosphate. In the second step, the acyl moiety is transferred to coenyzme A and AMP is generated. As written, the reactions are freely reversible: one high-energy bond is cleaved between PP_i and AMP, and one is formed between the fatty acid and CoA.

(1) Fatty acid+ATP↔Fatty acyl-AMP+PPi

(2) Fatty acyl-AMP+CoA↔Fatty acyl-CoA+AMP

In the third step, the reaction is driven in the direction of formation of fatty acyl-CoA by a ubiquitous pyrophosphatase that rapidly cleaves pyrophosphate to inorganic phosphate.

(3) PPi+H2O→2 Pi

NET: Fatty acid+ATP+CoA+H2O→Fatty acyl-CoA+AMP+2 Pi

As will be shown in other pathways in this chapter, the hydrolysis of pyrophosphate is a relatively common mechanism for driving reactions to completion.

Esterificaton of Fatty Acids

The glycerol 3-phosphate reaction pathway for the synthesis of triacylglycerols starts with fatty acyl-CoA and glycerol 3-phosphate, as shown in Figure 16-7. The acyl group of a fatty acyl-CoA is transferred to the sn-1 position of glycerol 3-phosphate in the reaction catalyzed by glycerol 3-phosphate acyltransferase. This is the first committed step in glycerolipid biosynthesis.

(1) Glycerol 3-phosphate+Fatty acyl-CoA→1-Acylglycerol 3-phosphate+CoA