21.1 Biosynthesis of Fatty Acids and Eicosanoids in Chapter 21 Lipid Biosynthesis

21.1 Biosynthesis of Fatty Acids and Eicosanoids

Even when compared to other major classes of metabolites, the division between fatty acid biosynthesis and breakdown is particularly striking. The two processes occur by different pathways, catalyzed by different sets of enzymes, and, in eukaryotes, occur in different cellular compartments. Fatty acid breakdown occurs in the mitochondria, whereas biosynthesis occurs in the cytosol. Moreover, biosynthesis requires the participation of a three-carbon intermediate, malonyl-CoA, that does not appear in the path of fatty acid breakdown.

Malonyl-Co A is C double bonded to O to the upper left, bonded to O minus to the lower left, and bonded to C H 2 to the right further bonded to C double bonded to O to the upper right and bonded to S to the lower right further bonded to Co A.

The general pathway of fatty acid synthesis and its regulation now take center stage. We consider the biosynthesis of longer-chain fatty acids, unsaturated fatty acids, and their eicosanoid derivatives at the end of this section.

Malonyl-CoA Is Formed from Acetyl-CoA and Bicarbonate

The formation of malonyl-CoA from acetyl-CoA is an irreversible three-step process, catalyzed by acetyl-CoA carboxylase. In animal cells, all three steps are catalyzed in the cytoplasm by a single multifunctional polypeptide (Fig. 21-1). The enzyme contains a biotin prosthetic group covalently bound in amide linkage to the ε-amino group of a Lys residue in one of the domains of the enzyme molecule. The reaction catalyzed by this enzyme is very similar to other biotin-dependent carboxylation reactions, such as those catalyzed by pyruvate carboxylase (see Fig. 16-16) and propionyl-CoA carboxylase (see Fig. 17-12). A carboxyl group, derived from bicarbonate $left-parenthesis HCO Subscript 3 Superscript minus Baseline right-parenthesis$ $left-parenthesis HCO Subscript 3 Superscript minus Baseline right-parenthesis$ , is first transferred to biotin in an ATP-dependent reaction. In a second step, the carboxyl group is carried by the biotin to a different active site, where the ${CO}_{2}$ $CO Subscript 2$ is transferred to acetyl-CoA in the third and final step to yield malonyl-CoA. As we will see, this carboxylation has the same function as the carboxylation of pyruvate by pyruvate carboxylase — it renders the next step in the reaction sequence much more favorable thermodynamically.

A figure shows the steps of the mammalian acetyl-Co A carboxylase reaction. — FIGURE 21-1 The acetyl-CoA carboxylase reaction. The mammalian acetyl-CoA carboxylase of the cytoplasm has three functional domains with distinct functions: biotin carrier protein; biotin carboxylase, which activates ${CO}_{2}$ $CO Subscript 2$ by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction; and transcarboxylase, which transfers activated ${CO}_{2}$ $CO Subscript 2$ (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA. Part of the biotin carrier protein domain and the long, flexible biotin arm rotate to carry the activated ${CO}_{2}$ $CO Subscript 2$ from the biotin carboxylase active site to the transcarboxylase active site. The active domain in each step is shaded in blue.

FIGURE 21-1 The acetyl-CoA carboxylase reaction. The mammalian acetyl-CoA carboxylase of the cytoplasm has three functional domains with distinct functions: biotin carrier protein; biotin carboxylase, which activates ${CO}_{2}$ $CO Subscript 2$ by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction; and transcarboxylase, which transfers activated ${CO}_{2}$ $CO Subscript 2$ (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA. Part of the biotin carrier protein domain and the long, flexible biotin arm rotate to carry the activated ${CO}_{2}$ $CO Subscript 2$ from the biotin carboxylase active site to the transcarboxylase active site. The active domain in each step is shaded in blue.

A light yellow half-circle is divided into two halves. It has an irregular top and a rounded bottom. The left side is labeled biotin carboxylase domain and has a cutout at the top in the shape of an inverted triangle, then a right side that slopes down to a rectangular cutout between the two halves above a circular biotin carrier protein domain. The right side is labeled transcarboxylase domain and has a small triangular peak just to the right of center, then a cutout in the shape of an inverted triangle, then a left slide that slopes down to the central rectangular cutout. The two halves are joined beneath the biotin carrier protein domain. A vertical zigzag chain extends up from the upper left of the biotin carrier protein domain. It begins as a three-carbon zigzag chain bonded to N H and this portion is labeled L y s side chain. N H is then further bonded to C double bonded to O to the left bonded to a vertical four-carbon zigzag chain above that joins the lower left vertex of a five-membered ring with S substituted for C at its bottom vertex and a top side shared with the bottom side of a five-membered ring above with C at its top vertex double bonded to O above and N H substituted for C at the upper left and right vertices. This top component is labeled biotin arm. A green rectangle angled above the left side contains C double bonded to O to the left, bonded to O H to the upper right, and bonded to O minus below. An arrow points down from this angled green rectangle to show that it fits into the cutout in the top of the left side biotin carboxylase domain. Accompanying text shows H C O 3 minus to the left of the green rectangle containing H C O 3 minus followed by plus A T P to its right. An arrow points down accompanied by a curved arrow showing the loss of A D P plus P subscript i end subscript. This yields a similar molecule in which the zigzag chain above the L y s side chain has bent to position the biotin arm against the green rectangle in the cutout on the left. N H at the top of the L y s side chain is bonded to C to the upper right double bonded to O to the right and bonded to a four-carbon zigzag chain that angles to the upper left to join the upper right vertex of a five-carbon ring with S substituted for C at its bottom vertex and an upper left side vertex shared with the lower right vertex of a five-membered ring above. This five-membered ring has C at the upper left vertex double bonded to O, H substituted for C at the upper right vertex, and N at the lower left vertex bonded to C below within the green rectangle and further double bonded to O the left and bonded to O minus below, all within the green rectangle fitted within to the cutout in the upper left of the left half of the half-circle, which is now highlighted in blue and labeled biotin carboxylase. An arrow points down to show that the piece of the zigzag chain above the L y s side chain has turned 180 degrees to orient the green rectangle in the cutout at the top of the right side. The left side is now yellow again and labeled biotin carboxylase domain. The right side is now highlighted in blue and labeled transcarboxylase domain. N H at the top of the L y s side chain is now bonded to C to the upper left double bonded to O to the left and bonded to a four-carbon zigzag chain that angles up to the right to meet the upper left vertex of a five-membered ring with S at its lower left vertex and an upper right side shared with the lower right side of a five-membered ring above with N H substituted for C at the upper left vertex, C at the upper right vertex double bonded to O, and N at the lower right vertex bonded to C in the green rectangle further double bonded to O to the right and bonded to O minus below, all within the green rectangle fitted into the cutout on the right side. Acetyl Co A, which is H 3 C bonded to C double bonded to O to the upper right and bonded to S to the lower right further bonded to Co A with all except for S bonded to Co A enclosed in a light red box, is shown with an arrow pointing to the flattened upper right edge of the transcarboxylase domain on the right side of the half-circle, just to the right of the cutout containing the green rectangle. An arrow points down to show that this yields a product with a yellow biotin carboxylase domain on the left, a blue transcarboxylase domain on the right, an a L y s side chain extending from a circular biotin carrier protein domain between them with its upper N H bonded to C to the left double bonded to O to the left and bonded to a four carbon zigzag chain that angles up to the right to join the upper left vertex of a five-membered ring with S at its lower left vertex and an upper right side shared with the lower right side of a five-membered ring above with N H substituted for C at the upper left and lower right vertices and C at the upper right vertex double bonded to O. To the right, malonyl-Co A is shown with a vertical green rectangle on the left and a light red chain on the right. C in the green rectangle is double bonded to O to the upper left, bonded to O minus to the lower right, and bonded to C H 2 to the right within the light red chain that is further bonded to C that is double bonded to O to the upper right within the light red region and bonded to S to the lower right outside of the light red region that is further bonded to Co A.

The bacterial version of acetyl-CoA carboxylase is similar but has three separate polypeptide subunits (including a separate biotin carrier protein) that catalyze the three steps. Plant cells contain both types of acetyl-CoA carboxylase.

Fatty Acid Synthesis Proceeds in a Repeating Reaction Sequence

The long carbon chains of fatty acids are assembled in the cytosol in a repeating four-step sequence (Fig. 21-2), catalyzed by a system collectively referred to as fatty acid synthase. A saturated acyl group produced by each four-step series of reactions becomes the substrate for subsequent condensation with an activated malonyl group. With each passage through the cycle, the fatty acyl chain is extended by two carbons.

A figure shows the addition of two carbons to a growing fatty acyl chain as a series of four steps. — FIGURE 21-2 Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl-CoA, with elimination of ${CO}_{2}$ $CO Subscript 2$ from the malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β-keto product of the condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: the β-keto group is reduced to an alcohol, elimination of $H_{2} O$ $upper H Subscript 2 Baseline upper O$ creates a double bond, and the double bond is reduced to form the corresponding saturated fatty acyl group.

FIGURE 21-2 Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl-CoA, with elimination of ${CO}_{2}$ $CO Subscript 2$ from the malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β-keto product of the condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: the β-keto group is reduced to an alcohol, elimination of $H_{2} O$ $upper H Subscript 2 Baseline upper O$ creates a double bond, and the double bond is reduced to form the corresponding saturated fatty acyl group.

At the top, a gray cylinder labeled fatty acid synthase consists of five vertical components and has a bond from the upper left of its left-hand component to S above that is further bonded to C in a yellow rectangle labeled acetyl group (first acetyl group). Within the yellow rectangle, this C is double bonded to O below and bonded to C H 3 to the left. A malonyl group above consists of a vertical green rectangle of C double bonded to O to the upper left and bonded to O minus to the lower left that is further bonded to C H 2 in a light red square that is further bonded to C that double bonded to O above within the same square and bonded to S to the right outside of the square. This S has a vertical bond down to C that begins an eleven-carbon zigzag chain that extends into a vertical brown oval that overlaps the center of the fatty acid synthase cylinder. Red arrows point from O minus in the green rectangle to the bond between the same O minus and C to its upper right within the same green rectangle, from the bond between C in the green rectangle and C H 2 in the light red square to C at the upper right of the yellow share below and from the bond between the same C at the upper right of the yellow square to S to its right. Step 1: Condensation. An arrow points downward accompanied by a curved arrow showing the loss of C O 2. This yields a similar structure in which the left side component of fatty acid synthase is bonded to H S and S connected by a zigzag chain to the oval overlapping the center of the fatty acid synthase is bonded to C within a light red box that is double bonded to O above and further bonded to C H 2 to its left with the C labeled alpha, all within the light red box. This C labeled alpha is bonded to a C labeled beta in yellow box, which is double bonded to O above and bonded to C H 3 to the left within the same yellow box. Step 2: Reduction. An arrow points down accompanied by a curved arrow showing the addition of an orange oval labeled N A D P H plus H plus and loss of N A D P plus. This yields a similar fatty acid synthase cylinder with its left side bonded to S H and a brown oval overlapping the center of the fatty acid synthase that contains a zigzag chain leading up to S bonded to C on the left within the light red square that is double bonded to O above and bonded to C H 2 to the left within the same square that is further bonded to C in the yellow square that is bonded to H above, C H 3 to the left, and O H below within the same yellow square. Step 3: Dehydration. An arrow points down accompanied by a curved arrow showing the loss of H 2 O. This yields a similar fatty acid synthase cylinder with its left side bonded to S H and a brown oval overlapping the center of the fatty acid synthase that contains a zigzag chain leading up to S bonded to C on the left that is double bonded to O above and bonded to C to the left bonded to H below and double bonded to C to the left within a yellow square that is bonded to H above and C H 3 to the left within the same yellow square. Step 4: Reduction. An arrow points down accompanied by a curved arrow showing the addition of a yellow oval labeled N A D P H plus H plus and the loss of N A D P plus. This yields a similar fatty acid synthase cylinder with its left side bonded to S H and a brown oval overlapping the center of the fatty acid synthase that contains a zigzag chain leading up to S bonded to C on the left within the light red square that is double bonded to O above and bonded to C H 2 to the left within the same square that is further bonded to C H 2 in the yellow square that is further bonded to C H 3 within the same yellow square. Accompanying text reads, saturated acyl group, lengthened by two carbons.

In the four-step pathway, a condensation reaction is followed by a reduction-dehydration-reduction sequence to convert the C-3 carbonyl to a methylene. The latter three steps are the chemical reverse of the oxidation-hydration-oxidation sequence in the β oxidation of fatty acids (Fig. 17-8a). However, both the electron-carrying cofactor and the activating groups in the reductive anabolic sequence differ from those in the oxidative catabolic process. Recall that in β oxidation, ${NAD}^{+}$ $NAD Superscript plus$ and FAD serve as electron acceptors and the activating group is the thiol $(— SH)$ $left-parenthesis em-dash SH right-parenthesis$ group of coenzyme A. By contrast, the reducing agent in the synthetic sequence is NADPH and the activating groups are two different enzyme-bound $— SH$ $em-dash SH$ groups, as described below.

In mammals, this synthetic system is called fatty acid synthase I (FAS I). There are seven active sites to catalyze the four-step cycle plus the charging steps described below. The active sites for different reactions lie in separate domains (Fig. 21-3a), all within a single multifunctional polypeptide chain $(M_{r} 240, 000)$ $left-parenthesis upper M Subscript r Baseline 240 comma 000 right-parenthesis$ . Two of these multifunctional polypeptides function as a homodimer ( $M_{r} 480, 000$ $upper M Subscript r Baseline 480 comma 000$ ; Fig. 21-3b). The two subunits seem to function independently. When all the active sites in one subunit are inactivated by mutation, fatty acid synthesis is only moderately reduced.

A two-part figure shows the structure of a fatty acid synthase type Roman numeral 1 system with the locations of enzymatic activities labeled in part a and with the native dimeric structure shown in part b. — FIGURE 21-3 The structure of a fatty acid synthase type I system. Shown here is the structure of a single (monomeric) polypeptide chain of the mammalian (porcine) enzyme system. (a) All of the active sites in the mammalian system are located in different domains within a single large polypeptide chain. The different enzymatic activities are β-ketoacyl-ACP synthase (KS), malonyl/acetyl-CoA–ACP transferase (MAT), β-hydroxyacyl-ACP dehydratase (DH), enoyl-ACP reductase (ER), and β-ketoacyl-ACP reductase (KR). ACP is the acyl carrier protein. The seventh domain is a thioesterase (TE) that releases the palmitate product from ACP when synthesis is completed. The ACP and TE domains are disordered in the crystal and are therefore not shown in the structure. (b) The native dimeric structure is shown, with one polypeptide transparent to show how the two independently operating subunits come together. The linear arrangement of the domains in the polypeptide is shown below the structure. [Data from PDB ID 2CF2, T. Maier et al., *Science* 311:1258, 2006.]

Part a shows a structure with two roughly linear halves with bumpy surface contours. The top half has a green, rounded component at the upper left labeled E R with a smaller, light green rounded piece below labeled D H. Just above the center of the place that these two green components meet, there is a small black circle labeled N A D P. Adjacent to the top right of the dark green upper component, there is a small yellow component labeled K R above a white piece that extends right and expands into a rounded right end of the overall structure. Some pieces of yellow are visible at its lower left center and to the right of the region where it curves down to become more rounded. The yellow piece that fits against the curve in the white piece has an open center and a c curved line extends from the bottom of this yellow piece to a brown circle labeled A C P from which a line continues to a larger purple oval labeled T E. At the lower left end of the light green piece at the left, a small white vertical piece connects the upper linear half with an orange, roughly oval piece labeled K S at the left side of the bottom linear half. To the right of this orange piece, there is a central white piece and then the bottom part ends with a red piece labeled M A T that protrudes slightly from its upper right side to produce a roughly triangular appearance. Part b shows two roughly linear pieces that are joined in the center. The right half is brighter in color and the left half is faded. The upper right part contains dark green cylinders on top in a rounded region labeled E R and lighter green cylinders below in a smaller rounded region labeled D H. In the center of the region where the two green pieces meet, there is a linear clump of red spheres with gray and blue spheres below and this clump is labeled N A D P. To the right of the dark green region, there is a narrower region with yellow cylinders labeled K R and this contains a similar N A D P in its center. An oval region to the right extends lower down and contains white cylinders. A thin vertical white piece joins the small piece with light green cylinders with a rounded region below labeled K S that contains orange cylinders. There is a slightly thinner region of white cylinders to its right, followed by a rounded region of red cylinders labeled M A T that protrudes slightly to the upper right to produce a roughly triangular appearance. The faded left side appears similar but with all of the elements presented in the reverse order. The top piece begins with dark green cylinders on top and light green cylinders below, then has region of yellow cylinders, then a region of white cylinders. The bottom piece begins with a region of orange cylinders, then a thinner region of white cylinders, then a larger piece with red cylinders. Components are shown horizontally across the bottom as a series of shapes joined by thin black lines. From left to right, the pieces are as follows: orange rectangle labeled K S; thin white vertical rectangle; light red rectangle labeled M A T; thinner white rectangle; small light green rectangle labeled D H 1; similar small light green rectangle labeled D H 2; white rectangle connected to another white rectangle and both labeled noncatalytic domains; long green rectangle labeled E R; yellow rectangle; brown vertical oval labeled A C P; long blue rectangle labeled T E. A C P and T E are not shown in the structure.

With FAS I systems, fatty acid synthesis leads to a single product. As it goes through the cycle, the acyl group is covalently linked to acyl carrier protein (ACP), which shuttles it from one active site to another in sequence. Acyl carrier protein is yet another contiguous part of the single FAS I polypeptide. No intermediates are released. When the chain length reaches 16 carbons, that product (palmitate, 16:0; see Table 10-1) leaves the cycle. Carbons C-16 and C-15 of the palmitate are derived from the methyl and carboxyl carbon atoms, respectively, of an acetyl-CoA used directly to prime the system at the outset (Fig. 21-4); the rest of the carbon atoms in the chain are derived from acetyl-CoA via malonyl-CoA.

A figure shows how the fatty acyl chain grows by two-carbon units donated by activated malonate during palmitate synthesis and that C O 2 is lost at each step. — FIGURE 21-4 The overall process of palmitate synthesis. The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of ${CO}_{2}$ $CO Subscript 2$ at each step. The initial acetyl group is shaded yellow; C-1 and C-2 of malonate, light red; and the carbon released as ${CO}_{2}$ $CO Subscript 2$ , green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0).

A gray cylinder labeled fatty acid synthase consists of a series of five pieces. The left-hand piece is bonded to S above further bonded to C in a yellow square that is double bonded to O to the right and bonded to C H 3 above, all within the yellow square. A vertical brown oval angled slightly to the right overlaps the tops of the second and third components of fatty acid synthase and an eleven-carbon zigzag chain that begins in the middle of this oval extends up to connect to S above. This S bonds to C in a light red square that is double bonded to O to the right and bonded to C H 2 above within the same light red square that is further bonded to C O O minus in a green rectangle. A series of four arrows points to the right. The first arrow in the series branches to show the loss of a green square labeled C O 2. 4 H plus plus 4 e minus above is shown with an arrow pointing right above the central two arrows. This yields a structure in which the upper left side fatty acid synthase is still bonded to S above. This S is further bonded to C in a light red rectangle that is double bonded to O to the right and bonded to C H 2 within the same light red rectangle that is further bonded to C H 2 in a vertical yellow rectangle that is further bonded to C H 3 above within the same yellow rectangle. A vertical brown oval angled slightly to the right overlaps the tops of the second and third components of fatty acid synthase and an eleven-carbon zigzag chain that begins in the middle of this oval extends up to connect to S above. This S bonds to C in a blue square that is double bonded to O to the right and bonded to C H 2 above within the same blue square that is further bonded to C O O minus in a green rectangle. A series of four arrows points to the right. The first arrow in the series branches to show the loss of a green square labeled C O 2. 4 H plus plus 4 e minus above is shown with an arrow pointing right above the central two arrows. This yields a similar structure in which S bonded to the upper left of fatty acid synthase is bonded to C in a blue rectangle that is double bonded to O to the right and bonded to C H 2 above within the same blue rectangle that is further bonded to C H 2 in a light red rectangle that is further bonded to C H 2 within the same light red rectangle that is bonded to C H 2 in a yellow rectangle that is further bonded to C H 3 within the same yellow rectangle. A series of four arrows points to the right. The first arrow in the series branches to show the loss of a green square labeled C O 2. 4 H plus plus 4 e minus above is shown with an arrow pointing right above the central two arrows. This yields a similar structure in which S bonded to the upper left of fatty acid synthase is bonded to C in a blue rectangle that is double bonded to O to the right and bonded to C H 2 above within the same blue rectangle that is further bonded to C H 2 in a blue rectangle further bonded to C H 2 within the same blue rectangle that is further bonded to C H 2 in a light red rectangle that is further bonded to C H 2 within the same light red rectangle that is bonded to C H 2 in a yellow rectangle that is further bonded to C H 3 within the same yellow rectangle. A series of four arrows points to the right. The first arrow in the series branches to show the loss of a green square labeled C O 2. A dashed arrow labeled four more additions points to a product in which fatty acid synthase is shown to the left with its upper left side bonded to S H and a vertical brown oval angled slightly to the right that overlaps the tops of its second and third components and has an eleven-carbon zigzag chain that begins in the middle of this oval extends up to connect to S H. Palmitate is also present. It has a blue box at the bottom containing C double bonded to O to the lower right, bonded to O minus to the lower left, and bonded to C H 2 above further bonded to CH 2 in a blue rectangle above further bonded to C H 2 within the same blue rectangle. This pattern continues through four additional blue rectangles before C H 2 at the top of the top blue rectangle bonds to C H 2 in a light red rectangle further bonded to C H 2 within the same light red rectangle that is bonded to C H 2 in a yellow rectangle further bonded to C H 3 above within the same yellow rectangle.

A somewhat different FAS I is found in yeast and other fungi, and is made up of two multifunctional polypeptides that form a complex with an architecture distinct from that of the vertebrate systems. Three of the seven required active sites are found on the α subunit and four on the β subunit. A different system, called FAS II, is found in plants and most bacteria. FAS II is a dissociated system; each step in the synthesis is catalyzed by a separate enzyme. Unlike FAS I, FAS II generates a variety of products, including saturated fatty acids of several lengths, as well as unsaturated, branched, and hydroxy fatty acids. An FAS II system is also found in vertebrate mitochondria, yet another indication of the bacterial origins of mitochondria in evolution.

The Mammalian Fatty Acid Synthase Has Multiple Active Sites

The multiple domains of mammalian FAS I function as distinct but linked enzymes. The active site for each enzyme is found in a separate domain within the larger polypeptide. Throughout the process of fatty acid synthesis, the intermediates remain covalently attached as thioesters to one of two thiol groups. One point of attachment is the $— SH$ $em-dash SH$ group of a Cys residue in one of the synthase domains (β-ketoacyl-ACP synthase; KS); the other is the $— SH$ $em-dash SH$ group of acyl carrier protein, a separate domain of the same polypeptide. Hydrolysis of thioesters is highly exergonic, and the energy released helps to make two steps ( and in Fig. 21-6) in fatty acid synthesis thermodynamically favorable.

Acyl carrier protein is the shuttle that holds the system together, containing the prosthetic group $4^{'}$ $bold 4 bold prime$ -phosphopantetheine, also found in coenzyme A (Fig. 21-5). The $4^{'}$ $4 prime$ -phosphopantetheine serves as a flexible arm, tethering the growing fatty acyl chain to the surface of the fatty acid synthase complex while carrying the reaction intermediates from one enzyme active site to the next. The same prosthetic group is used in FAS II systems.

A figure shows the structure of acyl carrier protein (A C P) compared with coenzyme A. — FIGURE 21-5 Acyl carrier protein (ACP). The prosthetic group is $4^{'}$ $4 prime$ -phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine, also found in the coenzyme A molecule, contains the B vitamin pantothenic acid. Its $— SH$ $em-dash SH$ group is the site of entry of malonyl groups during fatty acid synthesis. Coenzyme A, shown here for comparison, serves a similar chemical purpose in general metabolism.

FIGURE 21-5 Acyl carrier protein (ACP). The prosthetic group is $4^{'}$ $4 prime$ -phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine, also found in the coenzyme A molecule, contains the B vitamin pantothenic acid. Its $— SH$ $em-dash SH$ group is the site of entry of malonyl groups during fatty acid synthesis. Coenzyme A, shown here for comparison, serves a similar chemical purpose in general metabolism.

Two vertical molecules are shown side-by-side. The right-hand molecule is labeled coenzyme A. A vertical brown oval labeled A C P is bonded to C H 2 above that is connected to O above by a bond intersected by a dashed line to the right representing the bottom boundary of a bracket that extends to the top of the molecule and is labeled, 4 prime-phosphopantetheine. To the left of the bond between C H 2 and O, text reads, S e r side chain. Beginning at A C P, the vertical chain is C H 2 bonded to O bonded to P double bonded to O to the right, bonded to O minus to the left, and connected to O above by another bond intersected by a dashed line that marks the beginning of a bracket representing the bottom of pantothenic acid. This O is further bonded to C H 2 bonded to C bonded to C H 3 to the left and right and to C above bonded to C double bonded to O to the right and bonded to N H above further bonded to C H 2 bonded to C H 2 bonded to C double bonded to O to the right and bonded to N H above by a bond intersected by a dashed line to a bracket representing the top of pantothenic acid. This N H is further bonded to C H 2 bonded to C H 2 bonded to S H in a light red box. Accompanying text reads, malonyl groups are esterified to the further bonded S H group. Coenzyme A begins with 3 prime-phospho-A M P, which has a bond to O above intersected by the dashed vertical line marking the start of 4 prime-phosphopantetheine to the right. From that point, its structure is the same as the left-hand molecule except that the top S H is not in a light red box.

Fatty Acid Synthase Receives the Acetyl and Malonyl Groups

Before the condensation reactions that build up the fatty acid chain can begin, the two thiol groups on the enzyme complex must be charged with the correct acyl groups ( Fig. 21-6a). First, the acetyl group of acetyl-CoA is transferred to ACP in a reaction catalyzed by the malonyl/acetyl-CoA–ACP transferase (MAT) domain of the multifunctional polypeptide. The acetyl group is then transferred to the Cys $— SH$ $em-dash SH$ group of the β-ketoacyl-ACP synthase (KS). The second reaction, transfer of the malonyl group from malonyl-CoA to the $— SH$ $em-dash SH$ group of ACP, is also catalyzed by malonyl/acetyl-CoA–ACP transferase. In the charged synthase complex, the acetyl and malonyl groups are activated for the chain-lengthening process. We now consider the first four steps of this process in some detail; all step numbers refer to Figure 21-6.

A two-part figure shows the sequence events during the synthesis of a fatty acid by showing the mammalian F A S Roman numeral 1 complex in part a and the steps in part b. — FIGURE 21-6 Sequence of events during synthesis of a fatty acid. (a) The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight S shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but it is attached to the KR domain. The phosphopantetheine arm of ACP ends in an $— SH$ $em-dash SH$ . (b) The enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow; C-1 and C-2 of malonate, light red; and the carbon released as ${CO}_{2}$ $CO Subscript 2$ , green. Steps to are described in the text.

FIGURE 21-6 Sequence of events during synthesis of a fatty acid. (a) The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight S shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but it is attached to the KR domain. The phosphopantetheine arm of ACP ends in an $— SH$ $em-dash SH$ . (b) The enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow; C-1 and C-2 of malonate, light red; and the carbon released as ${CO}_{2}$ $CO Subscript 2$ , green. Steps to are described in the text.

Part a shows the structure of the mammalian F A S Roman numeral 1 complex. It is a crescent-shaped structure with a yellow piece labeled K R and beta-ketoacyl-A C P at the upper right. A strand extends right from this yellow piece to a brown oval labeled A C P below that contains a black 11-carbon zigzag chain that ends with a bond to S H. A green piece labeled E R and enoyl-A C P reductase it to the left of the yellow piece, then a light green piece labeled D H and beta-hydroxyacyl-A C P dehydratase forms the curved downward left side of the structure before joining to a long red piece labeled M A T and malonyl/acetyl-Co A- A C P transferase that curves back toward the left front to meet an orange piece labeled K S and beta-ketoacyl-A C P synthase that is rounded at its end, where it has a bond to H S. Acetyl Co A is shown to the lower left as C H 3 bonded to C double bonded to O to the upper right and bonded to S bonded to Co A to the lower right. Acetyl Co A is enclosed in a yellow rectangle except for S bonded to Co A. An arrow pointing right from acetyl Co A to Co A – S H crosses a downward arrow from the F A S Roman numeral 1 complex above that points down to show the same structure in part b, where it is at the upper right of a cyclic series of reactions and is shown in gray except for the brown oval labeled A C P and the red region labeled M A T. In this structure, A C P has bent left so that its zigzag chain ending with H S extends into the left-hand lobe labeled K S. This lobe, labeled K S, has a bond to S to the left further bonded to C in a yellow rectangle double bonded to O above and bonded to C H 3 to the left, all within the same yellow rectangle. An arrow points down from this structure to a similar structure below and is crossed by an arrow pointing left from malonyl-Co A to Co A – S H. Malonyl-Co A is shown as a green rectangle containing C double bonded to O to the upper left and bonded to O minus to the lower left, all within the green rectangle, and also bonded to C H 2 to the right in a light red box that is further bonded to C double bonded to O within the light red box and bonded to S below outside of the box further bonded to Co A. A dashed line indicating the place where these two arrows meet represents step 6 points from a version of the F A S Roman numeral 1 complex that is similar except that the segment labeled K S is bonded to S further bonded to C in a light red rectangle double bonded to O above and bonded to C H 2 to the left within the same light red rectangle that is bonded to C H 2 in a yellow rectangle that is further bonded to C H 3 within the same rectangle. Text accompanying the dashed line reads, Recharging of A C P with another malonyl group (M A T). This yields the center right molecule, which is similar except for the following differences: M A T is gray instead of red; K S is highlighted in orange and bonded to S to the left further bonded to C in a yellow rectangle that is double bonded to O below and bonded to C H 3 to the left within the same yellow rectangle; the brown oval labeled A C P has bent to be positioned horizontally across the top of K S with its zigzag chain extending left to S bonded to C in a light red box further double bonded to O above and bonded to C H 2 to the left within the same box further bonded to C in a green box double bonded to O to the upper left and bonded to O minus to the lower left within the same green box. Accompanying text reads, fatty acid synthase complex charged with an acetyl and a malonyl group. Red arrows point from the O minus int eh green box to the bond between the same O minus and C within the same green box, from the bond between C in the green box and C H 2 in the light red box to C in the yellow box bonded to S outside of the box to the right, and from the box between the same C and S to the same S. Step 1: Condensation (K S). An arrow points down accompanied by the loss of C O 2 in a green box. This yields a similar molecule in which K S is only bonded to S H, K R at the upper right is highlighted in yellow, and the brown oval labeled A C P has bent back across K R so that its zigzag chain leads to S just to the upper right of K R further bonded to C in a light red box double bonded to O above and bonded to C H 2 to the right within the same light red box further bonded to C in a yellow box double bonded to O below and bonded to C H 3 to the right within the same box. Text below reads beta-ketobutyryl-A C P. Step 2: Reduction of beta-keto group (K R). An arrow curves down to the left to the bottom center structure, labeled D-beta-hydroxybutyryl-A C P. An accompanying curved arrow shows the addition of an orange oval labeled N A D P H plus H plus and loss of N A D P plus. This structure no longer has K R highlighted. The brown oval labeled A C P has bent left to overlap D H at the left side, which is highlighted in green. The zigzag chain from A C P leads to S bonded to C in the light red box that is double bonded to O above and bonded to C H 2 to the left in the same light red box that is further bonded to C H in a yellow box that is further bonded to O H below and to C H 3 to the left in the same yellow box. Step 3: Dehydration (D H). An arrow curves up to italicized trans end italics-delta superscript 2-butenoyl-A C P with an arrow that branches away to show the loss of A C P. This is a similar molecule with only green E R at the top highlighted. The brown oval labeled A C P has curved to angle up over the bottom of E R with its zigzag chain extending to S to the upper left that is bonded to C in a light red box double bonded to O above and bonded to C H to the left within the same light red box that is further double bonded to C H in a yellow rectangle that is bonded to C H 3 within the same yellow rectangle. Step 4: Reduction of double bond (E R). This yields butyryl-A C P. Only the orange lobe labeled K S at the lower left is highlighted and it is bonded to H S. The brown oval labeled A C P overlaps the upper right of K S and its zigzag chain runs left to C in a light red box double bonded to O above and bonded to C H 2 to the left in the same box further bonded to C H 2 in a yellow rectangle further bonded to C H 3 in the same rectangle. Step 5: Translocation of butyryl group to C y s on beta-ketoacyl-A C P synthase. This yields a structure in which only the curved red piece labeled M A T is highlighted. The brown oval labeled A C P bends down to touch K S and its zigzag chain extends into K S to end with a bond to H S. K S has a bond to S to the left further bonded to C in a light red box double bonded to O above and bonded to C H 2 to the left in the same box further bonded to C H 2 in a yellow rectangle further bonded to C H 3 in the same rectangle. From this structure, the dashed arrow labeled step 6 points down to the right as previously described.

Step Condensation

The first reaction in the formation of a fatty acid chain is a formal Claisen condensation (see reaction class in Fig. 13-4) of the activated acetyl and malonyl groups to form acetoacetyl-ACP, an acetoacetyl group bound to ACP through the phosphopantetheine $— SH$ $em-dash SH$ group; simultaneously, a molecule of ${CO}_{2}$ $CO Subscript 2$ is produced. In this reaction, catalyzed by β-ketoacyl-ACP synthase, the acetyl group is transferred from the Cys $— SH$ $em-dash SH$ group of the enzyme to the malonyl group on the $— SH$ $em-dash SH$ of ACP, becoming the methyl-terminal two-carbon unit of the new acetoacetyl group.

The carbon atom of the ${CO}_{2}$ $CO Subscript 2$ formed in this reaction is the same carbon originally introduced into malonyl-CoA from ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ in the acetyl-CoA carboxylase reaction (Fig. 21-1). Thus, ${CO}_{2}$ $CO Subscript 2$ is only transiently in covalent linkage during fatty acid biosynthesis; it is removed as each two-carbon unit is added.

Why do cells go to the trouble of adding ${CO}_{2}$ $CO Subscript 2$ to make a malonyl group from an acetyl group, only to lose the ${CO}_{2}$ $CO Subscript 2$ during the formation of acetoacetate? The use of activated malonyl groups rather than acetyl groups makes the condensation reactions thermodynamically favorable. The methylene carbon (C-2) of the malonyl group, sandwiched between carbonyl and carboxyl carbons, forms a good nucleophile. In the condensation step, decarboxylation of the malonyl group facilitates nucleophilic attack of the methylene carbon on the thioester linking the acetyl group to β-ketoacyl-ACP synthase, displacing the enzyme’s $— SH$ $em-dash SH$ group. Coupling the condensation to the decarboxylation of the malonyl group renders the overall process highly exergonic. A similar carboxylation-decarboxylation sequence facilitates the formation of phosphoenolpyruvate from pyruvate in gluconeogenesis (see Figs. 14-17 and 14-18).

By using activated malonyl groups in the synthesis of fatty acids and activated acetate in their degradation, the cell makes both processes energetically favorable, although one is effectively the reversal of the other. The extra energy required to make fatty acid synthesis favorable is provided by the ATP used to synthesize malonyl-CoA from acetyl-CoA and ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ (Fig. 21-1).

Step Reduction of the Carbonyl Group

The acetoacetyl-ACP formed in the condensation step now undergoes reduction of the carbonyl group at C-3 to form d-β-hydroxybutyryl-ACP. This reaction is catalyzed by β-ketoacyl-ACP reductase (KR), and the electron donor is NADPH. Notice that the d-β-hydroxybutyryl group does not have the same stereoisomeric form as the l-β-hydroxyacyl intermediate in fatty acid oxidation (see Fig. 17-8).

Step Dehydration

The elements of water are now removed from C-2 and C-3 of d-β-hydroxybutyryl-ACP to yield a double bond in the product, trans- $Δ^{2}$ $bold upper Delta Superscript 2$ -butenoyl-ACP. The enzyme that catalyzes this dehydration is β-hydroxyacyl-ACP dehydratase (DH).

Step Reduction of the Double Bond

Finally, the double bond of trans- $Δ^{2}$ $upper Delta Superscript 2$ -butenoyl-ACP is reduced (saturated) to form butyryl-ACP by the action of enoyl-ACP reductase (ER); again, NADPH is the electron donor.

The Fatty Acid Synthase Reactions Are Repeated to Form Palmitate

Production of the four-carbon, saturated fatty acyl–ACP marks completion of one pass through the fatty acid synthase complex. In step , the butyryl group is transferred from the phosphopantetheine $— SH$ $em-dash SH$ group of ACP to the Cys $— SH$ $em-dash SH$ group of β-ketoacyl-ACP synthase, which initially bore the acetyl group (Fig. 21-6). To start the next cycle of four reactions that lengthens the chain by two more carbons (step ), another malonyl group is linked to the now unoccupied phosphopantetheine $— SH$ $em-dash SH$ group of ACP (Fig. 21-7). Condensation occurs as the butyryl group, acting like the acetyl group in the first cycle, is linked to two carbons of the malonyl-ACP group with concurrent loss of ${CO}_{2}$ $CO Subscript 2$ . The product of this condensation is a six-carbon acyl group, covalently bound to the phosphopantetheine $— SH$ $em-dash SH$ group. Its β-keto group is reduced in the next three steps of the synthase cycle to yield the saturated acyl group, exactly as in the first round of reactions — in this case forming the six-carbon product.

A figure shows the beginning of the second round of the fatty acid synthesis cycle. — FIGURE 21-7 Beginning of the second round of the fatty acid synthesis cycle. The butyryl group is on the Cys $— SH$ $em-dash SH$ group. The incoming malonyl group is first attached to the phosphopantetheine $— SH$ $em-dash SH$ group. Then, in the condensation step, the entire butyryl group on the Cys $— SH$ $em-dash SH$ is exchanged for the carboxyl group of the malonyl residue, which is lost as ${CO}_{2}$ $CO Subscript 2$ (green). This step is analogous to step in Figure 21-6. The product, a six-carbon β-ketoacyl group, now contains four carbons derived from malonyl-CoA and two derived from the acetyl-CoA that started the reaction. The β-ketoacyl group then undergoes steps through in Figure 21-6.

A mostly gray crescent-shaped structure has a piece labeled K R at the upper right. A strand extends right from this piece to a brown oval labeled A C P that is bent to contact a rounded orange structure below labeled K S. A C P contains a black 11-carbon zigzag chain that ends with a bond to S H. To the left of K R, the top left is formed by a curved piece labeled E R, then a piece labeled D H forms the curved downward left side of the structure before joining to a long red piece labeled M A T that curves back toward the left front to meet an orange piece labeled K S that is rounded at its end, where it has a bond to S that is further bonded to C in a light red box that is double bonded to O above and bonded to C H 2 in the same light red box further bonded to C H 2 in a yellow box further bonded to C H 3 in the same yellow box. Text below the yellow and light red boxes reads, butyryl group. An arrow points down to a similar structure below and is intersected by an almost horizontal arrow from malonyl-Co A on the left to Co A-S H on the right. Malonyl-Co A is shown as a green vertical rectangle with C double bonded to O to the upper left and bonded to O minus to the lower left within the same green rectangle and bonded to C H 2 to the right in a blue box that is further bonded to C double bonded to O to the upper right in the same light blue box and bonded to S to the lower right outside of the box that is further bonded to Co A. In the product, the crescent-shaped molecule is gray except for K S at the lower left, which is orange, and the brown oval labeled A C P. A C P has moved to overlap the top of K S and its zigzag chain extends left to bond to S that is further bonded to C in a light blue box double bonded to O above and bonded to C H 2 to the left in the same light blue box that is further bonded to C in a green rectangle that is double bonded to O to the upper left and bonded to O minus to the lower left within the same green rectangle. The orange lobe labeled K S has a bond from its left side to S further bonded to C in a light red box double bonded to O below and bonded to C H 2 to the left within the same light red box and further bonded to C H 2 in a yellow rectangle further bonded to C H 3 in the same yellow rectangle. Red arrows point from the negatively charged O in the green box to the bond between that O and C to its upper right, from the bond between C in the green box and C H 2 in the blue box to C in the light red box further bonded to S to the right, and from the bond between C in the light red box and S to the right to the same S. An arrow labeled condensation points down accompanied by a curved arrow showing the loss of C O 2 in a green box. This yields beta-ketoacyl-A C P, in which only yellow K R at the upper right is highlighted, K S is bonded to S H to the left, and the brown oval labeled A C P is bent across the front of K R with its zigzag chain bonded to S just to the right of K S and further bonded to C in a blue box double bonded to O above and bonded to C H 2 to the right in the same blue box further bonded to C in a light red box double bonded to O below and bonded to C H 2 to the right in the same light red box and further bonded to C H 2 in a yellow rectangle further bonded to C H 3 in the same yellow rectangle.

Seven cycles of condensation and reduction produce the 16-carbon saturated palmitoyl group, still bound to ACP. For reasons not well understood, chain elongation by the synthase complex generally stops at this point, and free palmitate is released from the ACP by a hydrolytic activity (thioesterase; TE) in the multifunctional protein.

We can consider the overall reaction for the synthesis of palmitate from acetyl-CoA in two parts. First, the formation of seven malonyl-CoA molecules:

7 Acetyl-CoA + 7 {CO}_{2} + 7 ATP \to 7 malonyl-CoA + 7 ADP + 7 P_{i}

$7 Acetyl hyphen CoA plus 7 CO Subscript 2 Baseline plus 7 ATP right-arrow 7 malonyl hyphen CoA plus 7 ADP plus 7 upper P Subscript i Baseline$

(21-1)

then seven cycles of condensation and reduction:

\begin{matrix} Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H^{+} \to \\ palmitate + 7 {CO}_{2} + 8 CoA + 14 {NADP}^{+} + 6 H_{2} O \end{matrix}

$StartLayout 1st Row Acetyl hyphen CoA plus 7 malonyl hyphen CoA plus 14 NADPH plus 14 upper H Superscript plus Baseline right-arrow 2nd Row palmitate plus 7 CO Subscript 2 Baseline plus 8 CoA plus 14 NADP Superscript plus Baseline plus 6 upper H Subscript 2 Baseline upper O EndLayout$

(21-2)

Notice that only six net water molecules are produced, because one is used to hydrolyze the thioester linking the palmitate product to the enzyme. The overall process (the sum of Eqns 21-1 and 21-2) is

\begin{matrix} 8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H^{+} \to \\ palmitate + 8 CoA + 7 ADP + 7 P_{i} + 14 {NADP}^{+} + 6 H_{2} O \end{matrix}

$StartLayout 1st Row 8 Acetyl hyphen CoA plus 7 ATP plus 14 NADPH plus 14 upper H Superscript plus Baseline right-arrow 2nd Row palmitate plus 8 CoA plus 7 ADP plus 7 upper P Subscript i Baseline plus 14 NADP Superscript plus Baseline plus 6 upper H Subscript 2 Baseline upper O EndLayout$

(21-3)

The biosynthesis of fatty acids such as palmitate thus requires acetyl-CoA and the input of chemical energy in two forms: the group transfer potential of ATP and the reducing power of NADPH. The ATP is required to attach ${CO}_{2}$ $CO Subscript 2$ to acetyl-CoA to make malonyl-CoA; the NADPH molecules are required to reduce the β-keto group and the double bond.

In nonphotosynthetic eukaryotes there is an additional cost to fatty acid synthesis, because acetyl-CoA is generated in the mitochondria and must be transported to the cytosol. As we will see, this extra step consumes two ATP per molecule of acetyl-CoA transported, increasing the energetic cost of fatty acid synthesis to three ATP per two-carbon unit.

Fatty Acid Synthesis Is a Cytosolic Process in Most Eukaryotes but Takes Place in the Chloroplasts in Plants

In most eukaryotes, the fatty acid synthase complex (FAS I) is found in the cytosol, as are the biosynthetic enzymes for nucleotides, amino acids, and glucose. This location segregates synthetic processes from degradative reactions, many of which take place in the mitochondrial matrix. There is a corresponding segregation of the electron-carrying cofactors used in anabolism (generally a reductive process) and those used in catabolism (generally oxidative).

Usually, NADPH is the electron carrier for anabolic reactions, and ${NAD}^{+}$ $NAD Superscript plus$ serves in catabolic reactions. The liver, the largest mammalian internal organ and a key metabolic center responding to large changes during feasting and fasting, provides a useful focus for this discussion. In hepatocytes, the $[NADPH] / [{NADP}^{+}]$ $left-bracket NADPH right-bracket slash left-bracket NADP Superscript plus Baseline right-bracket$ ratio is very high (~75) in the cytosol, furnishing a strongly reducing environment for the reductive synthesis of fatty acids and other biomolecules. The cytosolic $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ ratio is much lower $(\sim 8 \times 10^{- 4})$ $left-parenthesis tilde 8 times 10 Superscript negative 4 Baseline right-parenthesis$ , so the ${NAD}^{+}$ $NAD Superscript plus$ -dependent oxidative catabolism of glucose can take place in the same compartment, and at the same time, as fatty acid synthesis. The $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ ratio in the mitochondrion is much higher than that in the cytosol, because of the flow of electrons to ${NAD}^{+}$ $NAD Superscript plus$ from the oxidation of fatty acids, amino acids, pyruvate, and acetyl-CoA. This high mitochondrial $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ ratio favors the reduction of oxygen via the respiratory chain.

In hepatocytes and adipocytes, cytosolic NADPH is largely generated by the pentose phosphate pathway (Fig. 21-8a; also see Fig. 14-30) and by malic enzyme (Fig. 21-8b). The pyruvate produced by the action of malic enzyme reenters the mitochondrion.

A two-part figure shows the production of N A D P H by showing the pentose pathway in part a and a reaction catalyzed by malic enzyme in part b. — FIGURE 21-8 Production of NADPH. Two routes to NADPH, catalyzed by (a) the pentose phosphate pathway and (b) malic enzyme.

Part a shows glucose 6-phosphate on the left with a series of three arrows pointing to the right. This series of arrows is labeled pentose phosphate pathway. The first arrow is joined with a curved arrow showing the addition of N A D Plus and loss of an orange oval labeled N A D P H plus H plus. The third arrow is joined with a curved arrow showing the addition of N A D Plus and loss of an orange oval labeled N A D P H plus H plus. This yields ribulose 5-phosphate. Part b shows malate on the left as a four carbon vertical chain with C 1 forming C O O minus, C 2 bonded to H and O H, C 3 bonded to 2 H, and C 4 forming C O O minus in a light red box. Right- and left-pointing arrows above the words malic enzyme indicate a reversible reaction. A curved arrow shows the addition of N A D P plus and loss of an orange oval labeled N A D P H plus H plus. This yields pyruvate plus C O 2 in a red box. Pyruvate is a three-carbon vertical chain with C 1 forming C O O minus, C 2 double bonded to O to the right, and C 3 bonded to 3 H.

In the photosynthetic cells of plants, fatty acid synthesis occurs not in the cytosol but in the chloroplast stroma (Fig. 21-9). This makes sense, given that NADPH is produced in chloroplasts by the light-dependent reactions of photosynthesis:

H 2 O plus N A D P plus are shown with a horizontal arrow pointing to the right. A wavy arrow points from light to this horizontal arrow. The reaction yields one-half O 2 plus N A D P H plus H plus.

A figure shows structures involved in different types of lipid metabolism in animal and yeast cells and in plant cells. — FIGURE 21-9 Subcellular localization of lipid metabolism. Yeast and animal cells differ from higher plant cells in the compartmentation of lipid metabolism. Fatty acid synthesis takes place in the compartment in which NADPH is available for reductive synthesis (i.e., where the $[NADPH] / [{NADP}^{+}]$ $left-bracket NADPH right-bracket slash left-bracket NADP Superscript plus Baseline right-bracket$ ratio is high); this is the cytosol in animals and yeast, and the chloroplast in plants. Processes in red type are addressed in this chapter.

FIGURE 21-9 Subcellular localization of lipid metabolism. Yeast and animal cells differ from higher plant cells in the compartmentation of lipid metabolism. Fatty acid synthesis takes place in the compartment in which NADPH is available for reductive synthesis (i.e., where the $[NADPH] / [{NADP}^{+}]$ $left-bracket NADPH right-bracket slash left-bracket NADP Superscript plus Baseline right-bracket$ ratio is high); this is the cytosol in animals and yeast, and the chloroplast in plants. Processes in red type are addressed in this chapter.

The left-hand cell is labeled animal cells, yeast cells. The right-hand cell is labeled plant cell. Both cells are shown with an outer plasma membrane, beige cytosol surrounding the organelles, and gray structures representing organelles such as the Golgi complex. A nucleus is visible in each cell and a large vacuole is visible in the plant cell. Several orange mitochondria are shown. Beneath the word mitochondrion, a line pointing to a mitochondrion in the plant cell indicates that no fatty acid oxidation occurs there. A bracket pointing to a mitochondrion in the animal or yeast cell indicates that fatty acid oxidation, ketone body synthesis, and red highlighted fatty acid elongation occur there. Acetyl Co A production occurs in mitochondria in both types of cells. Endoplasmic reticulum is shown as curved membranes around the nucleus in each cell. Red highlighted text below the term endoplasmic reticulum indicates that phospholipid synthesis, sterol synthesis (late stages), fatty acid elongation, and fatty acid desaturation occur in both types of cells. A line points from the word cytosol to cytosol in the animal cell, between the organelles, and text below lists red highlighted N A D P H production (pentose phosphate pathway; malic enzyme), nonhighlighted [N A D P H]/[N A D P plus] high, highlighted isoprenoid and sterol synthesis (early stages), and fatty acid synthesis. Green chloroplasts in the plant cell are indicated by a line and text below reads, red highlighted N A D P H, A T P production; nonhighlighted [N A D P H]/[N A D P plus] high; and red highlighted fatty acid synthesis. A line points from the word peroxisomes to a small, round peroxisome in the plant cell and nonhighlighted text below reads, fatty acid oxidation (producing H 2 O 2); catalase, peroxidase: H 2 O 2 yields H 2 O.

Acetate Is Shuttled out of Mitochondria as Citrate

In nonphotosynthetic eukaryotes, nearly all the acetyl-CoA used in fatty acid synthesis is formed in mitochondria from pyruvate oxidation and from catabolism of the carbon skeletons of amino acids. Acetyl-CoA arising from the oxidation of fatty acids is not a significant source of acetyl-CoA for fatty acid biosynthesis in animals, because the two pathways are reciprocally regulated, as described below.

The inner mitochondrial membrane is impermeable to acetyl-CoA, so an indirect shuttle transfers acetyl group equivalents across the membrane (Fig. 21-10). Intramitochondrial acetyl-CoA first reacts with oxaloacetate to form citrate, in the citric acid cycle reaction catalyzed by citrate synthase (see Fig. 16-7). Citrate then passes through the inner membrane on the citrate transporter. In the cytosol, citrate cleavage by citrate lyase regenerates acetyl-CoA and oxaloacetate in an ATP-dependent reaction. Oxaloacetate cannot return to the mitochondrial matrix directly, as there is no oxaloacetate transporter. Instead, cytosolic malate dehydrogenase reduces the oxaloacetate to malate, which can return to the mitochondrial matrix on the malate–α-ketoglutarate transporter, in exchange for citrate. In the matrix, malate is reoxidized to oxaloacetate to complete the shuttle. However, most of the malate produced in the cytosol is used to generate cytosolic NADPH through the activity of malic enzyme (Fig. 21-8b). The pyruvate produced is transported into the mitochondria by the pyruvate transporter (Fig. 21-10), then converted back into oxaloacetate by pyruvate carboxylase in the matrix. In the resulting cycle, two ATP molecules are consumed (by citrate lyase and pyruvate carboxylase) for every molecule of acetyl-CoA delivered to fatty acid synthesis.

A figure shows a shuttle for the transfer of acetyl groups from mitochondria to the cytosol. — FIGURE 21-10 Shuttle for transfer of acetyl groups from mitochondria to the cytosol. The outer mitochondrial membrane is freely permeable to all these compounds. Pyruvate derived from amino acid catabolism in the mitochondrial matrix, or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the matrix. Acetyl groups pass out of the mitochondrion as citrate; in the cytosol they are delivered as acetyl-CoA for fatty acid synthesis. Oxaloacetate is reduced to malate, which can be returned to the mitochondrial matrix. The steps converting malate to oxaloacetate and oxaloacetate plus acetyl-CoA to citrate (indicated by blue arrows) are part of the citric acid cycle. The major fate of cytosolic malate, however, is oxidation by malic enzyme to generate cytosolic NADPH; the pyruvate produced returns to the mitochondrial matrix.

A cycle of reactions are shown with two vertical membranes across the center. The left-hand membrane is labeled inner membrane and contains three channels, one near the top and two near the bottom. The right-hand membrane is labeled outer membrane and is faded. It has three openings adjacent to the channels in the membrane to its left. The area to the left of the membranes is labeled mitochondrial matrix and the area to the right of the membranes is labeled cytosol. An arrow points from citrate at the eleven o’clock position through the top channel in the inner membrane, labeled citrate transporter, and through the opening in the outer membrane to citrate at the one o’clock position in the cytosol. An arrow labeled citrate lyase points from citrate to oxaloacetate at just above the three o’clock position. An outer curved arrow shows the addition of Co A-S H and the loss of a yellow box labeled acetyl Co A from which an arrow points to a yellow box labeled fatty acid synthesis and an inner curved arrow shows the addition of an orange oval labeled A T P and the loss of A D P plus P subscript i end subscript. A short arrow labeled malate dehydrogenase points from oxaloacetate to malate at just below the three o’clock position and is accompanied by a curved arrow showing the addition of an orange oval labeled N A D H plus H plus and the loss of N A D plus. Two arrows point away from malate. The first arrow is a dashed arrow that extends from malate through the middle opening in the outer membrane and then through the malate alpha-ketoglutarate transporter in the inner mitochondrial membrane to end at malate at just below the nine o’clock position in the matrix. A blue arrow labeled malate dehydrogenase points from malate to oxaloacetate at just past the nine o’clock position and is accompanied by a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H plus H plus. The second arrow from malate in the cytosol is labeled malic enzyme and points to pyruvate at the five o’clock position. It is accompanied by a curved arrow showing the addition of N A D P plus and loss of an orange oval labeled N A D P H plus H plus, followed by a branching arrow showing the loss of C O 2. An arrow points from pyruvate through the bottom opening in the outer membrane and the pyruvate transporter in the inner membrane to pyruvate at the seven o’clock position in the matrix. An arrow labeled pyruvate carboxylase points from pyruvate to oxaloacetate at just past the nine o’clock position and is accompanied by a curved line showing the addition of C O 2 and a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript. A blue arrow labeled citrate synthase points from oxaloacetate to citrate at the eleven o’clock position and is accompanied by a curved arrow to show the addition of a yellow box labeled acetyl-Co A (multiple sources) and loss of Co A-S H. All data are approximate.

Malate thus has two fates in metabolism. In the mitochondrial matrix, malate is part of the citric acid cycle. In the cytosol, malate degradation becomes a significant source of NADPH. After citrate cleavage to generate acetyl-CoA, conversion of the four remaining carbons to pyruvate and ${CO}_{2}$ $CO Subscript 2$ by malic enzyme generates about half the NADPH required for fatty acid synthesis. The pentose phosphate pathway contributes the rest of the needed NADPH.

Fatty Acid Biosynthesis Is Tightly Regulated

When a cell or an organism has more than enough metabolic fuel to meet its energy needs, the excess is generally converted to fatty acids and stored as lipids such as triacylglycerols. The reaction catalyzed by acetyl-CoA carboxylase is the rate-limiting step in the biosynthesis of fatty acids, and this enzyme is an important site of regulation. In vertebrates, palmitoyl-CoA, the principal product of fatty acid synthesis, is a feedback inhibitor of the enzyme; citrate is an allosteric activator (Fig. 21-11a), increasing $V_{max}$ $upper V Subscript max$ . Citrate plays a central role in diverting cellular metabolism from the consumption (oxidation) of metabolic fuel to the storage of fuel as fatty acids. When the concentrations of mitochondrial acetyl-CoA and ATP increase, citrate is transported out of mitochondria; it then becomes both the precursor of cytosolic acetyl-CoA and an allosteric signal for the activation of acetyl-CoA carboxylase. At the same time, citrate inhibits the activity of phosphofructokinase-1 (see Fig. 14-23), reducing the flow of carbon through glycolysis.

A two-part figure shows the regulation of fatty acid synthesis by showing where regulation occurs during a series of reactions from citrate to palmitoyl-Co A in part a and showing a micrograph of filaments of acetyl-Co A carboxylase in part b. — FIGURE 21-11 Regulation of fatty acid synthesis. (a) In the cells of vertebrates, both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA. In plants, acetyl-CoA carboxylase is activated by the changes in $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ and pH that accompany illumination (not shown here). (b) Filaments of acetyl-CoA carboxylase from chicken hepatocytes (the active, dephosphorylated form), as seen with the electron microscope.

FIGURE 21-11 Regulation of fatty acid synthesis. (a) In the cells of vertebrates, both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA. In plants, acetyl-CoA carboxylase is activated by the changes in $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ and pH that accompany illumination (not shown here). (b) Filaments of acetyl-CoA carboxylase from chicken hepatocytes (the active, dephosphorylated form), as seen with the electron microscope.

In part a, an arrow labeled citrate lyase points down from a yellow box labeled citrate to a yellow box labeled acetyl-Co A. An arrow labeled acetyl-Co A carboxylase points down from the yellow box labeled acetyl-Co A to a yellow box labeled malonyl-Co A. A dashed arrow points from citrate to a green triangle enclosed in a green circle next to the top of the arrow labeled acetyl-Co A carboxylase, just below acetyl-Co A. A series of two arrows points down from the yellow box labeled malonyl-Co A to a yellow box labeled palmitoyl-Co A. A dashed arrow points from palmitoyl-Co A to a red “X” in a red circle near the bottom of the arrow labeled acetyl-Co A carboxylase, just above malonyl-Co A. A red “X” in a red circle is indicated by a dashed arrow from text to the right reading glucagon, epinephrine, high trigger phosphorylation/inactivation. Part b shows a long vertical filament containing white subunits shown against a grainy background with a few darker circles.

Acetyl-CoA carboxylase is also regulated by covalent modification. Phosphorylation, triggered by the hormones glucagon and epinephrine or by high [AMP], inactivates the enzyme and reduces its sensitivity to activation by citrate, thereby slowing fatty acid synthesis. Phosphorylation occurs on at least three Ser residues and is catalyzed primarily by the AMP-activated protein kinase (AMPK). In its active (dephosphorylated) form, acetyl-CoA carboxylase polymerizes into long filaments (Fig. 21-11b); phosphorylation is accompanied by dissociation into monomeric subunits and loss of activity.

The acetyl-CoA carboxylase of plants and bacteria is not regulated by citrate or by a phosphorylation-dephosphorylation cycle. Instead, the plant enzyme is activated by an increase in stromal pH and $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ , which occurs on illumination of the plant (see Fig. 20-35). Bacteria do not use triacylglycerols as energy stores. In Escherichia coli, the primary role of fatty acid synthesis is to provide precursors for membrane lipids; the regulation of this process is complex, employing guanine nucleotides (such as ppGpp; see Fig. 8-42) that coordinate cell growth with membrane formation.

In addition to the moment-by-moment regulation of enzymatic activity, these pathways are regulated at the level of gene expression. For example, when animals ingest an excess of certain polyunsaturated fatty acids, the expression of genes encoding many lipogenic enzymes in the liver is suppressed. This gene regulation is mediated by a family of nuclear receptor proteins called PPARs, which we encountered in Section 17.2.

If fatty acid synthesis and β oxidation were to proceed simultaneously, the two processes would constitute a futile cycle, wasting energy. We noted earlier (see Fig. 17-13) that β oxidation is blocked by malonyl-CoA, which inhibits carnitine acyltransferase I. Thus, during fatty acid synthesis, production of the first intermediate, malonyl-CoA, shuts down β oxidation at the level of a transport system in the inner mitochondrial membrane. This control mechanism illustrates another advantage of segregating synthetic and degradative pathways in different cellular compartments.

Long-Chain Saturated Fatty Acids Are Synthesized from Palmitate

Palmitate, the principal product of the fatty acid synthase system in animal cells, is the precursor of other long-chain fatty acids (Fig. 21-12). It may be lengthened to form stearate (18:0) or even longer saturated fatty acids by further additions of acetyl groups, through the action of fatty acid elongation systems present in the smooth endoplasmic reticulum (smooth ER) and in mitochondria. The more active elongation system of the ER extends the 16-carbon chain of palmitoyl-CoA by two carbons, forming stearoyl-CoA. Although different enzyme systems are used, and coenzyme A rather than ACP is the acyl carrier in the reaction, the mechanism of elongation in the ER is otherwise identical to that in palmitate synthesis: donation of two carbons by malonyl-CoA, followed by reduction, dehydration, and reduction to the saturated 18-carbon product, stearoyl-CoA.

A figure shows routes of synthesis of unsaturated fatty acids and their derivatives. — FIGURE 21-12 Routes of synthesis of unsaturated fatty acids and their derivatives. Palmitate is the precursor of stearate and longer-chain saturated fatty acids, as well as the monounsaturated acids palmitoleate and oleate. Mammals cannot convert oleate to linoleate or α-linolenate (shaded), which are therefore required in the diet as essential fatty acids. Conversion of linoleate to other polyunsaturated fatty acids and eicosanoids is outlined. Unsaturated fatty acids are symbolized by indicating the number of carbons and the number and position of double bonds, as in Table 10-1. Linoleate and α-linolenate are important omega-6 and omega-3 fatty acids, respectively; they are also precursors for a wide range of unsaturated fatty acids that act as signaling molecules. Two-letter abbreviations specify the eicosanoid prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT). Particular classes of unsaturated fatty acids are further delineated by the number of double bonds, which defines subclasses referred to as series. For example, series 2 TX are thromboxanes with two double bonds in the hydrocarbon chain.

Palmitate, 16:0 is shown at the very top with two arrows pointing down. An arrow labeled desaturation points to palmitoleate, 16:1 (delta superscript 9 end superscript) to the lower right. A second arrow labeled elongation points down to stearate, 18.0. Two arrows point from stearate. An arrow labeled elongation points to longer saturated fatty acids to the lower right. An arrow labeled desaturation points down to oleate, 18:1 (delta superscript 9 end superscript) below. A series of three arrows point down from oleate to a light red box labeled linoleate, 18:2 (delta superscript 9, 12 end superscript). Text accompanying the series of three arrows reads, desaturation (in plants only). Linoleate is at the top of a lower right-hand box labeled Omega 6. Arrows point from linoleate to the lower right within the omega 6 box and to the lower left. An arrow labeled desaturation points from linoleate to gamma-linolenate, 18:3 (delta superscript 6, 9, 12 end superscript), from which an arrow labeled elongation points to eicosatrienoate, 20:3 (delta superscript 8, 11, 14 end superscript). From eicosatrienoate, an arrow labeled desaturation points down to a light red box labeled arachidonate, 20:4 (delta superscript 5, 8, 11, 14 end superscript). Four arrows point down from this light red box. From left to right, these arrows point to series 2 T X, series 2 P G, series 4 L T, and lipotoxins. The second arrow from linoleate is the first in a series of three arrows labeled desaturation (in plants only) that point to a light red box labeled alpha-linolenate, 18:3 (delta superscript 9, 12, 15 end superscript) in the right-hand box labeled omega 3. Within the omega 3 box, a series of three arrows point down from alpha-linolenate to eicosapentaenoic acid, (E P A; 20:5 (delta superscript 5, 8, 11, 14, 17) ). Five arrows point down from eicosapentaenoic acid. From left to right, these point to series 3 T X, series 3 P G, a light red box labeled docosahexaenoic acid (D H A; 22:6 (delta superscript 4, 7, 10, 13, 16, 19) ), series 5 L T, and resolvins. From the light red box labeled docosahexaenoic acid, three arrows point down. From left to right, these point to resolvins, protectins, and maresins.

Two key products of elongation pathways are linoleate, an omega-6 fatty acid (see Chapter 10 for the alternative nomenclature), and α-linolenate, an omega-3 fatty acid. These are precursors for two extensive families of derivative unsaturated fatty acids, the omega-6 and omega-3 families. Humans cannot synthesize linoleate and α-linolenate and must obtain them in the diet. The ratio of omega-6 to omega-3 fatty acids in the diet, if too high, can lead to cardiovascular disease. The importance of this ratio may reflect the multitude of signaling molecules in the omega-6 and omega-3 families (Fig. 21-12), with their equally complex physiological effects. Several of these derivative unsaturated fatty acids are considered below.

Desaturation of Fatty Acids Requires a Mixed-Function Oxidase

Palmitate and stearate serve as precursors of the two most common monounsaturated fatty acids of animal tissues: palmitoleate, $16 : 1 (Δ^{9})$ $16 colon 1 left-parenthesis upper Delta Superscript 9 Baseline right-parenthesis$ , and oleate, $18 : 1 (Δ^{9})$ $18 colon 1 left-parenthesis upper Delta Superscript 9 Baseline right-parenthesis$ ; both of these fatty acids have a single cis double bond between C-9 and C-10 (see Table 10-1). The double bond is introduced into the fatty acid chain by an oxidative reaction catalyzed by fatty acyl–CoA desaturase (Fig. 21-13), a mixed-function oxidase (Box 21-1). Two different substrates, the fatty acid and NADPH, simultaneously undergo two-electron oxidations. The path of electron flow includes a cytochrome (cytochrome $b_{5}$ $b 5$ ) and a flavoprotein (cytochrome $b_{5}$ $b 5$ reductase), both of which, like fatty acyl–CoA desaturase, are in the smooth ER. In plants, oleate is produced by a stearoyl-ACP desaturase (SCD) that uses reduced ferredoxin as the electron donor in the chloroplast stroma.

A figure shows electron transfer in the desaturation of fatty acids in vertebrates. — FIGURE 21-13 Electron transfer in the desaturation of fatty acids in vertebrates. Blue arrows show the path of electrons as two substrates — a fatty acyl–CoA and NADPH — undergo oxidation by molecular oxygen. These reactions take place on the lumenal face of the smooth ER. A similar pathway, but with different electron carriers, occurs in plants.

At the upper left, O 2 in a blue square is added to 2 H plus plus C H 3 bonded to (C H 2) italicized subscript n end italics end subscript bonded to C H 2 in a light red box bonded to C H 2 in the same light red bond bonded to (C H 2) italicized subscript m end italics end subscript bonded to C double bonded to O to the upper right and bonded to S to the lower right further bonded to Co A. Text below this hydrocarbon reads, saturated fatty acyl-Co A. A blue arrow labeled fatty acyl-Co A desaturated curves from this top molecule to yield products shown at the lower left: 2 H 3 O in a blue box plus C H 3 bonded to (C H 2) subscript italicized n end italics end subscript bonded to C H in a light red box double bonded to C H in the same light red box bonded to (C H 2) subscript italicized m end italics end subscript bonded to C double bonded to O to the upper right and bonded to S to the lower right further bonded to Co A. Text below this hydrocarbon reads, monounsaturated acyl-Co A. A curved downward arrow from 2 C y t italicized b end italics subscript 5 end subscript (F e 2 plus) meets the blue arrow labeled fatty acyl-Co A desaturase to the left then turns gray before reacting 2 C y t italicized b end italics subscript 5 end subscript (F e 3 plus) below. An arrow curves back up from 2 C y t italicized b end italics subscript 5 end subscript (F e 3 plus) to 2 C y t italicized b end italics subscript 5 end subscript (F e 2 plus) and turns blue as it meets a curved upward arrow on the right from C y t 2 b end italics subscript 5 end subscript reductase (F A D H 2) below to C y t italicized b end italics subscript 5 end subscript (F A D) above. This curved arrow is blue at the bottom then turns gray as it meets the curved arrow to its left. A curved arrow points down from C y t italicized b end italics subscript 5 end subscript (F A D) to C y t italicized b end italics subscript 5 end subscript (F A D H 2) below and turns blue as it meets a downward curved arrow to its right. This right-hand arrow begins at a red oval labeled N A D P H plus H plus and is blue at first, then meets the arrow to its left and turns gray as it reaches N A D P plus.

Box 21-1 MEDICINE

Oxidases, Oxygenases, Cytochrome P-450 Enzymes, and Drug Overdoses

In this chapter we encounter several enzymes that carry out oxidation-reduction reactions in which molecular oxygen is a participant. The stearoyl-CoA desaturase (SCD) that introduces a double bond into a fatty acyl chain (see Fig. 21-13) is one such enzyme.

The nomenclature for enzymes that catalyze reactions of this general type can be confusing. Oxidase is the general name for enzymes that catalyze oxidations in which molecular oxygen is the electron acceptor but oxygen atoms do not appear in the oxidized product. The enzyme that creates a double bond in fatty acyl–CoA during the oxidation of fatty acids in peroxisomes (see Fig. 17-14) is an oxidase of this type; a second example is the cytochrome oxidase of the mitochondrial respiratory chain (see Fig. 19-13). In the first case, the transfer of two electrons to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ produces hydrogen peroxide, $H_{2} O_{2}$ $upper H Subscript 2 Baseline upper O Subscript 2$ ; in the second, two electrons reduce $\frac{1}{2} O_{2}$ $one-half upper O Subscript 2$ to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . Many, but not all, oxidases are flavoproteins. Mixed-function oxidases oxidize two different substrates simultaneously; again, the molecular oxygen atoms do not appear in the oxidized products. Mixed-function oxidases act in fatty acid desaturation (fatty acyl–CoA desaturase; see Fig. 21-13) and in the last step of plasmalogen synthesis (see Fig. 21-30).

Oxygenases catalyze oxidative reactions in which oxygen atoms are directly incorporated into the product molecule, forming a new hydroxyl or carboxyl group, for example. Dioxygenases catalyze reactions in which both oxygen atoms of $O_{2}$ $upper O Subscript 2$ are incorporated into the organic product. An example of a dioxygenase is tryptophan 2,3-dioxygenase, which catalyzes the opening of the five-membered ring of tryptophan in the catabolism of this amino acid. When the reaction takes place in the presence of $^{18} O_{2}$ $Superscript 18 Baseline zero width space upper O Subscript 2 Baseline$ , the isotopic oxygen atoms are found in the two carbonyl groups of the product (shown in red):

Tryptophan is a benzene ring that shares its right side with a five-membered ring with N H substituted for C at its bottom vertex, a double bond at its upper right vertex, and an upper right vertex bonded to C H 2 bonded to C H bonded to N H 3 above with a positive charge on N and to C O O minus to the right. An arrow labeled tryptophan 2,3-dioxygenase points downward and is joined by a curved arrow showing the addition of red highlighted O 2. This yields italicized N end italics-formylkynurenine, which is a benzene ring with its upper right vertex bonded to C double bonded to red highlighted O above and bonded to C H 2 to the right bonded to C H bonded to N H 3 above with a positive charge on N and to C O O minus and with its lower right vertex bonded to N H bonded to C bonded to H to the upper right and double bonded to red highlighted O to the lower right.

Monooxygenases, more common and more complex in their action, catalyze reactions in which only one of the two oxygen atoms of $O_{2}$ $upper O Subscript 2$ is incorporated into the organic product, the other being reduced to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ ; an example is squalene monooxygenase (see Fig. 21-37). Monooxygenases require two substrates to serve as reductants of the two oxygen atoms of $O_{2}$ $upper O Subscript 2$ . The main substrate accepts one of the two oxygen atoms, and a cosubstrate furnishes hydrogen atoms to reduce the other oxygen atom to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . The general reaction equation for monooxygenases is

A H + B H_{2} + O — O \to A — OH + B + H_{2} O

$upper A upper H plus upper B upper H Subscript 2 Baseline plus upper O em-dash upper O right-arrow upper A em-dash OH plus upper B plus upper H Subscript 2 Baseline upper O$

where AH is the main substrate and ${BH}_{2}$ $BH Subscript 2$ is the cosubstrate. Because most monooxygenases catalyze reactions in which the main substrate becomes hydroxylated, they are also called hydroxylases. They are also sometimes called mixed-function oxygenases to indicate that they oxidize two different substrates simultaneously.

Monooxygenases are divided into several classes, depending on the nature of the cosubstrate. Some use reduced flavin nucleotides ( ${FMNH}_{2}$ $FMNH Subscript 2$ or ${FADH}_{2}$ $FADH Subscript 2$ ), others use NADH or NADPH, and still others use α-ketoglutarate as cosubstrate. The enzyme that hydroxylates the phenyl ring of phenylalanine to form tyrosine is a monooxygenase that uses tetrahydrobiopterin as cosubstrate (see Fig. 18-23). (This is the enzyme that is defective in the human genetic disease phenylketonuria.)

The most numerous and most complex monooxygenation reactions are those employing a type of heme protein called cytochrome P-450. Like mitochondrial cytochrome oxidase, enzymes containing a cytochrome P-450 domain can react with $O_{2}$ $upper O Subscript 2$ and bind carbon monoxide, but they can be differentiated from cytochrome oxidase because the carbon monoxide complex of their reduced form absorbs light strongly at 450 nm — thus the name P-450.

Cytochrome P-450 enzymes catalyze hydroxylation reactions in which an organic substrate, RH, is hydroxylated to $R — OH$ $upper R em-dash OH$ , incorporating one oxygen atom of $O_{2}$ $upper O Subscript 2$ ; the other oxygen atom is reduced to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ by reducing equivalents that are furnished by NADH or NADPH but are usually passed to cytochrome P-450 by an iron-sulfur protein. Figure 1 shows a simplified outline of the action of cytochrome P-450.

FIGURE 1 Simplified cytochrome P-450 reaction cycle.

A figure shows two reaction cycles in the center. The left-hand cycle shows blue highlighted cytochrome P-450 reductase (F e – S) in the center with oxidized at the top and reduced at the bottom. The right-hand cycle shows cytochrome P-450 in its center with reduced at the top and oxidized at the bottom. At the upper left, an orange oval labeled N A D P H has a blue curved arrow down to N A D P plus below that becomes gray as it meets a downward arrow from the cycle to its right. The right-hand arrow forms the left side of the first cycle. It begins from oxidized above and curves down to become blue where it meets the left-hand arrow before reaching the word reduced below. A blue arrow curves up from reduced and becomes gray as it meets an arrow to the right before reaching the word oxidized on top. This right-hand upward arrow begins at oxidized at the bottom of the second cycle and becomes blue as it meets the upward arrow of the cycle to its left. To the right, a blue arrow curves down from reduced to oxidized and turns gray as it meets two curved arrows to the right. The outer curved right-hand arrow begins at R H and turns blue as it meets the downward arrow to its left before ending at R red highlighted O H. The inner curved right-hand arrow begins at red highlighted O 2 and turns red as it meets the downward arrow to its left before ending at R red highlighted O H.

One large family of P-450–containing proteins consists of two general types: those highly specific for a single substrate (like typical enzymes) and those with more promiscuous binding sites that accept a variety of substrates, generally similar in being hydrophobic. In the adrenal cortex, for example, a specific cytochrome P-450 participates in the hydroxylation of steroids to yield the adrenocortical hormones (see Fig. 21-49). There are dozens of P-450 enzymes that act on specific substrates in the biosynthetic pathways to steroid hormones and eicosanoids (Fig. 2). Cytochrome P-450 enzymes with broader specificity are important in the hydroxylation of many different drugs, such as barbiturates and other xenobiotics (substances foreign to the organism), particularly if they are hydrophobic and relatively insoluble. The environmental carcinogen benzo[a]pyrene, found in cigarette smoke, undergoes cytochrome P-450–dependent hydroxylation during detoxification. Hydroxylation of xenobiotics, sometimes combined with the attachment of a polar compound such as glucuronic acid to the hydroxyl group, makes them more soluble in water and allows their excretion in urine. Hydroxylation (and glucuronidation) inactivates most drugs, and the rate at which it occurs can determine how long a given dose of a medication remains in the blood at therapeutic levels.

FIGURE 2 Pathways of sterol biosynthesis, showing the steps requiring cytochrome P-450 enzymes.

A box labeled acetyl-Co A at the upper left has an arrow labeled P-450 that points down to a box labeled 7-dehydroxycholesterol. Arrows point to the right and down from 7-dehydroxycholesterol. A series of five arrows point to the right from 7-dehydroxycholesterol. The first two are short, the third has three P-450 listed vertically above it, and the fourth and fifth each have one P-450 listed above. This yields a box labeled vitamin D subscript 3 degradation products. Two arrows point down from 7-dehydroxycholesterol to a box labeled cholesterol. A left-hand series of two arrows, each labeled P-450, point down from cholesterol to 7 alpha, 27-dihydroxycholesterol at the left side of a long box. A central arrow labeled P-450 points down from cholesterol to 7 alpha-hydroxycholesterol in the center of the long box. A right-hand series of two arrows point down from cholesterol to 7 alpha, 24-dihydroxycholesterol at the right side of the long box. Lines from each of the three molecules in this long box come together to form an arrow labeled P-450 that points to a second arrow labeled P-450 that points to a box labeled primary bile acids. An arrow labeled P-450 points right from the box labeled cholesterol to a box labeled pregnenolone. Two parallel series of four arrows each point right from pregnenolone to a box labeled testosterone (androgen). In the top series, the first two arrows are labeled P-450. In the bottom series, the middle two arrows are labeled P-450. An arrow labeled P-450 points down from the space between the third and four arrows in the bottom series to a box labeled estradiol (estrogen). An arrow labeled P-450 also points from the box labeled testosterone (androgen) to the box labeled estradiol (estrogen). A series of four vertical arrows all labeled P-450 point down from the space between the first and second arrows in the bottom horizontal series from pregnenolone to testosterone (androgen) to a box labeled aldosterone (mineralocorticoid). A series of two vertical arrows each labeled P-450 point down from the space between the third and fourth arrows of the bottom horizontal series from pregnenolone to testosterone (androgen) to a box labeled cortisol (glucocorticoid).

Humans differ in their levels of drug-metabolizing enzymes, both because of their genetics and because past exposure to substrates can induce the synthesis of higher levels of P-450 enzymes. Ethanol and barbiturate drugs share a P-450 enzyme. Long-term heavy drinking induces synthesis of this enzyme. Then, because the barbiturate is inactivated and cleared faster, larger doses are required to get the same therapeutic effect. If an individual takes this larger-than-usual dose of barbiturate and then also drinks alcohol, competition between the alcohol and the barbiturate for the limited amount of enzyme means that both alcohol and barbiturate are cleared more slowly. The resulting high levels of these two central nervous system depressants can be lethal. Similar complications arise when an individual takes two drugs that happen to be inactivated by the same P-450 enzyme; each drug increases the effective dose of the other by slowing its inactivation. It is therefore essential for physicians and pharmacists to know about all of a patient’s prescribed and over-the-counter drugs and supplements, as well as a history of heavy drinking, or smoking, or exposure to environmental toxins.

The SCD of animals (as studied in mice) has an important role in the development of obesity and the insulin resistance that often accompanies obesity and precedes development of type 2 diabetes mellitus. Mice have four isozymes, SCD1 through SCD4, of which SCD1 is the best understood. Its synthesis is induced by dietary saturated fatty acids, and also by the action of SREBP and LXR, two protein regulators of lipid metabolism that activate transcription of lipid-synthesizing enzymes (described in Section 21.4). Mice with mutant forms of SCD1 are resistant to diet-induced obesity and do not develop diabetes under conditions that cause both obesity and diabetes in mice with normal SCD1.

Mammalian hepatocytes can readily introduce double bonds at the $Δ^{9}$ $upper Delta Superscript 9$ position of fatty acids but cannot introduce additional double bonds between C-10 and the methyl-terminal end. Thus, as noted above, mammals cannot synthesize the omega-6 family precursor linoleate, $18 : 2 (Δ^{9,12})$ $18 colon 2 left-parenthesis upper Delta Superscript 9 comma 12 Baseline right-parenthesis$ , or the omega-3 family precursor α-linolenate, $18 : 3 (Δ^{9, 12, 15})$ $18 colon 3 left-parenthesis upper Delta Superscript 9 comma 12 comma 15 Baseline right-parenthesis$ . Plants, however, can synthesize both; the desaturases that introduce double bonds at the $Δ^{12}$ $upper Delta Superscript 12$ and $Δ^{15}$ $upper Delta Superscript 15$ positions are located in the ER and in chloroplasts. The ER enzymes act not on free fatty acids but on a phospholipid, phosphatidylcholine, that contains at least one oleate linked to the glycerol (Fig. 21-14). Both plants and bacteria must synthesize polyunsaturated fatty acids to ensure membrane fluidity at reduced temperatures.

A figure shows the action of plant desaturases by showing the reactions that convert phosphatidylcholine containing oleate to phosphatidylcholine containing alpha-linoleate. — FIGURE 21-14 Action of plant desaturases. Desaturases in plants oxidize phosphatidylcholine-bound oleate to polyunsaturated fatty acids. Some of the products are released from the phosphatidylcholine by hydrolysis.

The top molecule has a vertical three-carbon chain with the top carbon bonded to 2 H and to O to the right bonded to C double bonded to O above further bonded to a zigzag chain of 15 additional carbons to the right. The middle carbon is bonded to H and to O to the right bonded to C in a light red box that is numbered 1, double bonded to O above, and further bonded to a zigzag chain of 17 additional carbons with the final carbon numbered as 18. There is a cis double bond between C 9, which is numbered, and C 10 to the right. The entire eighteen-carbon sequence from C double bonded to O to C 18 is enclosed in a light red box. The bottom C is bonded to 2 H and to O on the right further bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right further bonded to C H 2 bonded to C H 2 bonded to N plus (C H 3) 3. This molecule is labeled phosphatidylcholine containing oleate, 18:1 (delta superscript 9 end superscript), with oleate and its structural abbreviation shown in a light red box. An arrow labeled desaturase points down to a similar molecule in which the eighteen-carbon chain now has cis double bonds between C 9 and C 10 and between C 11 and C 13. This molecule is labeled phosphatidylcholine containing linoleate, 18:2 (delta superscript 9, 12 end superscript), with linoleate and its structural abbreviation shown in a light red box. An arrow labeled desaturase points down to a similar molecule in which the eighteen-carbon chain now has cis double bonds between C 9 and C 10, C 11 and C 13, and C 15 and C 16. This molecule is labeled phosphatidylcholine containing alpha-linoleate, 18:3 (delta superscript 9, 12, 15 end superscript), with alpha-linoleate and its structural abbreviation shown in a light red box.

Because they are necessary precursors for the synthesis of other products, linoleate and α-linolenate are essential fatty acids for mammals; they must be obtained from dietary plant material. Once ingested, linoleate may be converted to certain other polyunsaturated acids, particularly γ-linolenate, eicosatrienoate, and arachidonate (eicosatetraenoate), all of which can be made only from linoleate (Fig. 21-12). Similarly, α-linolenate is converted to two important derivatives, eicosapentaenoic acid (EPA) and docosa-hexaenoic acid (DHA). Arachidonate, $20 : 4 (Δ^{5, 8, 11, 14} )$ $20 colon 4 left-parenthesis upper Delta Superscript 5 comma 8 comma 11 comma 14 Baseline zero width space right-parenthesis$ , EPA, $20 : 5 (Δ^{5, 8, 11, 14, 17} )$ $20 colon 5 left-parenthesis upper Delta Superscript 5 comma 8 comma 11 comma 14 comma 17 Baseline zero width space right-parenthesis$ , and DHA, $22 : 6 (Δ^{4, 7, 10, 13, 16, 19} )$ $22 colon 6 left-parenthesis upper Delta Superscript 4 comma 7 comma 10 comma 13 comma 16 comma 19 Baseline zero width space right-parenthesis$ , are essential precursors of distinct classes of eicosanoids, lipids with important regulatory functions. The 20- and 22-carbon fatty acids are synthesized from linoleate and -linolenate by fatty acid elongation reactions analogous to those described on page 753.

Eicosanoids Are Formed from 20- and 22-Carbon Polyunsaturated Fatty Acids

Eicosanoids are a family of very potent biological signaling molecules that act as short-range messengers, affecting tissues near the cells that produce them.

In response to hormonal or other stimuli, phospholipase $A_{2}$ $upper A Subscript 2$ , present in most types of mammalian cells, attacks membrane phospholipids, releasing arachidonate from the middle carbon of glycerol. Enzymes of the smooth ER then convert arachidonate to prostaglandins, beginning with the formation of prostaglandin $H_{2} ({PGH}_{2})$ $upper H Subscript 2 Baseline left-parenthesis PGH Subscript 2 Baseline right-parenthesis$ , the immediate precursor of many other prostaglandins and thromboxanes (Fig. 21-15a). The two reactions that lead to ${PGH}_{2}$ $PGH Subscript 2$ are catalyzed by a bifunctional enzyme, cyclooxygenase (COX), also called $H_{2}$ $upper H Subscript 2$ prostaglandin synthase. In the first step, the cyclooxygenase activity introduces molecular oxygen to convert arachidonate to ${PGG}_{2}$ $PGG Subscript 2$ . The second step, catalyzed by the peroxidase activity of COX, converts ${PGG}_{2}$ $PGG Subscript 2$ to ${PGH}_{2}$ $PGH Subscript 2$ .

FIGURE 21-15 The “cyclic” pathway from arachidonate to prostaglandins and thromboxanes. (a) After arachidonate is released from phospholipids by the action of phospholipase $A_{2}$ $upper A Subscript 2$ , the cyclooxygenase and peroxidase activities of COX (also called prostaglandin $H_{2}$ $upper H Subscript 2$ synthase) catalyze the production of ${PGH}_{2}$ $PGH Subscript 2$ , the precursor of other prostaglandins and of thromboxanes. (b) Aspirin inhibits the first reaction by acetylating an essential Ser residue on the enzyme. Ibuprofen and naproxen inhibit the same step, probably by mimicking the structure of the substrate or an intermediate in the reaction.

Part a begins with text at the upper left reading, phospholipid containing arachidonate. An arrow labeled phospholipidase A subscript 2 points down to arachidonate, 20:4 (delta superscript 5, 8, 11, 14), accompanied by a curved arrow showing the loss of lysophospholipid. Arachidonate has C O O minus at the upper right that begins a chain of 20 carbons with a curve at one end to form a looped structure that is open on the right side. C O O minus at the top right has a zigzag chain of three carbons to its left before reaching a cis double bond between the fifth and sixth carbons, which forms the top of a structure that resembles a hexagon with sides that don’t come together to form left and right vertices. From C 6, there is a bond to C H 2 and then a bond C 8, which is cis double bonded to C 9 at the upper left corner. C 9 has a bond to C H 2 at the lower left forming the left end, from which a bond extends to C 11 at the lower right that forms a double bond with C 12 beneath the double bond between C 8 and C 9. This gives the appearance of a partial ring that is open on its right side. C 12 is bonded to C H 2 to the upper right, which is bonded to C 14. C 14 is double bonded to C 15, forming the bottom side of the second partial ring-like structure. From C 15, a zigzag chain of five additional carbons completes the bottom half of the structure. An arrow labeled cyclooxygenase activity of C O X points down accompanied by a curved arrow showing the addition of 2 red highlighted O 2. A red “X” in a red circle next to the downward arrow is indicated by a dashed horizontal arrow from aspirin, ibuprofen, naproxen. This points down to a similar structure below labeled P G G 2. It has C O O minus at the upper right bonded to a zigzag chain of four carbons with the fourth carbon in this chain, C 5, forming a cis double bond to C 6. C 6 is bonded to C 7 to the upper left, forming the top vertex of a partial double six-membered ring missing its right side, with a double bond at its lower right side, and with a left side shared with a five-membered ring that has a left side vertex and upper and lower left vertices each bonded to red highlighted O that are joined together by a vertical bond. The lower right vertex of the partial six-membered ring has a bond down to a vertex from which a bond extends down to red highlighted O bonded to red highlighted O bonded to nonhighlighted H. From here, a zigzag chain of five carbons completes the bottom half of the molecule. An arrow labeled peroxidase activity of C O X points down to P G H 2, which is similar except that the bond to O bonded to O H at the bottom center is now a bond to red highlighted O bonded to nonhighlighted H. Arrows point down to the lower left and right. The left-hand arrow points to other series 2 prostaglandins and the right-hand arrow points to series 2 thromboxanes. Part b begins with a blue oval labeled S e r with a bond to O H to the right. Text below reads, C O X. This is added to aspirin (acetylsalicylate), which is a benzene ring with the top vertex bonded to CO O minus and the upper right vertex bonded to O further connected by a red bond to red highlighted C connected by a red double bond to red highlighted O to the upper right and by a red bond to red highlighted C H 3 to the lower right. An arrow points right to show that this yields a blue oval labeled S e r bonded to O to the right further connected by a red bond to red highlighted C connected by a red highlighted double bond to O to the upper right and by a red bond to red highlighted C H 3 to the lower right. Text below reads, acetylated, inactivated C O X. Salicylate is also present and is a benzene ring with the top vertex bonded to C O O minus and the upper right vertex bonded to O H. Below, ibuprofen is shown as a benzene ring with its top vertex bonded to C H 2 bonded to C H 2 bonded to C H 3 to the upper right and left and with its bottom vertex bonded to C H bonded to C H 3 to the lower left and bonded to C O O minus to the lower right. Naproxen is shown as a benzene ring with its top vertex bonded to O bonded to C H 3 above and with its lower right side shared with the upper let side of a second benzene ring with its bottom vertex bonded to C H bonded to C H 3 to the lower left and to C O O minus to the lower right.

Key Convention

Prostaglandins with different functional groups on the ring are given different letter designations: A, B, C, D, E, F, G, H, and R. The subscript number following the letter, as in ${PGH}_{2}$ $PGH Subscript 2$ and ${PGG}_{2}$ $PGG Subscript 2$ , indicates the number of double bonds. Prostaglandins with two double bonds, all of which are derived from arachidonate, are referred to as series 2 prostaglandins; those with three double bonds, derived from EPA, as series 3 (Fig. 21-12). Similar naming patterns are used for other classes of eicosanoids described below.

Series 2 prostaglandins have important roles in the immediate response to stress or injury, including inflammation, pain, swelling, and dilation of blood vessels. Series 3 prostaglandins, in general, act more slowly and usually moderate the responses associated with series 2 prostaglandins.

Mammals have two isozymes of prostaglandin $H_{2}$ $upper H Subscript 2$ synthase, COX-1 and COX-2. These have different functions but closely similar amino acid sequences (60% to 65% sequence identity) and similar reaction mechanisms at both of their catalytic centers. COX-1 is responsible for synthesis of the prostaglandins that regulate the secretion of gastric mucin, and COX-2 is responsible for synthesis of the prostaglandins that mediate inflammation, pain, and fever.

Pain can be relieved by inhibiting COX-2. The first drug widely marketed for this purpose was aspirin (acetylsalicylate; Fig. 21-15b). The name “aspirin” (from a for acetyl and spir for Spirsaüre, the German word for the salicylates prepared from the plant Spiraea ulmaria) appeared in 1899 when the drug was introduced by the Bayer company. Aspirin irreversibly inactivates the cyclooxygenase activity of both COX isozymes, by acetylating a Ser residue and blocking each enzyme’s active site. The synthesis of prostaglandins and thromboxanes is thereby inhibited. Additional widely used nonsteroidal anti-inflammatory drugs (NSAIDs; Fig. 21-15b), ibuprofen and naproxen, inhibit the same pair of enzymes. However, the inhibition of COX-1 can result in undesired side effects, including stomach irritation and more serious conditions. In the 1990s, NSAID compounds that had a greater specificity for COX-2 were developed as advanced therapies for severe pain. Three of these drugs were approved for use worldwide: rofecoxib (Vioxx), valdecoxib (Bextra), and celecoxib (Celebrex). Though initially considered a success, Vioxx and Bextra were withdrawn as field reports and clinical studies connected the drugs with an increased risk of heart attack and stroke. Celebrex is still on the market but is being used with increased caution. The detailed reasons for the problems with these drugs are still not clear but serve as a cautionary note. We are increasingly aware of the complexity of the web of these signaling interactions, and predicting the consequences of targeting specific components with pharmaceutical agents remains an imperfect process.

Thromboxane synthase, present in blood platelets (thrombocytes), converts ${PGH}_{2}$ $PGH Subscript 2$ to thromboxane $A_{2}$ $upper A Subscript 2$ , from which other series 2 thromboxanes are derived (Fig. 21-15a). The series 2 thromboxanes induce constriction of blood vessels and platelet aggregation, early steps in blood clotting. Low doses of aspirin, taken regularly, reduce the probability of heart attacks and strokes by reducing thromboxane production.

Thromboxanes, like prostaglandins, contain a ring of five or six atoms; the pathway from arachidonate to the series 2 prostaglandins and thromboxanes is sometimes called the “cyclic” pathway, to distinguish it from the “linear” pathway that leads from arachidonate to the leukotrienes, which are linear compounds (Fig. 21-16). Leukotriene synthesis begins with the action of several lipoxygenases that catalyze the incorporation of molecular oxygen into arachidonate. These enzymes, found in leukocytes and in heart, brain, lung, and spleen, are mixed-function oxidases of the cytochrome P-450 family (see Box 21-1). The various leukotrienes differ in the position of the peroxide group introduced by the lipoxygenases. The linear pathway from arachidonate, unlike the cyclic pathway, is not inhibited by aspirin or other NSAIDs.

FIGURE 21-16 The “linear” pathway from arachidonate to leukotrienes.

Arachidonate is shown at the top as a horizontal zigzag chain with carbons 1, 5, 8, 11, 14, and 20 numbered. C 1 at the right forms C O O minus, C 5 forms a cis double bond with C 6, C 8 forms a cis double bond with C 9, C 11 forms a cis double bond with C 12, C 14 forms a cis double bond with C 15, and C 20 is the terminal carbon on the left. Arrows point down to the lower left and right. The left-hand arrow is labeled lipoxygenase and a curved arrows shows the addition of red highlighted O 2. This yields 12-hydroperoxyeicosatetraenoate (12-H P T E). This has a similar structure to the reactant except that there is a double bond between C 10 and C 11, a single bond between C 11 and C 12, and a bond from C 12 to red highlighted O bonded to red highlighted O bonded to nonhighlighted H. An arrow labeled multistep points down to text reading, other leukotrienes. The right-hand arrow is labeled lipoxygenase and a curved arrows shows the addition of red highlighted O 2. This yields 5-hydroperoxyeicosatetraenoate (5-H P T E). This has a similar structure to the reactant except that C 5 is bonded to red highlighted O bonded to red highlighted O bonded to nonhighlighted H, there is no double bond between C 5 and C 6, and there is a double bond between C 6 and C 7. An arrow labeled multistep points down to leukotriene A subscript 4 end subscript (L T A subscript 4 end subscript), from which an arrow points right to L T C subscript 4 end subscript, from which an arrow points right to L T D subscript 4 and subscript.

Pathogenic organisms, as well as irritants such as air pollution and tobacco smoke, trigger an inflammatory response in the affected tissue, which consists of two phases: initiation and resolution. Eicosanoids of the omega-6 family are critical to initiation — playing key roles in recruiting leukocytes, making blood vessels more permeable, and stimulating chemotaxis and migration of immune system cells. As the source of tissue damage is brought under control, the inflammation must be resolved and the tissue brought back to its normal state. Resolution of inflammation is called catabasis, and it is promoted by several classes of signaling molecules; prominent among these are several leukotrienes and prostaglandins. Many eicosanoids of the omega-3 family (including series 3 prostaglandins and thromboxanes) are anti-inflammatory, although the classification is not absolute; individual eicosanoids can be inflammatory in one tissue and anti-inflammatory in another.

Catabasis is also promoted by a set of eicosanoids termed specialized pro-resolving mediators (SPMs). The first family of SPMs to be discovered was the lipoxins, followed more recently by resolvins, protectins, and maresins. All SPMs are derived from essential fatty acids (Fig. 21-12). They affect different target cells and tissues in different ways. The sum of their action is to promote removal of debris, microbes, and dead cells, to restore blood vessel integrity, and to regenerate tissue. Particular SPMs also reduce pain and fever, and play roles in resolving the tissue inflammation leading to diabetes, obesity, and asthma. Further research on SPMs thus has potential for the development of new pharmaceutical targets.

Plants also derive important signaling molecules from fatty acids. As in animals, a key step in the initiation of signaling is activation of a specific phospholipase. In plants, the fatty acid substrate released by phospholipase action is α-linolenate. A lipoxygenase then catalyzes the first step in a pathway that converts α-linolenate to jasmonate, a substance known to have signaling roles in defense against insects, resistance to fungal pathogens, and maturation of pollen. Jasmonate also affects seed germination, root growth, and fruit and seed development.

SUMMARY 21.1 Biosynthesis of Fatty Acids and Eicosanoids

Malonyl-CoA, a key precursor of fatty acids, is synthesized by the action of acetyl-CoA carboxylase.

Beginning with malonyl-CoA and acetyl-CoA, fatty acids are synthesized in a repeating cycle of four steps.

Long-chain saturated fatty acids are synthesized from acetyl-CoA by a cytosolic system of six enzymatic activities plus acyl carrier protein (ACP). There are two types of fatty acid synthase. FAS I, found in vertebrates and fungi, consists of multifunctional polypeptides. FAS II is a dissociated system found in bacteria and plants. Both contain two types of OSH groups (one furnished by the phosphopantetheine of ACP, the other by a Cys residue of β-ketoacyl-ACP synthase) that function as carriers of the fatty acyl intermediates.

Malonyl-ACP, formed from acetyl-CoA (shuttled out of mitochondria) and ${CO}_{2}$ $CO Subscript 2$ , condenses with an acetyl bound to the Cys OSH to yield acetoacetyl-ACP, with release of ${CO}_{2}$ $CO Subscript 2$ . This is followed by reduction to the d-β-hydroxy derivative, dehydration to the trans- $Δ^{2}$ $upper Delta Superscript 2$ -unsaturated acyl-ACP, and reduction to butyryl-ACP. NADPH is the electron donor for both reductions. Fatty acid synthesis is regulated at the level of malonyl-CoA formation.

Six more molecules of malonyl-ACP react successively at the carboxyl end of the growing fatty acid chain to form palmitoyl-ACP — the end product of the fatty acid synthase reaction. Free palmitate is released by hydrolysis.

Fatty acid synthesis occurs in the cytosol of animal cells, and in chloroplasts in plants.

Acetate is exported from the mitochondria as citrate.

Fatty acid synthesis is tightly regulated, principally by regulation of acetyl-CoA carboxylase.

Palmitate may be elongated to the 18-carbon stearate.

Palmitate and stearate can be desaturated to yield palmitoleate and oleate, respectively, by the action of mixed-function oxidases.

Mammals cannot make linoleate and must obtain it from plant sources; they convert exogenous linoleate to arachidonate, the parent compound of eicosanoids (prostaglandins, thromboxanes, leukotrienes, and specialized pro-resolving mediators), a family of very potent signaling molecules. The synthesis of prostaglandins and thromboxanes is inhibited by NSAIDs that act on the cyclooxygenase activity of prostaglandin $H_{2}$ $upper H Subscript 2$ synthase.