804 Chapter 25 ● Lipid Biosynthesis
There are three principal sources of acetyl-CoA (Figure 25.1):
1.Amino acid degradation produces cytosolic acetyl-CoA.
2.Fatty acid oxidation produces mitochondrial acetyl-CoA.
3.Glycolysis yields cytosolic pyruvate, which (after transport into the mitochondria) is converted to acetyl-CoA by pyruvate dehydrogenase.
The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetylCoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)
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FIGURE 25.1 ● |
The citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing |
equivalents (electrons) for fatty acid synthesis. The shuttle collects carbon substrates, primarily from glycolysis but also from fatty acid oxidation and amino acid catabolism. Most of the reducing equivalents are glycolytic in origin. Pathways that provide carbon for fatty acid synthesis are shown in blue; pathways that supply electrons for fatty acid synthesis are shown in red.
25.1 ● The Fatty Acid Biosynthesis and Degradation Pathways Are Different |
805 |
NADPH can be produced in the pentose phosphate pathway as well as by malic enzyme (Figure 25.1). Reducing equivalents (electrons) derived from glycolysis in the form of NADH can be transformed into NADPH by the combined action of malate dehydrogenase and malic enzyme:
Oxaloacetate NADH H 88n malate NAD
Malate NADP 88n pyruvate CO2 NADPH H
How many of the 14 NADPH needed to form one palmitate (Eq. 25.1) can be made in this way? The answer depends on the status of malate. Every citrate entering the cytosol produces one acetyl-CoA and one malate (Figure 25.1). Every malate oxidized by malic enzyme produces one NADPH, at the expense of a decarboxylation to pyruvate. Thus, when malate is oxidized, one NADPH is produced for every acetyl-CoA. Conversion of 8 acetyl-CoA units to one palmitate would then be accompanied by production of 8 NADPH. (The other 6 NADPH required [Eq. 25.1] would be provided by the pentose phosphate pathway.) On the other hand, for every malate returned to the mitochondria, one NADPH fewer is produced.
Acetate Units Are Committed to Fatty Acid Synthesis by
Formation of Malonyl-CoA
Rittenberg and Bloch showed in the late 1940s that acetate units are the building blocks of fatty acids. Their work, together with the discovery by Salih Wakil that bicarbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetylCoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 25.2). The reaction is catalyzed by acetylCoA carboxylase, which contains a biotin prosthetic group. This carboxylase is the only enzyme of fatty acid synthesis in animals that is not part of the multienzyme complex called fatty acid synthase.
Acetyl-CoA Carboxylase Is Biotin-Dependent and
Displays Ping-Pong Kinetics
The biotin prosthetic group of acetyl-CoA carboxylase is covalently linked to the -amino group of an active-site lysine in a manner similar to pyruvate carboxylase (Figure 23.3). The reaction mechanism is also analogous to that of pyruvate carboxylase (Figure 23.4): ATP-driven carboxylation of biotin is followed by transfer of the activated CO2 to acetyl-CoA to form malonyl-CoA. The enzyme from Escherichia coli has three subunits: (1) a biotin carboxyl carrier protein (a dimer of 22.5-kD subunits); (2) biotin carboxylase (a dimer of 51kD subunits), which adds CO2 to the prosthetic group; and (3) transcarboxylase (an 2 2 tetramer with 30-kD and 35-kD subunits), which transfers the activated CO2 unit to acetyl-CoA. The long, flexible biotin–lysine chain (biocytin) enables the activated carboxyl group to be carried between the biotin carboxylase and the transcarboxylase (Figure 25.3).
Acetyl-CoA Carboxylase in Animals Is a Multifunctional Protein
In animals, acetyl-CoA carboxylase (ACC) is a filamentous polymer (4 to 8106 D) composed of 230-kD protomers. Each of these subunits contains the biotin carboxyl carrier moiety, biotin carboxylase, and transcarboxylase activities, as well as allosteric regulatory sites. Animal ACC is thus a multifunctional protein. The polymeric form is active, but the 230-kD protomers are inactive. The activity of ACC is thus dependent upon the position of the equilibrium between these two forms:
Inactive protomers y8888z active polymer
806 Chapter 25 ● Lipid Biosynthesis
(a)
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Step 1 |
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Step 2 The transcarboxylation of biotin |
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FIGURE 25.2 ● (a) The acetyl-CoA carboxylase reaction produces malonyl-CoA for fatty acid synthesis. (b) A mechanism for the acetyl-CoA carboxylase reaction. Bicarbonate is activated for carboxylation reactions by formation of N-carboxybiotin. ATP drives the reaction forward, with transient formation of a carbonylphosphate intermediate (Step 1). In a typical biotin-dependent reaction, nucleophilic attack by the acetyl-CoA carbanion on the carboxyl carbon of N-carboxybiotin—a transcarboxylation—yields the carboxylated product (Step 2).
Because this enzyme catalyzes the committed step in fatty acid biosynthesis, it is carefully regulated. Palmitoyl-CoA, the final product of fatty acid biosynthesis, shifts the equilibrium toward the inactive protomers, whereas citrate, an important allosteric activator of this enzyme, shifts the equilibrium toward the active polymeric form of the enzyme. Acetyl-CoA carboxylase shows the kinetic behavior of a Monod–Wyman–Changeux V-system allosteric enzyme (Chapter 15).
FIGURE 25.6
808 Chapter 25 ● Lipid Biosynthesis
Dephospho-acetyl-CoA carboxylase
(Low [citrate] activates, high [fatty acyl-CoA] inhibits)
ATP P
Kinases Phosphatases
ADP
H2O
Phospho-acetyl-CoA carboxylase
(High [citrate] activates, low [fatty acyl-CoA] inhibits)
P P P P P P P
FIGURE 25.5 ● The activity of acetyl-CoA carboxylase is modulated by phosphorylation and dephosphorylation. The dephospho form of the enzyme is activated by low [citrate] and inhibited only by high levels of fatty acyl-CoA. In contrast, the phosphorylated form of the enzyme is activated only by high levels of citrate, but is very sensitive to inhibition by fatty acyl-CoA.
ulatory, whereas others are “silent” and have no effect on enzyme activity. Unphosphorylated acetyl-CoA carboxylase binds citrate with high affinity and thus is active at very low citrate concentrations (Figure 25.5). Phosphorylation of the regulatory sites decreases the affinity of the enzyme for citrate, and in this case high levels of citrate are required to activate the carboxylase. The inhibition by fatty acyl-CoAs operates in a similar but opposite manner. Thus, low levels of fatty acyl-CoA inhibit the phosphorylated carboxylase, but the dephosphoenzyme is inhibited only by high levels of fatty acyl-CoA. Specific phosphatases act to dephosphorylate ACC, thereby increasing the sensitivity to citrate.
Acyl Carrier Proteins Carry the Intermediates
in Fatty Acid Synthesis
The basic building blocks of fatty acid synthesis are acetyl and malonyl groups, but they are not transferred directly from CoA to the growing fatty acid chain. Rather, they are first passed to acyl carrier protein (or simply ACP), discovered by P. Roy Vagelos. This protein consists (in E. coli) of a single polypeptide chain of 77 residues to which is attached (on a serine residue) a phosphopantetheine group, the same group that forms the “business end” of coenzyme A. Thus, acyl carrier protein is a somewhat larger version of coenzyme A, specialized for use in fatty acid biosynthesis (Figure 25.6).
The enzymes that catalyze formation of acetyl-ACP and malonyl-ACP and the subsequent reactions of fatty acid synthesis are organized quite differently in different organisms. We first discuss fatty acid biosynthesis in bacteria and plants, where the various reactions are catalyzed by separate, independent proteins. Then we discuss the animal version of fatty acid biosynthesis, which involves a single multienzyme complex called fatty acid synthase.
Fatty Acid Synthesis in Bacteria and Plants
The individual steps in the elongation of the fatty acid chain are quite similar in bacteria, fungi, plants, and animals. The ease of purification of the separate enzymes from bacteria and plants made it possible in the beginning to sort out each step in the pathway, and then by extension to see the pattern of biosynthesis in animals. The reactions are summarized in Figure 25.7. The elongation reactions begin with the formation of acetyl-ACP and malonyl-ACP, which
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Phosphopantetheine group of coenzyme A |
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Phosphopantetheine prosthetic group of ACP
● Fatty acids are conjugated both to coenzyme A and to acyl carrier protein through the sulfhydryl of phosphopantetheine prosthetic groups.
810 Chapter 25 ● Lipid Biosynthesis
A D E E P E R L O O K
Choosing the Best Organism for the Experiment
The selection of a suitable and relevant organism is an important part of any biochemical investigation. The studies that revealed the secrets of fatty acid synthesis are a good case in point.
The paradigm for fatty acid synthesis in plants has been the avocado, which has one of the highest fatty acid contents in the plant kingdom. Early animal studies centered primarily on
pigeons, which are easily bred and handled and which possess high levels of fats in their tissues. Other animals, richer in fatty tissues, might be even more attractive but more challenging to maintain. Grizzly bears, for example, carry very large fat reserves but are difficult to work with in the lab!
are formed by acetyl transacylase (acetyl transferase) and malonyl transacylase (malonyl transferase), respectively. The acetyl transacylase enzyme is not highly specific—it can transfer other acyl groups, such as the propionyl group, but at much lower rates. (Fatty acids with odd numbers of carbons are made beginning with a propionyl group transfer by this enzyme.) Malonyl transacylase, on the other hand, is highly specific.
Decarboxylation Drives the Condensation of
Acetyl-CoA and Malonyl-CoA
Another transacylase reaction transfers the acetyl group from ACP to -keto- acyl-ACP synthase (KSase), also known as acyl-malonyl-ACP condensing enzyme. The first actual elongation reaction involves the condensation of acetylACP and malonyl-ACP by the -ketoacyl-ACP synthase to form acetoacetyl-ACP (Figure 25.7). One might ask at this point: Why is the three-carbon malonyl group used here as a two-carbon donor? The answer is that this is yet another example of a decarboxylation driving a desired but otherwise thermodynamically unfavorable reaction. The decarboxylation that accompanies the reaction with mal- onyl-ACP drives the synthesis of acetoacetyl-ACP. Note that hydrolysis of ATP drove the carboxylation of acetyl-CoA to form malonyl-ACP, so, indirectly, ATP is responsible for the condensation reaction to form acetoacetyl-ACP. MalonylCoA can be viewed as a form of stored energy for driving fatty acid synthesis.
It is also worth noting that the carbon of the carboxyl group that was added to drive this reaction is the one removed by the condensing enzyme. Thus, all the carbons of acetoacetyl-ACP (and of the fatty acids to be made) are derived from acetate units of acetyl-CoA.
Reduction of the -Carbonyl Group Follows a Now-Familiar Route
The next three steps—reduction of the -carbonyl group to form a -alcohol, followed by dehydration and reduction to saturate the chain (Figure 25.7)— look very similar to the fatty acid degradation pathway in reverse. However, there are two crucial differences between fatty acid biosynthesis and fatty acid oxidation (besides the fact that different enzymes are involved): First, the alcohol formed in the first step has the D configuration rather than the L form seen in catabolism, and, second, the reducing coenzyme is NADPH, although NAD and FAD are the oxidants in the catabolic pathway.
The net result of this biosynthetic cycle is the synthesis of a four-carbon unit, a butyryl group, from two smaller building blocks. In the next cycle of the process, this butyryl-ACP condenses with another malonyl-ACP to make a