As is the case for fatty acid degradation, fatty acid synthesis occurs as three stages of processing:

The first biochemical hurdle in fatty acid synthesis is that synthesis takes place in the cytoplasm, but acetyl CoA, the raw material for fatty acid synthesis, is formed in mitochondria. The mitochondria are not readily permeable to acetyl CoA. How are acetyl CoA molecules transferred to the cytoplasm? The problem is solved by the transport of acetyl CoA out of the mitochondria in the form of citrate. Citrate is formed in the mitochondrial matrix by the condensation of acetyl CoA with oxaloacetate (Figure 28.1). This reaction begins the citric acid cycle when energy is required. When the energy needs of a cell have been met, citrate is relocated to the cytoplasm by a transport protein, where it is cleaved by ATP-citrate lyase at the cost of a molecule of ATP to yield cytoplasmic acetyl CoA and oxaloacetate:

This reaction occurs in three steps: (1) the formation of a phospho-enzyme with the donation of a phosphoryl group from ATP, (2) binding of citrate and CoA followed by the formation of citroyl CoA and release of the phosphate, and (3) cleavage of citroyl CoA to yield acetyl CoA and oxaloacetate. The transport and cleavage reactions must take place eight times to provide all of the carbon atoms required for palmitate synthesis. ATP-citrate lyase is stimulated by insulin, which initiates a signal transduction pathway that ultimately results in the phosphorylation and activation of the lyase by Akt (also called protein kinase B).

In addition to being a precursor for fatty acid synthesis, citrate serves as a signal molecule. It inhibits phosphofructokinase, which controls the rate of glycolysis. Citrate in the cytoplasm indicates an energy-rich state, signaling that there is no need to oxidize glucose.

The synthesis of palmitate requires 14 molecules of NADPH as well as the expenditure of ATP. Some of the reducing power is generated when oxaloacetate, formed in the transfer of acetyl groups to the cytoplasm, is returned to the mitochondria. The inner mitochondrial membrane is impermeable to oxaloacetate. Hence, a series of bypass reactions are needed. First, oxaloacetate is reduced to malate by NADH. This reaction is catalyzed by a cytoplasmic malate dehydrogenase:

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Second, malate is oxidatively decarboxylated by an NADP⁺-linked malate enzyme (also called malic enzyme).

The pyruvate formed in this reaction readily enters mitochondria, where it is carboxylated to oxaloacetate by pyruvate carboxylase:

The sum of these three reactions is

Thus, one molecule of NADPH is generated for each molecule of acetyl CoA that is transferred from mitochondria to the cytoplasm. Hence, eight molecules of NADPH are formed when eight molecules of acetyl CoA are transferred to the cytoplasm for the synthesis of palmitate. The additional six molecules of NADPH required for the synthesis of palmitate come from the pentose phosphate pathway (Chapter 26).

The accumulation of the precursors for fatty acid synthesis is a wonderful example of the coordinated use of multiple pathways. The citric acid cycle, the transport of citrate from mitochondria, and the pentose phosphate pathway provide the carbon atoms and reducing power, whereas glycolysis and oxidative phosphorylation provide the ATP to meet the needs for fatty acid synthesis (Figure 28.2).

Like the synthesis of all biopolymers, fatty acid synthesis requires an activation step. Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA, the activated form of acetyl CoA. This reaction is catalyzed by acetyl CoA carboxylase 1, a regulatory enzyme in fatty acid metabolism. As we will see shortly, malonyl CoA is the actual carbon donor for all but two of the carbon atoms of palmitic acid.

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Acetyl CoA is combined with HCO₃⁻, the form of CO₂ in aqueous solutions. This irreversible reaction is the committed step in fatty acid synthesis:

The synthesis of malonyl CoA is a two-step process:

The enzyme system that catalyzes the synthesis of saturated long-chain fatty acids from acetyl CoA, malonyl CoA, and NADPH is called fatty acid synthase. The synthase is actually a complex of distinct enzymes, each of which has a different function in fatty acid synthesis. In bacteria, the enzyme complex catalyzes all but the activation step of fatty acid synthesis (Table 28.1). The elongation phase of fatty acid synthesis in bacteria starts when acetyl CoA and malonyl CoA react with a scaffold protein called acyl carrier protein (ACP), forming acetyl ACP and malonyl ACP. Just as most construction projects require foundations on which the structures are built, fatty acid synthesis needs a molecular foundation. The intermediates in fatty acid synthesis are linked to the sulfhydryl end of a phosphopantetheine group of ACP—the same “business end” as that of CoA—which is, in turn, attached to a serine residue of the acyl carrier protein (Figure 28.3).

Acyl carrier protein, a single polypeptide chain of 77 residues, can be regarded as a giant prosthetic group, a “macro CoA.” Acetyl transacylase and malonyl transacylase catalyze the formation of acetyl ACP and malonyl ACP, respectively:

The synthesis of fatty acids with an odd number of carbon atoms starts with propionyl ACP, which is formed from propionyl CoA by acetyl transacylase.

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Acetyl ACP and malonyl ACP react to form acetoacetyl ACP (Figure 28.4). β-Ketoacyl synthase, also called the condensing enzyme, catalyzes this condensation reaction.

In the condensation reaction, a four-carbon unit is formed from a two-carbon unit and a three-carbon unit, and CO₂ is released. Why is the four-carbon unit not formed from two two-carbon units—say, two molecules of acetyl ACP? The equilibrium for the synthesis of acetoacetyl ACP from two molecules of acetyl ACP is highly unfavorable. In contrast, the equilibrium is favorable if malonyl ACP, the activated form of acetyl CoA, is a reactant, because its decarboxylation contributes to a substantial decrease in free energy. In effect, ATP drives the condensation reaction, though ATP does not directly participate in that reaction. Instead, ATP is used to carboxylate acetyl CoA to malonyl CoA, the activated form of acetyl CoA. The free energy thus stored in malonyl CoA is released in the decarboxylation accompanying the formation of acetoacetyl ACP. Although HCO₃^– is required for fatty acid synthesis, its carbon atom does not appear in the product. Rather, all the carbon atoms of fatty acids containing an even number of carbon atoms are derived from acetyl CoA.

The next three steps in fatty acid synthesis reduce the keto group (shown in the margin) at C-3 to a methylene group (–CH₂–; Figure 28.4). First, acetoacetyl ACP is reduced to d-3-hydroxybutyryl ACP. This reaction differs from the corresponding oxidation in fatty acid degradation in two respects: (1) the d rather than the l isomer is formed and (2) NADPH is the reducing agent, whereas NAD⁺ is the oxidizing agent in β oxidation. This difference exemplifies the general principle that NADPH is consumed in biosynthetic reactions, whereas NADH is generated in energy-yielding reactions. d-3-Hydroxybutyryl ACP is then dehydrated to form crotonyl ACP, which is a trans-Δ²-enoyl ACP. The final step in the cycle reduces crotonyl ACP to butyryl ACP. NADPH is again the reductant, whereas FAD is the oxidant in the corresponding reaction in β oxidation. The enzyme that catalyzes this step, enoyl ACP reductase, is inhibited by triclosan, a broad-spectrum antibacterial agent that is added to a variety of products such as toothpaste, soaps, and skin creams. These last three reactions—a reduction, a dehydration, and a second reduction—convert acetoacetyl ACP into butyryl ACP, which completes the first elongation cycle.

In the second round of fatty acid synthesis, butyryl ACP condenses with another malonyl ACP to form a C₆-β-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C₄-β-ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C₆-β-ketoacyl ACP into a C₆-acyl ACP, which is ready for a third round of elongation. The elongation cycles continue until C₁₆-acyl ACP is formed. This intermediate is a good substrate for a thioesterase that hydrolyzes C₁₆-acyl ACP to release the fatty acid chain from the carrier protein. Because it acts selectively on C₁₆-acyl ACP, the thioesterase acts as a ruler to determine fatty acid chain length. The synthesis of longer-chain fatty acids is discussed in Section 28.2.

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Now that we have seen all of the individual reactions of fatty acid synthesis, let us determine the overall reaction for the synthesis of the C₁₆ fatty acid palmitate. The stoichiometry of the synthesis of palmitate is

The equation for the synthesis of the malonyl CoA used in the preceding reaction is

Hence, the overall stoichiometry for the synthesis of palmitate is

Although the basic biochemical reactions in fatty acid synthesis are similar in E. coli and eukaryotes, the structure of the synthase varies considerably. The component enzymes of animal fatty acid synthases, in contrast with those of E. coli and plants, are linked in a large polypeptide chain.

The structure of a large part of the mammalian fatty acid synthase has recently been determined, with the acyl carrier protein and thioesterase remaining to be resolved. The enzyme is a dimer of identical 270-kDa subunits. Each chain contains all of the active sites required for activity, as well as an acyl carrier protein tethered to the complex (Figure 28.5A). Despite the fact that each chain possesses all of the enzymes required for fatty acid synthesis, the monomers are not active. A dimer is required.

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The two component chains interact such that the enzyme activities are partitioned into two distinct compartments (Figure 28.5B). The selecting and condensing compartment binds the acetyl and malonyl substrates and condenses them to form the growing chain. Interestingly, the mammalian fatty acid synthase has one active site, malonyl-acetyl transacylase, that adds both acetyl CoA and malonyl CoA. In contrast, most other fatty acid synthases have two separate enzyme activities, one for acetyl CoA and one for malonyl CoA. The modification compartment is responsible for the reduction and dehydration activities that result in the saturated fatty acid product.

Many eukaryotic multienzyme complexes are multifunctional proteins in which different enzymes are linked covalently. An advantage of this arrangement is that the synthetic activity of different enzymes is coordinated. In addition, intermediates can be efficiently handed from one active site to another without leaving the assembly.

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