Lipids (‘fats’) are essential for the formation of a cell membrane that contains the cell contents, as well as for other cell functions. The cell membrane, comprised of several different complex lipids, is an essential part of a free-living cell that can reproduce itself. 4
Lipids have much higher energy density than sugars or amino acids, so their formation in any chemical soup is a problem for origin of life scenarios (high energy compounds are thermodynamically much less likely to form than lower energy compounds).
The fatty acids that are the primary component of all cell membranes have been very difficult to produce, even assuming the absence of oxygen (a ‘reducing’ atmosphere). Even if such molecules were produced, ions such as magnesium and calcium, which are themselves necessary for life and have two charges per atom (++, i.e. divalent), would combine with the fatty acids, and precipitate them, making them unavailable.
Biological lipids are a chemically diverse group of compounds, the common and defining feature of which is their insolubility in water. The biological functions of the lipids are as diverse as their chemistry. Fats and oils are the principal stored forms of energy in many organisms. Phospholipids and sterols are major structural elements of biological membranes. Other lipids, although present in relatively small quantities, play crucial roles as enzyme cofactors, electron carriers, light-absorbing pigments, hydrophobic anchors for proteins, “chaperones” to help membrane proteins fold, emulsifying agents in the digestive tract, hormones, and intracellular messengers.
Lipids play a variety of cellular roles, some only recently recognized. They are the principal form of stored energy in most organisms and major constituents of cellular membranes. Specialized lipids serve as
pigments (retinal, carotene)
cofactors (vitamin K)
detergents (bile salts)
hormones (vitamin D derivatives, sex hormones)
extracellular and intracellular messengers (eicosanoids, phosphatidylinositol derivatives)
anchors for membrane proteins (covalently attached fatty acids, prenylgroups, and phosphatidylinositol)
The fats and oils used almost universally as stored forms of energy in living organisms are derivatives of fatty acids. The fatty acids are hydrocarbon derivatives, at about the same low oxidation state (that is, as highly reduced) as the hydrocarbons in fossil fuels. The cellular oxidation of fatty acids (to CO2 and H2O), like the controlled, rapid burning of fossil fuels in internal combustion engines, is highly exergonic.
Among the most biologically significant properties of lipids are their hydrophobic properties. These properties are mainly due to a particular component of lipids: fatty acids, or simply fats. Fatty acids are hydrocarbon chains of various lengths and degrees of unsaturation that terminate with a carboxylic acid group. The fatty acid chains in membranes usually contain between 14 and 24 carbon atoms; they may be saturated or unsaturated. Short chain length and unsaturation enhance the fluidity of fatty acids and their derivatives by lowering the melting temperature.
Structural Lipids in Membranes
The central architectural feature of biological membranes is a double layer of lipids, which acts as a barrier to the passage of polar molecules and ions. Membrane lipids are amphipathic: one end of the molecule is hydrophobic, the other hydrophilic. Their hydrophobic interactions with each other and their hydrophilic interactions with water direct their packing into sheets called membrane bilayers.
■ Lipids are water-insoluble cellular components, of diverse structure, that can be extracted by nonpolar solvents.
■ Almost all fatty acids, the hydrocarbon components of many lipids, have an even number of carbon atoms (usually 12 to 24); they are either saturated or unsaturated, with double bonds almost always in the cis configuration.
■ Triacylglycerols contain three fatty acid molecules esterified to the three hydroxyl groups of glycerol. Simple triacylglycerols contain only one type of fatty acid; mixed triacylglycerols, two or three types. Triacylglycerols are primarily storage fats; they are present in many foods.
■ Partial hydrogenation of vegetable oils in the food industry converts some cis double bonds to the trans configuration. Trans fatty acids in the diet are an important risk factor for coronary heart disease.
There are five general types of membrane lipids:
■ Glycerophospholipids, in which the hydrophobic regions are composed of two fatty acids joined to glycerol; galactolipids and sulfolipids, which also contain two fatty acids esterified to glycerol, but lack the characteristic phosphate of phospholipids
■ Archaeal tetraether lipids, in which two very long alkyl chains are ether-linked to glycerol at both ends; sphingolipids, in which a single fatty acid is joined to a fatty amine
■ Sphingosine and sterols, compounds characterized by a rigid system of four fused hydrocarbon rings.
■ The polar lipids, with polar heads and nonpolar tails, are major components of membranes. The most abundant are the glycerophospholipids, which contain fatty acids esterified to two of the hydroxyl groups of glycerol, and a second alcohol, the head group, esterified to the third hydroxyl of glycerol via a phosphodiester bond. Other polar lipids are the sterols.
Glycerophospholipids differ in the structure of their head group; common glycerophospholipids are phosphatidylethanolamine and phosphatidylcholine. The polar heads of the
glycerophospholipids are charged at pH near 7.
Chloroplast membranes are rich in galactolipids, composed of a diacylglycerol with one or two linked galactose residues, and sulfolipids, diacylglycerols with a linked sulfonated sugar residue and thus a negatively charged head group.
Archaea have unique membrane lipids, with long-chain alkyl groups ether-linked to glycerol at both ends and with sugar residues and/or phosphate joined to the glycerol to provide a polar or charged head group. These lipids are stable under the harsh conditions in which archaea live.
The sphingolipids contain sphingosine, a long-chain aliphatic amino alcohol, but no glycerol. Sphingomyelin has, in addition to phosphoric acid
Lipids as Signals,Cofactors,and Pigments
The two functional classes of lipids considered thus far (storage lipids and structural lipids) are major cellular components; membrane lipids make up 5% to 10% of the dry mass of most cells, and storage lipids more than 80% of the mass of an adipocyte. With some important exceptions, these lipids play a passive role in the cell; lipid fuels are stored until oxidized by enzymes, and membrane lipids form impermeable barriers around cells and cellular compartments. Another group of lipids, present in much smaller amounts, have active roles in the metabolic traffic as metabolites and messengers. Some serve as potent signals—as hormones, carried in the blood from one tissue to another, or as intracellular messengers generated in response to an extracellular signal (hormone or growth factor). Others function as enzyme cofactors in electron-transfer reactions in chloroplasts and mitochondria, or in the transfer of sugar moieties in a variety of glycosylation reactions. A third group consists of lipids with a system of conjugated double bonds: pigment molecules that absorb visible light. Some of these act as light-capturing pigments in vision and photosynthesis; others produce natural colorations, such as the orange of
pumpkins and carrots and the yellow of canary feathers. Finally, a very large group of volatile lipids produced in plants serve as signals that pass through the air, allowing
plants to communicate with each other, and to invite animal friends and deter foes. We describe in this section a few representatives of these biologically active lipids. In
later chapters, their synthesis and biological roles are considered in more detail.
Some types of lipids, although present in relatively small quantities, play critical roles as cofactors or signals.
■ Phosphatidylinositol bisphosphate is hydrolyzed to yield two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate. Phosphatidylinositol 3,4,5-trisphosphate is a nucleation point for supramolecular protein complexes involved in biological signaling.
■ Prostaglandins, thromboxanes, and leukotrienes (the eicosanoids), derived from arachidonate, are extremely potent hormones.
■ Steroid hormones, derived from sterols, serve as powerful biological signals, such as the sex hormones.
■ Vitamins D, A, E, and K are fat-soluble compounds made up of isoprene units. All play essential roles in the metabolism or physiology of animals. Vitamin D is precursor to a hormone that regulates calcium metabolism. Vitamin A furnishes the visual pigment of the vertebrate eye and is a regulator of gene expression during epithelial cell growth. Vitamin E functions in the protection of membrane lipids from oxidative damage, and vitamin K is essential in the blood-clotting process.
■ Ubiquinones and plastoquinones, also isoprenoid derivatives, are electron carriers
The ability to synthesize a variety of lipids is essential to all organisms. It will be described the biosynthetic pathways for some of the most common cellular lipids, illustrating the strategies employed in assembling these water-insoluble products from water-soluble precursors such as acetate. Like other biosynthetic pathways, these reaction sequences are endergonic and reductive. They use ATP as a source of metabolic energy and a reduced electron carrier (usually NADPH) as a reductant. We first describe the biosynthesis of fatty acids, the primary components of both triacylglycerols and phospholipids, then examine the assembly of fatty acids into triacylglycerols and the simpler membrane phospholipids.
Synthesis of most lipids in microorganisms can be viewed as having two essential components—fatty acid synthesis and glycerol synthesis. Synthesis starts with transfer of the acetyl group of acetyl-CoA to a carrier protein called acyl carrier protein (ACP). This carrier serves to hold the fatty acid chain as it is elongated by progressively adding 2-carbon units. When the newly synthesized fatty acid reaches its requiredlength, usually 14, 16, or 18 carbons long, it is released from ACP. The glycerol component of the fat is synthesized from dihydroxyacetone phosphate.
Fatty acids are important for energy storage, phospholipid membrane formation, and signaling pathways. Fatty acid metabolism consists of catabolic processes that generate energy and primary metabolites from fatty acids, and anabolic processes that create biologically important molecules from fatty acids and other dietary sources.3
Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and malonyl-CoA precursors through action of enzymes called fatty acid synthases. It is an important part of the lipogenesis process, which – together with glycolysis – functions to create fats from blood sugar in living organisms. 1
The synthesis of long-chain fatty acids (lipogenesis) is carried out by two enzyme systems: acetyl-CoA carboxylase and fatty acid synthase.
Fatty acids are essential nutrients for all organisms, except archaea. 2 A fatty acid is a carboxylic acid with a long, unbranched aliphatic tail that is either saturated or unsaturated.
Within cells, fatty acids serve many vital functions:
· As major components of cell membranes, including internal organelle membranes (each phospholipid contains two fatty acid tails)
· For energy storage - (yields significantly more energy than carbohydrates, for the same mass)
· As messenger substances (e.g. ceramide is a fatty acid-containing messenger in cytokine-induced apoptosis)
· For the post-translational modification of certain proteins
The synthesis of fatty acids is essentially the reverse chemistry of its degradation by oxidation, both pathways involving an activated two-carbon intermediate, acetyl-CoA. Therefore, a cell needs a means of separating the two opposing pathways in order to allow their independent control. In eukaryotes, this is achieved both physically and chemically:
· Fatty acid synthesis occurs in the cytoplasm, while its oxidation occurs in mitochondria
· Fatty acid synthesis requires the oxidation of the co-factor NADPH, while fatty acid oxidation requires the reduction of FAD+ and NAD+
In addition, distinct enzymes control the two pathways, permitting a further level of control. Fatty acid synthesis is carried out by fatty acid synthase.
Fatty Acids Are Hydrocarbon Derivatives
Fatty acids are carboxylic acids with hydrocarbon chains ranging from 4 to 36 carbons long (C4 to C36). In some fatty acids, this chain is unbranched and fully saturated (contains no double bonds); in others the chain contains one or more double bonds ( See picture below ). A few contain three-carbon rings, hydroxyl groups, or methylgroup branches. At room temperature (25 C), the saturated fatty acids from 12:0 to 24:0 have a waxy consistency, whereas unsaturated fatty acids of these lengths are oily liquids. This difference in melting points is due to different degrees of packing of the fatty acid molecules. In the fully saturated compounds, free rotation around each carbon–carbon bond gives the hydrocarbon chain great flexibility; the most stable conformation is the fully extended form, in which the steric hindrance of neighboring atoms is minimized. These molecules can pack together tightly in nearly crystalline arrays, with atoms all along their lengths in van der Waals contact with the atoms of neighboring molecules. In unsaturated fatty acids, a cis double bond forces a kink in the hydrocarbon chain. Fatty acids with one or several such kinks cannot pack together as tightly as fully saturated fatty acids, and their interactions with each other are therefore weaker. Because less thermal energy is needed to disorder these poorly ordered arrays of unsaturated fatty acids, they have markedly lower melting points than saturated fatty acids of the same chain length.
The most commonly occurring fatty acids have even numbers of carbon atoms in an unbranched chain of 12 to 24 carbons . the even number of carbons results from the mode of
synthesis of these compounds, which involves successive condensations of two-carbon (acetate) units.
Fatty acids are carboxylic acids with hydrocarbon chains ranging from 4 to 36 carbons long (C4 to C36). In some fatty acids, this chain is unbranched and fully saturated (contains no double bonds); in others the chain contains one or more double bonds . A few contain three-carbon rings, hydroxyl groups, or methylgroup branches.
Fatty acid synthesis
The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to malonyl-CoA. The ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid. The spontaneous decarboxylation drives the condensation.
Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and malonyl-CoA precursors through action of enzymes called fatty acid synthases. It is an important part of the lipogenesis process, which – together with glycolysis – functions to create fats from blood sugar in living organisms. 1
The Reaction Sequence for the Biosynthesis of Fatty Acids
Biosynthesis of Fatty Acids and Eicosanoids
After the discovery that fatty acid oxidation takes place by the oxidative removal of successive two-carbon (acetyl-CoA) units, biochemists thought the biosynthesis of fatty acids might proceed by a simple reversal of the same enzymatic steps. However, as they were to find out, fatty acid biosynthesis and breakdown occur by different pathways, are catalyzed by different sets of enzymes, and take place in different parts of the cell. Moreover, biosynthesis requires the participation of a three-carbon intermediate,
malonyl-CoA, that is not involved in fatty acid breakdown.
The first step :
Malonyl-CoA Is Formed from Acetyl-CoA and Bicarbonate
The formation of malonyl-CoA from acetyl-CoA is an irreversible process, catalyzed by acetyl-CoA carboxylase. The bacterial enzyme has three separate polypeptide subunits;
The acetyl-CoA carboxylase reaction. Acetyl-CoA carboxylase has three functional regions: biotin carrier protein (gray); biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction; and transcarboxylase, which transfers activated CO2 (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA. The long, flexible biotin arm carries the activated CO2 from the biotin carboxylase region to the transcarboxylase active site. The active enzyme in each step is shaded blue.
In animal cells, all three activities are part of a single multifunctional polypeptide. Plant cells contain both types of acetyl- CoA carboxylase. In all cases, the enzyme contains a
biotin prosthetic group covalently bound in amide linkage to the e-amino group of a Lys residue in one of the three polypeptides or domains of the enzyme molecule. The two-step reaction catalyzed by this enzyme is very similar to other biotin-dependent carboxylation reactions, such as those catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase . A carboxyl group, derived from bicarbonate (HCO3 ), is first transferred to biotin in an ATP-dependent reaction. The biotinyl group serves as a temporary carrier of CO2, transferring it to acetyl-CoA in the second step to yield malonyl-CoA.
The second step:
Fatty acid synthase
Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. 5
A battery of enzymes are required to synthesise fatty acids, however their organisation differs among species. Fatty acid synthetase (FAS) can be divided into two groups based on the organisation of their catalytic units: fatty acid synthase I (FAS I), found in vertebrates and fungi, and fatty acid synthase II (FAS II), found in plants and bacteria. The FAS I found in vertebrates consists of a single multifunctional polypeptide chain. The mammalian FAS I is the prototype. Seven active sites for different reactions lie in separate domains.
Type I FAS systems
Type I FAS systems are multi-enzyme complexes that contain all the catalytic units as distinct domains covalently linked into one (alpha) or two (alpha and beta) polypeptides. Type I systems include eukaryotic, as well as a few bacterial, FAS enzymes. These systems can be further divided into subgroups according to the organisation of individual polypeptides and the domains within these polypeptides:
· Animal FAS enzymes consist of (alpha)2 homodimers
· Fungal FAS enzymes consist of (alpha)6(beta)6 dodecamers
· A few bacterial FAS enzymes consist of (alpha)6 hexamers
Type I FAS systems carry out multiple steps of fatty acid synthesis in each sterically isolated reaction chamber. Mammalian FAS is thought to have evolved through gene fusion.
Type II FAS systems
In type II FAS systems, the enzymes exist as distinct, individual proteins, where each protein catalyses a single step in the reaction pathway. Most prokaryotic FAS systems fall into this category, as well as certain plant FAS systems.
Fatty Acid Synthesis Proceeds in a Repeating Reaction Sequence
In all organisms, the long carbon chains of fatty acids are assembled in a repeating four-step sequence. 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. 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 oxidation, NAD and FAD serve as electron acceptors and the activating group is the thiol (—SH) 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 groups. There are two major variants of fatty acid synthase: fatty acid synthase I (FAS I), found in vertebrates and fungi, and fatty acid synthase II (FAS II), found in plants and bacteria. The FAS I found in vertebrates consists of a single multifunctional polypeptide chain. The mammalian FAS I is the prototype. Seven active sites for different reactions lie in separate domains. The mammalian polypeptide functions as a homodimer. The subunits appear to function independently. When all the active sites in one subunit are inactivated by mutation, fatty acid synthesis is only modestly reduced. 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 the vertebrate systems (Fig. 21–3b). Three of the seven required active sites are found on the subunit and four on the subunit.
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 described later in the text.
1 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 CO2 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 -keto product of this condensation is then reduced in three more steps nearly identical to the reactions of oxidation, but in the reverse sequence:
2 the -keto group is reduced to an alcohol
3 elimination of H2O creates a double bond, and
4 the double bond is reduced to form the corresponding saturated fatty acyl group.
The structure of fatty acid synthase type I systems. The low-resolution structures of (a) the mammalian and (b) fungal enzyme systems are shown. (a) All of the active sites in the mammalian system are located in different domains within a single large polypeptide chain. The different enzymatic activites are:
-ketoacyl-ACP synthase (KS),
–ACP transferase (MAT),
-hydroxyacyl-ACP dehydratase (DH),
-enoyl-ACP reductase (ER),
-ketoacyl-ACP reductase (KR).
ACP is the acyl carrier protein. The linear arrangement of the domains in the polypeptide is shown in the lower panel. The seventh domain (TE) is a thioesterase that releases the palmitate product from ACP when the synthesis is completed. The ACP and TE domains are disordered in the crystal and are therefore not shown in the structure. (b) In the structure of the FAS I from the fungus Thermomyces lanuginosus, the same active sites are divided between two multifunctional polypeptide chains that function together. Six copies of each polypeptide are found in the heterododecameric complex. A wheel of sixsubunits, which include ACP as well as the KS and KR active sites, is found at the center of the complex. In the wheel three subunits are found on one face, three on the other. On either side of the wheel are domes formed by trimers of the subunits (containing the ER and DH active sites, as well as two domains with active sites analogous to MAT in the mammalian enzyme). The domains of one of each type of subunit are colored according to the active site colors of the mammalian enzyme in (a).
With FAS I systems, fatty acid synthesis leads to a single product, and no intermediates are released. When the chain length reaches 16 carbons, that product (palmitate, 16:0) 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
The rest of the carbon atoms in the chain are derived from acetyl-CoA via malonyl-CoA. FAS II, in plants and bacteria, is a dissociated system; each step in the synthesis is catalyzed by a separate and freely diffusible enzyme. Intermediates are also diffusible and may be diverted into other pathways (such as lipoic acid synthesis). 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. The discussion to follow will focus on the mammalian FAS I.
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 group of a Cys residue in one of the synthase domains (-ketoacyl-ACP synthase; KS); the other is the —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 different steps ( 1 and 5 in Fig. below) in fatty acid synthesis (condensation) thermodynamically favorable.
Acyl carrier protein (ACP) is the shuttle that holds the system together. The Escherichia coli ACP is a small protein containing the prosthetic group 4-phosphopantetheine The 4-phosphopantetheine prosthetic group of E. coli ACP is believed to serve 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 ACP of mammals has a similar function and the same prosthetic group; as we have seen, however, it is embedded as a domain in a much larger multifunctional polypeptide.
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: ( Figure below, top ) First, the acetyl group of acetyl-CoA is transferred to ACP in a reaction catalyzed by the malonyl /acetyl-CoA–ACP transferase (MAT in Fig. below) domain of the multifunctional polypeptide. The acetyl group is then transferred to the Cys —SH group of the -ketoacyl-ACP synthase (KS). The second reaction, transfer of the malonyl group from malonyl-CoA to the —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.
The first four steps of this process are now considered in some detail; all step numbers refer to figure above:
Step 1 Condensation The first reaction in the formation of a fatty acid chain is a formal Claisen condensation involving the activated acetyl and malonyl groups to form acetoacetyl-ACP, an acetoacetyl group bound to ACP through the phosphopantetheine —SH group; simultaneously, a molecule of CO2 is produced. In this reaction, catalyzed by -ketoacyl-ACP synthase, the acetyl group is transferred from the Cys —SH group of the enzyme to the malonyl group on the —SH of ACP, becoming the methyl-terminal two-carbon unit of the new acetoacetyl group. The carbon atom of the CO2 formed in this reaction is the same carbon originally introduced into malonyl- CoA from HCO3 by the acetyl-CoA carboxylase reaction. Thus CO2 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 CO2 to make a malonyl group from an acetyl group, only to lose the CO2 during the formation of acetoacetate? Recall that in the oxidation of fatty acids, cleavage of the bond between two acyl groups (cleavage of an acetyl unit from the acyl chain) is highly exergonic, so the simple condensation of two acyl groups (two acetyl-CoA molecules, for example) is highly endergonic. The use of activated malonyl groups rather than acetyl groups is what makes the condensation reactions thermodynamically favorable. The methylene carbon (C-2) of the malonyl group, sandwiched between carbonyl and carboxyl carbons, is chemically situated to act as a good nucleophile. In the condensation step (step 1 ), decarboxylation of the malonyl group facilitates the nucleophilic attack of the methylene carbon on the thioester linking the acetyl group to -ketoacyl-ACP synthase, displacing the enzyme’s —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. 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 HCO3
Step 2 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--hydroxybutyryl-ACP. This reaction is catalyzed by-ketoacyl-ACP reductase (KR) and the electron donor is NADPH. Notice that the D--hydroxybutyryl group does not have the same stereoisomeric form as the L--hydroxyacyl intermediate in fatty acid oxidation.
Step 3 Dehydration The elements of water are now removed from C-2 and C-3 of D--hydroxybutyryl-ACP to yield a double bond in the product, trans-2- butenoyl-ACP. The enzyme that catalyzes this dehydration is -hydroxyacyl-ACP dehydratase (DH).
Step 4 Reduction of the Double Bond. Finally, the double bond of trans-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. The butyryl group is now transferred from the phosphopantetheine —SH group of ACP to the Cys —SH group of -ketoacyl-ACP synthase, which initially bore the acetyl group (Fig. above). To start the next cycle of four reactions that lengthens the chain by two more carbons, another malonyl group is linked to the now unoccupied phosphopantetheine —SH group of ACP
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 CO2. The product of this condensation is a six-carbon acyl group, covalently bound to the phosphopantetheine—SH group. Its -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. 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: then seven cycles of condensation and reduction: Note that only six net water molecules are produced, because one is used to hydrolyze the thioester linking the palmitate product to the enzyme. 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 CO2 to acetyl-CoA to make malonyl-CoA; the NADPH is required to reduce the double bonds. 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 ATPs per molecule of acetyl-CoA transported, increasing the energetic cost of fatty acid synthesis to three ATPs per two-carbon unit.
Fatty Acid Synthesis Occurs in the Cytosol of Many Organisms but in the Chloroplasts of Plants
In most higher eukaryotes, the fatty acid synthase complex is found exclusively 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 serves in catabolic reactions. In hepatocytes, the [NADPH]/[NADP] ratio is very high (about 75) in the cytosol, furnishing a strongly reducing environment for the reductive synthesis of fatty acids and other biomolecules. The cytosolic [NADH]/[NAD] ratio is much smaller (only
about 8 104), so the NAD-dependent oxidative catabolism of glucose can take place in the same compartment, and at the same time, as fatty acid synthesis. The [NADH]/[NAD] ratio in the mitochondrion is much higher than in the cytosol, because of the flow of electrons to NAD from the oxidation of fatty acids, amino acids, pyruvate, and acetyl-CoA. This high mitochondrial [NADH]/[NAD] ratio favors the reduction of oxygen via the respiratory chain.
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 the catabolism of the carbon skeletons of amino acids.
That means, fatty acid synthesis in eukaryotes is interdependent with mitochondria and 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. The mitochondrial inner membrane is impermeable to acetyl-CoA, so an indirect shuttle transfers acetyl group equivalents across the inner membrane
Intramitochondrial acetyl-CoA first reacts with oxaloacetate to form citrate, in the citric acid cycle reaction catalyzed by citrate synthase
That means the citric acid cycle must also be in place already
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–-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.a).
The pyruvate produced is transported to the mitochondria by the pyruvate transporter, and converted back into oxaloacetate by pyruvate carboxylase in the matrix. The resulting cycle results in the consumption of two ATPs (by citrate lyase and pyruvate carboxylase) for every molecule of acetyl-CoA delivered to fatty acid synthesis. After citrate cleavage to generate acetyl-CoA, conversion of the four remaining carbons to pyruvate and CO2 via malic enzyme generates about half the NADPH required for fatty acid synthesis. The pentose phosphate pathway contributes the rest of the needed NADPH.
Citrate Shuttle 6
• FAs are synthesized in the cytoplasm from acetylCoA
• AcetylCoA generated from pyruvate by the action of PDH
and by β-oxidation of fatty acids is in the mitochondria.
• For fatty acid biosynthesis, acetylCoA has to be transported
from the mitochondria to the cytoplasm. This is done via a
shuttle system called the Citrate Shuttle.
• AcetylCoA reacts with oxaloacetate to give citrate. A
tricarboxylate translocase transports citrate from
mitochondria to cytosol.
• In the cytosol, citrate is cleaved back to oxaloacetate and
acetylCoA. This reaction is catalyzed by ATP-citrate lyase
and requires the hydrolysis of one molecule of ATP.
Fatty Acid Biosynthesis Is Tightly Regulated
When a cell or 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. a) increasing Vmax.
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, reducing the flow of carbon through glycolysis. Acetyl-CoA carboxylase is also regulated by covalent modification. Phosphorylation, triggered by the hormones glucagon and epinephrine, inactivates the enzyme and reduces its sensitivity to activation by citrate, thereby slowing fatty acid synthesis. In its active (dephosphorylated) form, acetyl-CoA carboxylase polymerizes into long filaments (Fig.b above); 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 phosphorylationdephosphorylation cycle. The plant enzyme is activated by an increase in stromal pH and [Mg2], which occurs on illumination of the plant. Bacteria do not use triacylglycerols as energy stores. In E. coli, the primary role of fatty acid synthesis is to provide precursors for membrane lipids; the regulation of this process is complex, involving guanine nucleotides (such as ppGpp) 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 a wide range of lipogenic enzymes in the liver is suppressed. The detailed mechanism by which these genes are regulated is not yet clear.
If fatty acid synthesis and oxidation were to proceed simultaneously, the two processes would constitute a futile cycle, wasting energy. Oxidation is blocked by malonyl-CoA, which inhibits carnitine acyltransferase I. Thus during fatty acid synthesis, the production of the first intermediate, malonyl-CoA, shuts down oxidation at the level of a transport system in the mitochondrial inner membrane. This control mechanism illustrates another advantage of segregating synthetic and degradative pathways in different cellular compartments.
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