Biochemistry, 4th Edition P77 ppsx

24.1 How Are Fatty Acids Synthesized? 723 (Levels of free fatty acids are very low in the typical cell. The palmitate made in this process is rapidly converted to CoA esters in preparation for the formation of tri- acylglycerols and phospholipids.) Cells Must Provide Cytosolic Acetyl-CoA and Reducing Power for Fatty Acid Synthesis Eukaryotic cells face a dilemma in providing suitable amounts of substrate for fatty acid synthesis. Sufficient quantities of acetyl-CoA, malonyl-CoA, and NADPH must be generated in the cytosol for fatty acid synthesis. Malonyl-CoA is made by carboxylation of acetyl-CoA, so the problem reduces to generating suf- ficient acetyl-CoA and NADPH. There are three principal sources of acetyl-CoA (Figure 24.1): 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. Fatty acyl- carnitine Citrate Fatty acyl- carnitine Citrate Amino acid catabolism Malate Malate Oxaloacetate Oxaloacetate Fatty acid oxidation Mitochondrial matrix Cytosol Fatty acids Fatty acids Amino acids + + Inner mitochondrial membrane Pyruvate Pyruvate Glycolysis Glucose TCA cycle ATP CO 2 CO 2 CO 2 NAD + NAD + NAD + ADP P i NADH NADH NADH NAD + NADH NADP + NADPH Acetyl-CoA Acetyl-CoA Fatty acyl-CoA Malic enzyme Malate dehydrogenase Malate dehydrogenase Pyruvate carboxylase Pyruvate dehydrogenase ATP-citrate lyase Citrate synthase FIGURE 24.1 The citrate–malate–pyruvate shuttle provides cytosolic acetate units and some reducing equiva- lents (electrons) for fatty acid synthesis.The shuttle collects carbon substrates, primarily from glycolysis but also from fatty acid oxidation and amino acid catabolism. Pathways that provide carbon for fatty acid synthe- sis are shown in blue; pathways that supply electrons for fatty acid synthesis are shown in red. 724 Chapter 24 Lipid Biosynthesis 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 (Fig- ure 24.1). Here it can be converted back into acetyl-CoA 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.) NADPH can be produced in the pentose phosphate pathway as well as by malic enzyme (Figure 24.1). 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 ϩ NADH ϩ H ϩ ⎯⎯→malate ϩ NAD ϩ Malate ϩ NADP ϩ ⎯⎯→pyruvate ϩ CO 2 ϩ NADPH ϩ H ϩ How many of the 14 NADPH needed to form one palmitate (see equation on page 722) 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 24.1). Every malate oxidized by malic enzyme produces one NADPH, at the expense of a de- carboxylation to pyruvate. Thus, when malate is oxidized, one NADPH is pro- duced 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, as shown in the equation on page 722, 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 bi- carbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetyl-CoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 24.2). The reaction is catalyzed by acetyl-CoA 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 (see Figure 22.2). The reaction mechanism is also analogous to that of pyruvate car- boxylase (see Figure 22.3): ATP-driven carboxylation of biotin is followed by trans- fer of the activated CO 2 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 51-kD subunits), which adds CO 2 to the prosthetic group; and (3) carboxyltransferase (an ␣ 2 ␤ 2 tetramer with 30- and 35-kD subunits), which transfers the activated CO 2 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 carboxyltransferase (Figure 24.3). Biotin carboxylase domain of human acetyl-CoA carboxylase 2 ( p db id = 2HJW) The biotin carboxylase domain from human acetyl-CoA carboxylase 2, with the A-subdomain in blue, the B-subdomain in red, the A–B linker in green, and the C-subdomain in yellow. 24.1 How Are Fatty Acids Synthesized? 725 Acetyl-CoA Carboxylase in Animals Is a Multifunctional Protein In animals, acetyl-CoA carboxylase (ACC) is a filamentous polymer (4 to 8 ϫ 10 6 D) composed of 265-kD protomers. Each of these subunits contains the biotin carboxyl carrier moiety, biotin carboxylase, and carboxyltransferase activities, as well as al- losteric regulatory sites. Animal ACC is thus a multifunctional protein. The polymeric form is active, but the 265-kD protomers are inactive. The activity of ACC is thus de- pendent upon the position of the equilibrium between these two forms: Inactive protomers 34 active polymer 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 in which allosteric effectors shift the T/R equi- librium between active R conformers and inactive T conformers. Carboxyltransferase domain of yeast acetyl-CoA carboxylase ( p db id = 1OD2) The carboxyltransferase domain dimer of acetyl-CoA carboxylase-1 from Saccharomyces cerevisiae. The N- and C-subdomains of one monomer are cyan and yel- low, whereas those of the other monomer are purple and green. CoA is shown as a ball-and-stick model in one subunit. + – HCO 3 – – O C O O P O O – O – O NNH S O HN NH S – O C O O NNH S – O C O C SCoA O H 2 C H 2 C C SCoA O COO – + HN NH S Lys Lys Lys Lys O CH 2 – O – C C SCH 3 CoA O ++HCO 3 O C S CoA O + + + H + P i P i ATP ATP ADP ADP Step 1 The carboxylation of biotin Step 2 The transcarboxylation reaction Biotin (a) (b) ACTIVE FIGURE 24.2 (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 car- boxylation reactions by formation of N-carboxybiotin. ATP drives the reaction forward, with transient forma- tion 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). Test yourself on the concepts in this figure at www.cengage.com/ login. 726 Chapter 24 Lipid Biosynthesis Phosphorylation of ACC Modulates Activation by Citrate and Inhibition by Palmitoyl-CoA The regulatory effects of citrate and palmitoyl-CoA are dependent on the phosphorylation state of acetyl-CoA carboxylase. The animal enzyme is phosphorylated at eight to ten sites on each enzyme subunit (Figure 24.4). Some of these sites are regulatory, whereas oth- ers 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 con- O HN NH S O C N H O C SCoA CH 3 C O – O O PO 3 2 – O N NH S O C N H O C SCoA CH 2 C – O O N NH S C N H C O – O O N NH S C N C O – O O N NH S C N H C O – O – P O O O Biotin carboxyl carrier protein Biotin carboxylase Carboxyltransferase FIGURE 24.3 In the acetyl-CoA carboxylase reaction, the biotin ring, on its flexible tether, acquires carboxyl groups from carbonylphosphate on the biotin carboxylase subunit and transfers them to acyl-CoA molecules on the carboxyltransferase subunits. Colors of the domains correspond to those in Figure 24.4. P P P P P P P 1 83 621 661 821 1200 1574 2346 cAMP-dependent protein kinase (PKA), AMP-dependent protein kinase (AMPK) 95 Protein kinase C (PKC) Carboxyl- transferase BCCP Biotin carboxylase 77 AMP-dependent protein kinase (AMPK) 76 cAMP-dependent protein kinase (PKA), protein kinase C (PKC) 29 25 23 Casein kinase II Calmodulin-dependent protein kinase Residue number FIGURE 24.4 Schematic of the acetyl-CoA carboxylase polypeptide, with domains and phosphorylation sites indicated, along with the protein kinases responsible. Phosphorylation at Ser 1200 is primarily responsible for decreasing the affinity for citrate. 24.1 How Are Fatty Acids Synthesized? 727 centrations (Figure 24.5). Phosphorylation of the regulatory sites decreases the affin- ity 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 op- posite manner. Thus, low levels of fatty acyl-CoA inhibit the phosphorylated carboxy- lase, but the dephosphoenzyme is inhibited only by high levels of fatty acyl-CoA. Spe- cific 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 ACP. This protein consists (in E. coli) of a single polypeptide chain of 77 residues to which is attached (on a serine residue) a phosphopante- theine group, the same group that forms the “business end” of coenzyme A. Thus, ACP is a somewhat larger version of coenzyme A, specialized for use in fatty acid biosynthesis (Figure 24.6). In Some Organisms, Fatty Acid Synthesis Takes Place in Multienzyme Complexes The enzymes that catalyze formation of acetyl-ACP and malonyl-ACP and the subse- quent reactions of fatty acid synthesis are organized quite differently in different or- ganisms. Fatty acid synthesis in mammals occurs on homodimeric fatty acyl synthase I (FAS I), each 270-kD polypeptide of which contains all reaction centers required to produce a fatty acid. In lower eukaryotes, such as yeast and fungi, the enzymatic P P P P P P P P P ATP Dephospho-acetyl-CoA carboxylase (Low [citrate] activates, high [fatty acyl-CoA] inhibits) H 2 O P i PhosphatasesKinases Phospho-acetyl-CoA carboxylase (High [citrate] activates, low [fatty acyl-CoA] inhibits) ADP FIGURE 24.5 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 sensi- tive to inhibition by fatty acyl-CoA. CH 2 H CH 3 O O P O – HS CH 2 N H C O CH 2 CH 2 N H C O C HO C CH 3 CH 2 O O O P O – CH 2 HH HH O Adenine OH 2– O 3 PO CH 2 H CH 3 O O P O – HS CH 2 N H C O CH 2 CH 2 N H C O C HO C CH 3 CH 2 O CH 2 Ser Acyl carrier protein Phosphopantetheine group of coenzyme A Phosphopantetheine prosthetic group of ACP FIGURE 24.6 Fatty acids are conjugated both to coenzyme A and to acyl carrier protein through the sulfhydryl of phosphopantetheine prosthetic groups. A DEEPER LOOK 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 avo- cado, 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! 728 Chapter 24 Lipid Biosynthesis activities of FAS are distributed on two multifunctional peptide chains, which form 2.6-megadalton ␣ 6 ␤ 6 complexes. In plants, most bacteria, and parasites, the enzymes of fatty acid synthesis are separated and independent, and this collection of enzymes is referred to as fatty acyl synthase II (FAS II). The individual steps in the elongation of the fatty acid chain are quite similar across all organisms. The mammalian pathway (Figure 24.7) is a cycle of elongation that involves six enzyme activities. The elongation cycle is initiated by transfer of the ␤-Ketoacyl synthase MAT MAT Thioesterase ␤-Ketoacyl-ACP reductase Palmitate ␤-Hydroxyacyl- ACP dehydratase ␤-Enoyl-ACP reductase CoASH KR KR DH DH ER ER TE KS KS KS 56 234 7 COO – 1 Acetyl-CoA O CH 3 C S-CoA O CH 3 CO 2 CO 2 C S-KSase Malonyl-CoA COO – ACP-SH O CH 2 C S-CoA COO – O CH 2 C S-ACP O CH 3 C S-ACP S ACP Acetoacetyl-ACP O CH 3 C O CH 2 C CH 3 C O CH 2 C OH H S ACP D-␤-Hydroxybutyryl-ACP CH 3 C H C H O C S ACP Crotonyl-ACP CH 3 O CH 2 CH 2 C S ACP Butyryl-ACP NADP + NADPH + H + H 2 O H 2 O NADP + NADPH + H + ␤-Hydroxyacyl-ACP ␤-Ketoacyl-ACP ␤-Enoyl-ACP Acyl (C n+2 )-ACP NADP + NADPH + H + NADP + NADPH + H + FIGURE 24.7 The pathway of palmitate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl building blocks are introduced as ACP conjugates. Decarboxylation drives the ␤-ketoacyl-ACP synthase and results in the addition of two-carbon units to the growing chain. The first turn of the cycle begins at ➊ and goes to butryrl-ACP; subse- quent turns of the cycle are indicated as ➋ through ➏. 24.1 How Are Fatty Acids Synthesized? 729 acyl moiety of acetyl-CoA to the acyl carrier protein by the malonyl-CoA–acetyl-CoA- ACP transacylase (MAT), which also transfers the malonyl group of malonyl-CoA to ACP. Decarboxylation Drives the Condensation of Acetyl-CoA and Malonyl-CoA The ␤-ketoacyl-ACP synthase (KS) catalyzes the decarboxylative condensation of the acyl group with malonyl-ACP to produce a ␤-ketoacyl-ACP intermediate (acetoacetyl- ACP in the first cycle). The mechanism (Figure 24.8) begins with acetyl group trans- fer to MAT, followed with attack by the ACP thiol sulfur to form an acetyl-ACP. The acetyl group is transferred to a cysteine sulfur on KS, freeing the ACP thiol to acquire the malonyl group. In the condensation reaction that follows, decarboxylation of the malonyl group creates a transient, highly nucleophilic carbanion that can attack the acetate group. The net reaction for each turn of this cycle (see Figure 24.7) is addition of a two- carbon unit to the acyl group. 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 de- carboxylation that accompanies the reaction with malonyl-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 re- action to form acetoacetyl-ACP. Malonyl-CoA can be viewed as a form of stored en- ergy 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 car- bons 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 by ␤-ketoacyl-ACP re- ductase (KR) to form a ␤-alcohol, then dehydration by ␤-hydroxyacyl-ACP dehy- dratase (DH) and reduction by 2,3-trans-enoyl-ACP reductase (ER) to saturate the chain (see Figure 24.7)—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 biosynthesis has the D-configuration rather than the L-form MAT H 3 CC C CH 3 SH ACP O MAT O S ACP SCoA H 3 CO HS KS C OO – MAT OH – OOC CH 2 C SCoA O C CH 2 O MAT O COO – C CH 3 SH ACP O KS S C CH 2 O C S ACP C CH 2 CH 3 O C KS SH S ACP O C CH 3 O KS S OH O CO 2 FIGURE 24.8 A mechanism for mammalian ketoacyl synthase. An acetyl group is transferred from CoA to MAT, then to the acyl carrier protein, and then to ketoacyl synthase. Next, a malonyl group is transferred to MAT and then to the acyl carrier protein. Decarboxylation of the malonyl group creates a transient carbanion on the acyl group of ACP, which attacks the KS acetyl group to form a ketoacyl-ACP. A cycle (see Figure 24.7) of keto group reduction (by KR), water removal (by DH), and double bond reduction (by ER; see next section) will finally produce an acyl group increased in length by two carbons. 730 Chapter 24 Lipid Biosynthesis seen in catabolism; second, the reducing coenzyme is NADPH, whereas NAD ϩ and FAD are the oxidants in the catabolic pathway. The net result of the first turn of the 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 six- carbon ␤-ketoacyl-ACP and CO 2 . Subsequent reduction to a ␤-alcohol, dehydration, and another reduction yield a six-carbon saturated acyl-ACP. This cycle continues with the net addition of a two-carbon unit in each turn until the chain is 16 carbons long (see Figure 24.7). The KS cannot accommodate larger substrates, so the reac- tion cycle ends with a 16-carbon chain. Hydrolysis of the C 16 -acyl-ACP yields a palmitic acid and the free ACP. In the end, seven malonyl-CoA molecules and one acetyl-CoA yield a palmitate (shown here as palmitoyl-CoA): Acetyl-CoA ϩ 7 malonyl-CoA Ϫ ϩ 14 NADPH ϩ 14 H ϩ ⎯⎯→ palmitoyl-CoA ϩ 7 HCO 3 Ϫ ϩ 14 NADP ϩ ϩ 7 CoASH The formation of seven malonyl-CoA molecules requires 7 Acetyl-CoA ϩ 7 HCO 3 Ϫ ϩ 7 ATP 4Ϫ ⎯⎯→ 7 malonyl-CoA Ϫ ϩ 7 ADP 3Ϫ ϩ 7 P i 2Ϫ ϩ 7 H ϩ Thus, the overall reaction of acetyl-CoA to yield palmitic acid is 8 Acetyl-CoA ϩ 7 ATP 4Ϫ ϩ 14 NADPH ϩ 7 H ϩ ⎯⎯→ palmitoyl-CoA ϩ 14 NADP ϩ ϩ 7 CoASH ϩ 7 ADP 3Ϫ ϩ 7 P i 2Ϫ Note: These equations are stoichiometric and are charge balanced. See problem 1 at the end of the chapter for practice in balancing these equations. Eukaryotes Build Fatty Acids on Megasynthase Complexes The multiple enzyme domains of eukaryotic fatty acyl synthases are arrayed on large protein structures termed megasynthases. The individual enzyme domains of these KS KS 2 ␣ MAT MAT MAT ER A C P TEKR KR KR DH DH DH DH DH ␣ PPTKR KR 2 KR 2 ␤ AT AT AT MPT MPT MPT MPT MPT ER ER DH DH DH KS KS 2 Reaction chamber Reaction chamber ER ER 2 Fungal fatty acid synthase Mammalian fatty acid synthase Reaction chamber Reaction chamber ϭ Active sites A T: Acetyl transferase MPT: Malonyl/palmitoyl transferase MAT: Malonyl-CoA–acetyl-CoA-ACP transacylase TE: Thioesterase A CP: Acyl carrier protein PPT: Phosphopantetheinyl transferase KR: ␤-Ketoacyl reductase KS: ␤-Ketoacyl synthase ER: ␤-Enoyl reductase DH: Dehydratase A C P FIGURE 24.9 Organization of enzyme functions on two eukaryotic fatty acid synthases. (left) Fungal FAS is a closed barrel 260 Å high and 230 Å wide. (right) Mammalian (pig) FAS is an asymmetric X-shape 210 Å high, 180 Å wide, and 90 Å deep.The arrangement of functional domains along the FAS polypeptides is shown at the bottom of the figure. KS domains form dimers in both structures. KR domains form dimers in the fungal enzyme, whereas ER and DH domains form dimers in the mammalian complex. 24.1 How Are Fatty Acids Synthesized? 731 structures in all eukaryotes are homologous to the corresponding small, discrete en- zymes of bacterial FAS pathways. Remarkably, however, lower eukaryotes such as fungi and higher eukaryotes such as mammals have evolved entirely different megasynthase architectures for fatty acid synthesis. Mammalian homodimeric FAS has a flattened X-shape, whereas the fungal dodecameric FAS is a large, closed bar- rel, with two reaction chambers separated by equatorial stabilizing struts (Figure 24.9). In the fungal structure, the six ␣-subunits form a central ring that is a “trimer of dimers” (Figure 24.10a,b). Each ␣-subunit contributes an extended ␣-helical seg- ment to the center of the structure. Pairs of these helices form three coiled-coil struts anchored by a six-helix bundle in the center of the barrel. Each ␣-subunit (b) Central wheel(a) (c) Tilted side view Lower reaction chamber KS dimer Upper reaction chamber Interchamber opening L1 L2 ACP PPT Central anchor Peripheral anchor KR dimer 90° FIGURE 24.10 (a) FAS from S. cerevisiae possesses two trimeric reaction chambers separated by equatorial stabilizing struts. ACP domains are located at the equa- torial base of each reaction chamber, close to the catalytic site of KS. (b) The equatorial base of this structure is an ␣ 6 trimer of dimers, with alternating KS and KR domains.The central stabilizing struts are ␣-helical extensions of the function- al domains arranged around the outside of the ring. A central six-helix bundle stabilizes the structure. (c) The ACP domains (red) are tethered on flexible linkers (yellow) so that they can move from one active site to the next in the catalytic cycle. Comparison of the ACP domains of S.cerevisiae and E.coli reveals that the ACP domains probably extend the acyl-phosphopantetheine group to active sites but then retract the acyl group into a hydrophobic cleft while moving from one site to the next (pdb ϭ 2UV8; images courtesy of Marc Leibundgut and Nenad Ban, ETH [Zurich]). 732 Chapter 24 Lipid Biosynthesis contains KR and KS domains. Three KR and three KS active sites are oriented to- ward the upper reaction chamber, and three of each face the lower chamber. The ␤-subunit trimers form rounded caps over the upper and lower reaction chambers. Each chamber contains three pores that allow substrates (acetyl-CoA and malonyl- CoA) to diffuse in and palmitoyl-CoA to exit. On each end of the structure, the ac- tive sites of the four ␤-subunit enzyme domains (see Figure 24.9) are oriented to- ward the interior of the reaction chamber. Three ACP domains in each chamber shuttle growing acyl chains from site to site during the catalytic cycle. Each ACP is tethered by two flexible linker peptides, which facilitate its site-to-site movement (Figure 24.10c). The phosphopantetheine arm on each ACP can extend outward to reach into active sites or may retract to insert its acyl chain in a protective hy- drophobic cavity during intersite transport. The homodimeric mammalian FAS contains all six functional enzyme domains on each subunit (Figures 24.9 and 24.11). In the X-shaped dimer, three of the domains—including KS, ER, and DH—form dimeric structures, whereas the KR and MAT domains are separated and lie near the ends of the extended “arms.” The arms form reaction chambers on either side of the structure. The flexible ACP do- mains do not appear in this structure (probably because they are not fixed in any one position in the crystals used for the structural studies). However, since it follows the KR domain in the polypeptide sequence, the ACP domain probably lies at the end of each KR arm, where it can rotate to interact with the adjacent active sites. In both the fungal and the mammalian FAS structures, the close association of enzymic domains within one large complex permits efficient transfer of intermedi- ates from one active site to the next. In addition, the presence of all these enzyme domains on one or two polypeptides allows the cell to coordinate synthesis of all en- zymes needed for fatty acid synthesis. KR KR DH DH KS 2 MAT MAT ER 2 FIGURE 24.11 Structural studies reveal that mammalian FAS homodimer is X-shaped. The ACP domains are probably located adjacent to the KR domains at the ends of the arms (pdb id ϭ 2VZ8; image courtesy of Marc Leibundgut and Nenad Ban, ETH [Zurich]).