Energy Storage Fatty acid synthesis is regulated, both in plants and animals.

Excess carbohydrate and protein in the diet are converted into fat. Only a relatively small amount of energy is stored in animals as glycogen or other carbohydrates, and the level of glycogen is closely regulated.Protein storage doesnt take place in animals. Except for the small amount that circulates in the cells, amino acids exist in the body only in muscle or other proteincontaining tissues. If the animal or human needs specific amino acids, they must either be synthesized or obtained from the breakdown of muscle protein. Adipose tissue serves as the major storage area for fats in animals. A normal human weighing 70 kg contains about 160 kcal of usable energy. Less than 1 kcal exists as glycogen, about 24 kcal exist as amino acids in muscle, and the balancemore than 80 percent of the totalexists as fat. Plants make oils for energy storage in seeds. Because plants must synthesize all their cellular components from simple inorganic compounds, plantsbut usually not animalscan use fatty acids from these oils to make carbohydrates and amino acids for later growth after germination. Fatty Acid Biosynthesis The biosynthetic reaction pathway to a compound is usually not a simple opposite of its breakdown. Chapter 12 of Volume 1 discusses this concept in regard to carbohydrate metabolism and gluconeogenesis.In fatty acid synthesis, acetyl-CoA is the direct precursor only of the methyl end of the growing fatty acid chain. All the other carbons come from the acetyl group of acetyl-CoA but only after it is modified to provide the actual substrate for fatty acid synthase, malonyl-CoA. Malonyl-CoA contains a 3-carbon dicarboxylic acid, malonate, bound to Coenzyme A. Malonate is formed from acetyl-CoA by the addition of CO2 using the biotin cofactor of the enzyme acetyl-CoA carboxylase. HCO3 Acetyl-CoA + HCO3 + ATP Malonyl-CoA + ADP + Pi Formation of malonyl-CoA is the commitment step for fatty acid synthesis, because malonyl-CoA has no metabolic role other than serving as a precursor to fatty acids. Fatty acid synthase (FAS) carries out the chain elongation steps of fatty acid biosynthesis. FAS is a large multienzyme complex. In mammals, FAS contains two subunits, each containing multiple enzyme activities. In bacteria and plants, individual proteins, which associate into a large complex, catalyze the individual steps of the synthesis scheme.
HO C C C SCoA H H OO

BIOCHEMISTRY II 21 LIPID BIOSYNTHESIS

Initiation Fatty acid synthesis starts with acetyl-CoA, and the chain grows from the tail end so that carbon 1 and the alpha-carbon of the complete fatty acid are added last. The first reaction is the transfer of the acetyl group to a pantothenate group of acyl carrier protein (ACP), a region of the large mammalian FAS protein. (The acyl carrier protein

is a small, independent peptide in bacterial FAS, hence the name.) The pantothenate group of ACP is the same as is found on Coenzyme A, so the transfer requires no energy input: Acetyl~S-CoA + HS-ACP HS-CoA + Acetyl~S-ACP In the preceding reaction, the S and SH refer to the thio group on the end of Coenzyme A or the pantothenate groups. The ~ is a reminder that the bond between the carbonyl carbon of the acetyl group and the thio group is a high energy bond (that is, the activated acetyl group is easily donated to an acceptor). The second reaction is another transfer, this time, from the pantothenate of the ACP to cysteine sulfhydral (SH) group on FAS. Acetyl~ACP + HS-FAS HS-ACP + Acetyl~S-FAS Elongation The pantothenate SH group is now ready to accept a malonyl group from malonyl-CoA: Figure 2-1
H+ (acyl carrier protein) acetyltransferase (1) CH3 C SCoA ACP ACP S O + + ACP CH3 C S ACP O + CH3 C CoA O + -ketoacyl(acyl carrier protein) synthase (3) (a) CH3 C SH SACP SACP SACP SACP O + CoA (acyl carrier protein) malonyltransferase OOC (2) CH2 C SCoA O CH2 C O OOC CH2 Enz Enz CH2 OOC S Enz + -ketoacyl(acyl carrier protein) reductase (4) CH3 C O C O SACP + CO2 + Enz + NADPH + H+ enoyl(acyl carrier protein) reductase

NADPH + -hydroxyacyl(acyl carrier protein) dehydratase (5) CH3 CH OH + -ketoacyl(acyl carrier protein) synthase (b) CH3 C O CH3 C CH2 SH O C O SACP NADP + + + H2O CH3 CH CH2 OH CH2 C O SACP NADP + CH3 CH2 CH2 C + O C SACP O C O CH3 CH CH C SACP O CH3 CH CH C SACP + O (6)

22 CLIFFSQUICKREVIEW
LIPID BIOSYNTHESIS

Note that at this point, the FAS has two activated substrates, the acetyl group bound on the cysteine SH and the malonyl group bound on the pantothenate SH. Transfer of the 2-carbon acetyl unit from Acetyl~S-cysteine to malonyl-CoA has two features: s Release of the CO2 group of malonyic acid that was originally put on by acetyl-CoA carboxylase s Generation of a 4-carbon -keto acid derivative, bound to the pantothenate of the ACP protein The ketoacid is now reduced to the methylene (CH2) state in a three-step reaction sequence. 1. Reduction by NADPH to form the -hydroxy acid derivative: 2. Dehydration, that is, the removal of water to make a transdouble bond: 3. Reduction by NADPH to make the saturated fatty acid:
H3 C C C C H H H H SACP O HC C C C HH HH SACP O

HCCC H H H H O C S ACP S ACP O C O HCCC H H H H H NADPH NADP OH

BIOCHEMISTRY II 23 LIPID BIOSYNTHESIS

The elongated 4-carbon chain is now ready to accept a new 2carbon unit from malonyl-CoA. The 2-carbon unit, which is added to the growing fatty acid chain, becomes carbons 1 and 2 of hexanoic acid (6-carbons). Release The cycle of transfer, elongation, reduction, dehydration, and reduction continues until palmitoyl-ACP is made. Then the thioesterase activity of the FAS complex releases the 16-carbon fatty acid palmitate from the FAS. Note that fatty acid synthesis provides an extreme example of the phenomenon of metabolic channeling: neither free fatty acids with more than four carbons nor their CoA derivatives can directly participate in the synthesis of palmitate. Instead they must be broken down to acetyl-CoA and reincorporated into the fatty acid. Fatty acids are generated cytoplasmically while acetyl-CoA is made in the mitochondrion by pyruvate dehydrogenase.This implies that a shuttle system must exist to get the acetyl-CoA or its equivalent out of the mitochondrion. The shuttle system operates in the following way: Acetyl-CoA is first converted to citrate by citrate synthase in the TCA-cycle reaction. Then citrate is transferred out of the mitochondrion by either of two carriers, driven by the electroosmotic gradient: either a citrate/phosphate antiport or a citrate/malate antiport as shown in Figure 2-2. 24 CLIFFSQUICKREVIEW
LIPID BIOSYNTHESIS BIOCHEMISTRY II 25 LIPID BIOSYNTHESIS

Figure 2-2 After it is in the cytosol, citrate is cleaved to its 2- and 4-carbon components by citrate lyase to make acetyl-CoA and oxaloacetate. Citrate lyase requires ATP.
citrate 3 phosphate 3 phosphate 3 citrate 3

Matrix Intermembrane citrate 3 malate 2 malate 2 citrate 3

Fatty acid biosynthesis (and most biosynthetic reactions) requires NADPH to supply the reducing equivalents. Oxaloacetate is used to generate NADPH for biosynthesis in a two-step sequence. The first step is the malate dehydrogenase reaction found in the TCA cycle. This reaction results in the formation of NAD from NADH (the NADH primarily comes from glycolysis). The malate formed is a substrate for the malic enzyme reaction, which makes pyruvate, CO2, and NADPH. Pyruvate is transported into the mitochondria where pyruvate carboxylase uses ATP energy to regenerate oxaloacetate. Palmitate is the starting point for other fatty acids that use a set of related reactions to generate the modified chains and head groups of the lipid classes. Microsomal enzymes primarily catalyze these chain modifications. Desaturation uses O2 as the ultimate electron acceptor to introduce double bonds at the nine, six, and five positions of an acyl-CoA. Elongation is similar to synthesis of palmitate because it uses malonyl-CoA as an intermediate. See Figure 2-3. 26 CLIFFSQUICKREVIEW
LIPID BIOSYNTHESIS

Figure 2-3
R CH2 C SCoA O R CH2 C CH2 O C SCoA O CH3 C SCoA CoASH O -ketothiolase NADH + H + NAD + -hydroxyacyl CoA dehydrogenase NADPH + H + NADP + enoyl CoA reductase CH2 CH OH R CH2 H2 O C SCoA O R CH2 CH CH C SCoA O R CH2 CH2 CH2 C SCoA O enoyl CoA hydratase

BIOCHEMISTRY II 27

LIPID BIOSYNTHESIS

Synthesis of Triacylglycerols Glycerol accepts fatty acids from acyl-CoAs to synthesize glycerol lipids. Glycerol phosphate comes from glycolysisspecifically from the reduction of dihydroxyacetone phosphate using NADH as a cofactor. Then the glycerol phosphate accepts two fatty acids from fatty acyl-CoA. The fatty acyl-CoA is formed by the expenditure of two high-energy phosphate bonds from ATP. Fatty acyl-CoA is the donor of the fatty acyl group to the two nonphosphorylated positions of glycerol phosphate to make a phosphatidic acid. The third fatty acid can be added after the removal of the phosphate of the phosphatidic acid. This scheme results in a triacylglycerol, although other phosphatidic acids can be used as precursors to various membrane lipids.
H2 C C R1 HC O C O R2 O O O H2 C O P O O R C OH + ATP + CoASH + AMP + PPi O R C SCoA O

28 CLIFFSQUICKREVIEW
LIPID BIOSYNTHESIS

Cholesterol Biosynthesis and its Control Despite a lot of bad press, cholesterol remains an essential and important biomolecule in animals. As much as half of the membrane lipid in a cellular membrane is cholesterol, where it helps maintain constant fluidity and electrical properties. Cholesterol is especially prominent in membranes of the nervous system. Cholesterol also serves as a precursor to other important molecules. Bile acids aid in lipid absorption during digestion. Steroid hormones all derive from cholesterol, including the adrenal hormones that maintain fluid balance; Vitamin D, which is an important regulator of calcium status; and the male and female sex hormones. Although humans wouldnt survive in one sense or another without cholesterol metabolites, cholesterol brings with it some well-known side effects. Doctors find cholesterol derivatives, being essentially insoluble in water, in the deposits (plaque) that characterize diseased arteries. Isoprenoid Compounds Cholesterol is synthesized from acetyl-CoA in the liver. Cholesterol and a number of natural products from plants (including rubber) are isoprenoid compounds. The isoprenoid unit is a 5-carbon structure. Isoprenoid compounds are synthesized from a common intermediate, mevalonic acid. Mevalonate is synthesized from acetyl-CoA and then serves as the precursor to isoprenoid units.
CCCC

BIOCHEMISTRY II 29 LIPID BIOSYNTHESIS

acetyl-CoA mevalonate The key enzyme in this pathway is HMG-CoA reductase in connection with ketone body formation. The reactions leading to HMGCoA are shared with that pathway. Acetyl-CoA can be derived directly from metabolism, especially lipid degradation, or by the acetate thiokinase reaction: Two acetyl-CoA molecules condense to form acetoacetyl-CoA. Finally, the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) adds a third acetyl-CoA.
CH3 + CoASH O C OH C CH2 C CH3 SCoA O OC O CH2 C CH2 SCoA C SCoA HO O H3 C H2 O
H3C C SCoA + + CoASH O H3C C SCoA O C SCoA O H3C C CH2 O

H3C C OH + ATP + CoASH + AMP + PPi O H3C C SCoA O

30 CLIFFSQUICKREVIEW
LIPID BIOSYNTHESIS

HMG CoA Reductase HMG-CoA reductase is the committed and therefore the regulatory step in cholesterol biosynthesis. If HMG-CoA is reduced to mevalonate, cholesterol is the only product that can result. The reduction is a two-step reaction, which releases the Coenzyme A cofactor and converts the thiol-bound carboxylic group of HMG-CoA to a free alcohol. Two NADPH molecules supply the reducing equivalents because the thioester must first be reduced to the level of an aldehyde

and then to an alcohol. Mevalonate Squalene Mevalonate molecules are condensed to a 30-carbon compound, squalene. The alcohol groups of mevalonate are first phosphorylated. Then they multiply phosphorylated mevalonate decarboxylates to make the two compounds isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). mevalonate phosphomevalonate pyrophosphomevalonate This is a simple sequence of two phosphorylations using ATP as the donor:
C O O CH2 SCoA 2 NADPH 2 NADP CoASH + H2 C C CH3 C OH HO H2COH O CH2 H2 C C CH3 C OH HO

BIOCHEMISTRY II 31 LIPID BIOSYNTHESIS 32 CLIFFSQUICKREVIEW LIPID BIOSYNTHESIS

pyrophosphomevalonate isopentenyl pyrophosphate dimethylallyl pyrophosphate. First, the other hydroxyl group of mevalonate accepts a phosphate from ATP. The resulting compound rearranges in an enzyme-catalyzed reaction, eliminating both CO2 and phosphate. The 5-carbon compound that results, IPP, is rapidly isomerized with DMAPP. geranyl and farnesyl pyrophosphates squalene In plants and fungi, IPP and DMAPP are the precursors to many so-called isoprenoid compounds, including natural rubber. In animals, they are mainly precursors to sterols, such as cholesterol. The first step is condensation of one of each to geranyl pyrophosphate, which then condenses with another molecule of IPP to make farnesyl pyrophosphate. Some important membrane-bound proteins have a farnesyl group added on to them; however, the primary fate of farnesyl pyrophosphate is to accept a pair of electrons from NADPH and
CH2 C CH3 H2C CH3

COO O CH2 O CH3 CH2 C IPP CO2 Pi P P P CH2 O P P H3C CH3 CH C DMAPP CH2 O P P
P CH2 CH2OH C CH2 HO COOH CH3 CH2 CH2O C CH2 HO COOH CH3 ATP ADP ATP ADP P CH2 CH2O C CH2 HO COOH CH3 P

condense with another molecule of itself to release both pyrophosphate groups. The resulting 30-carbon compound is squalene; it folds into a structure that closely resembles the structure of the steroid rings, although the rings are not closed yet. Squalene Lanosterol The first recognizable steroid ring system is lanosterol; it is formed first by the epoxidation of the double bond of squalene that was originally derived from a DMAPP through farnesyl pyrophosphate, and
C C CH2 C HH CH2 C CH2 CH2 C H H3 C H3 C C CH2 CH2

H C CH3 CH3 C CH3 CH2 CH2 H C C CH3 CH3 CH2 CH2 H CC CH3

C C CH2 C H CH2 C CH2 CH2 C H H3 C H3 C C CH2O P P ( 2) CH3 H CH3 C C CH2 C + H CH2 C PPi H H3 C H3 C CH2O P P CH3 DMAPP PP C C CH2O P P + H C H H3 C H H3 C C CH2O P P CH2 H3 C

BIOCHEMISTRY II 33 LIPID BIOSYNTHESIS 34 CLIFFSQUICKREVIEW LIPID BIOSYNTHESIS

then by the cyclization of squalene epoxide. The enzyme that forms the epoxide uses NADPH to reduce molecular oxygen to make the epoxide. See Figure 2-4. Figure 2-4
CH3 CH3 CH3 CH3 Squalene 2,3-epoxide cyclase CH3 CH3

CH3 CH3 O Lanosterol H+ CH3 CH3 CH3 CH3 CH3 CH3 HO CH3 H CH3
14

Lanosterol Cholesterol This sequence of reactions is incompletely understood but involves numerous oxidations of carbon groups, for example, the conversion of methyl groups to carboxylic acids, followed by decarboxylation. The end product, cholesterol, is the precursor to cholesterol esters in the liver and is transported to the peripheral tissues where it is a precursor to membranes (all cells), bile salts (liver), steroid hormones (adrenals and reproductive tissues), and vitamin D (skin, then liver, and finally kidney). Cholesterol Transport, Uptake, and Control Cholesterol is exported to the peripheral tissues in LDL and VLDL (see Chapter 1). About 70 percent of the cholesterol molecules in LDL are esterified with a fatty acid (for example, palmitate) on the OH group (at Carbon 3; see Figure 2-5). Cells take up cholesterol from the LDL by means of LDL receptors in the outer cell membrane. Figure 2-5
H3C(CH2)14 CO O cholesterol ester

BIOCHEMISTRY II 35 LIPID BIOSYNTHESIS

The pathway for uptake involves several steps, including the following: 1. The assembly of the receptor-LDL complexes into a coated pit on the cell surface. 2. The pit folds into a spherical endosome, which is a small vesicle of cell membrane with receptor-LDL complexes on the inside. 3. The endosome fuses with a lysosome containing a large number of degradative enzymes and a low pH on the inside. 4. The receptors separate from the endosome-lysosome and return to the cell surface. 5. The cholesterol esters are hydrolyzed to free cholesterol. 6. The free cholesterol inhibits the synthesis and/or causes the degradation of HMG-CoA reductase and of LDL receptor. This last step ensures that more cholesterol will not be taken up or made than is needed. 7. Cholesterol is re-esterified with a fatty acid for storage inside

the cell. 36 CLIFFSQUICKREVIEW

Figure 2-6 A close connection exists between the regulation of cholesterol biosynthesis and uptake. When HMG-CoA reductase is inhibited, the cell responds by synthesizing more LDL receptors to ensure the uptake of cholesterol from the serum. When cholesterol is present in a high enough concentration in the cell, LDL receptors are not exported to the cell surface, an example of the phenomenon of down regulation. The tight regulation of cholesterol metabolism helps explain the pathology of coronary artery disease, a major killer in developed countries. Clearly, diet affects coronary artery disease: Individuals with high intake of saturated fat and cholesterol are most at risk. Furthermore, a high serum concentration of LDL cholesterol is associated with an increased risk of coronary artery disease.
LDL receptors LDL binding Internalization Lysosomal hydrolysis Regulatory actions LDL Protein Amino acids HMG-CoA reductase 1. LDL receptors Cholesterol 3. 2. ACAT Cholesteryl oleate Cholesteryl linoleate

The capability of LDL receptors to remove LDL cholesterol from the circulation can rationalize these clinical observations. If little cholesterol is available in the diet, the cells of the peripheral tissues respond by up-regulating the number of LDL receptors on the cell surface. The higher concentration of receptors means that more of the cholesterol will be removed from the circulatory system. Because the inappropriate deposition of cholesterol is a major contributor to blocked arteries, if the cholesterol is removed from the circulation, less risk of blockage exists. On the other hand, if a large amount of cholesterol exists in the diet, and the cells have enough for their needs, they will synthesize fewer LDL receptors, less cholesterol will be removed from the circulatory system, and the risk of artery disease increases further. Several therapies are used to treat individuals with high serum cholesterol levels. The first is a low-fat, low-cholesterol diet. If the diet provides less cholesterol, then the cells will synthesize more

LDL receptors to meet their needs, which means that more cholesterol will be removed from the circulation. The second therapy which goes hand in hand with the firstis to decrease the reabsorption of bile acids in the gut, for example, by increasing the amount of soluble fiber in the diet or by administering synthetic bile acid binders. Bile acids bind to these agents. Fiber and the synthetic binders cannot be absorbed by the intestines and are excreted, carrying the bile acids with them. Excess cholesterol is then converted to bile acids and ultimately excreted. The third method is to inhibit HMG-CoA synthesis with any of several drugs on the market. HMGCoA reductase carries out the committed step of cholesterol biosynthesis. Inhibiting this enzyme decreases the amount of cholesterol synthesized intracellularly, and the cells compensate by increasing the number of LDL receptors on the cell surface. This helps remove LDL cholesterol from the circulation.