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Transcript
CHAPTER 19 LIPID METABOLISM Introduction: Fats are much more efficient forms of energy storage than carbohydrates for two main reasons. They are much more highly reduced than carbohydrates, indicated by a preponderance of C-H bonds for which the electrons “belong” to C, rather than C-O bonds, in which the electrons “belong” to O. Thus, during their catabolism there will be more electrons transferred to O2 per gram of fat than per gram of carbohydrate, thus more energy released (9 kcal/g fat compared to 4 kcal/g carbo). Since fats are highly insoluble in water, there is little or no water of hydration associated with stored fat, unlike the case for glycogen. A gram of hydrated fat yields more than six times as much energy as a gram of hydrated carbohydrate. A typical 70 kg man with about 11% body fat stored in the abdominal cavity, under the skin, will have about 100,000 kcal of energy stored as fat, as compared with about 600 kcal in glycogen. Initial phase of digestion digestion and absorption On the average, 40% or more of the daily energy requirements of humans in highly industrialized countries is supplied by dietary fat, although most nutritional guidelines recommend that figure be no more than 30%. Dietary fats are emulsified in the gut as mixed micelles of bile salts, which are synthesized from cholesterol (see Figure 19-1) in the liver and stored in the gallbladder. Water soluble, pancreatic lipase catalyzes the hydrolysis of dietary triglycerides to form monoand di-acyl glycerols, thereby liberating the fatty acids as their Na+ and K+ salts. Micelle formation greatly increases the accessibility of the enzyme to substrate. The hydrolysis products diffuse into the epithelial cells lining the intestinal mucosa (surface), where they are re-esterified and packaged into the core of chylomicrons, which deliver dietary triglycerides (and cholesterol) to skeletal muscle and adipose cells. Note that fatty acids can form micelles above a certain concentration (critical micelle concentration), which can have a detergent affect, desirable in the case of bile salts in the intestine, but very undesirable in a cell. Fatty acids are prevented from forming micelles by complexing with intestinal fatty acid binding protein. An extracellular lipase, lipoprotein lipase, hydrolyzes the core triglyceride as the chylomicrons bind to the endothelium (inner surface) of the capillaries in skeletal muscle and adipose tissue. Lipoprotein lipase is activated by a protein component of chylomicrons, apo CII. These tissues take up the hydrolyzed fatty acids, where they are re-esterified and either stored in adipocytes or catabolized in muscle. Very low density lipoprotein (VLDL), is synthesized in the liver and transports endogenous triglycerides and cholesterol to adipose and muscle. It delivers its core triglycerides much in the same way that chylomicrons delivers their dietary counterparts; that is, lipoprotein lipase, activated by apo C-II, hydrolyzes the triglycerides, whereupon they enter their target cell either for storage (adipocytes) or catabolism (myocytes). VLDL remnants appear first as intermediate density lipoproteins (IDL), then as low density lipoproteins (LDL). The core of an LDL particle is largely cholesteryl ester, and it is via LDL (“bad” cholesterol) that cholesterol is transported from liver to extrahepatic tissue. High density lipoprotein (HDL) removes cholesterol from extrahepatic cells, thus is involved in reverse cholesterol transport (“good” cholesteryl). (Cellular) Fatty acid oxidation Triglycerides in adipocytes are mobilized in a hormone-initiated (epinephrine) process involving cAMP much in the same way that cAMP initiates glycogen breakdown in muscle cells by activating protein kinase. (cAMP-dependent protein kinase). In this case, hormonesensitive lipase liberates the fatty acids by hydrolyzing the stored triglycerides. Once formed by the action of hormone-sensitive lipase, the fatty acids diffuse across the plasma membrane of an adipocyte and are released into the blood, where they are bound by serum albumin and transported to their target cells (see chapter 20). Fatty acids are primed, or activated, in the cytoplasm of the target cell by their conversion to the corresponding fatty acyl CoA, catalyzed by a member of the fatty acyl CoA synthetase family: O C O ATP 2 Pi AMP + PPi C S CoA O The activated fatty acyl CoA enters the mitochondria where catabolism takes place via a three step process (See Figure 19-11): 1. A transportable species, an acyl carnitine derivative, is formed via the catalytic action of transferase I; 2. The acyl carnitine is transported across the inner mitochondrial membrane by a carnitine carrier protein. Transferase II reforms the fatty acyl CoA once inside the mitochondrial matrix. + C SCoA HO O Transferases I CH3 N CH3 CH3 C O2 II C O O CH3 N CH3 CH3 C O2 The catabolism of the fatty acyl CoA to acetyl CoA occurs in the mitochondrial matrix via beta-oxidation in a series of reactions very similar to reactions in the citric acid cycle: O O C CH2 CH2 C O R Succinate C S CoA O FAD O FAD FADH2 FADH2 H O O C C C C Fumarate O O H H2O O C CH O CH2 OH R C S CoA O O C Malate O R C S CoA OH O NAD+ NAD+ NADH O C C O CH2 O H2O NADH O C Oxaloacetate O R C S CoA O O The final step in beta-oxidation involves the action of thiolase, which catalyzes the cleavage of the fatty acyl CoA into another fatty acyl CoA, whose acyl chain is shortened by two methylene groups, and acetyl CoA: B Enzyme CoASH C S CoA O Thiolase O + H+ C S CoA + H3C C S CoA O O For a 16-carbon fully saturated fatty acid (16:0 palmitate), this process will be repeated six more times (for a total of seven times) to yield 8 acetyl CoA. Beta oxidation is sometimes referred to as a cyclic process because it starts with and yields a fatty acyl CoA. However this is not a catalytic cycle, and the product fatty acyl CoA has its chained shortened by two carbons. The acetyl CoA generated by beta oxidation enter the citric acid cycle, where they are converted into CO2 and yield 12 ATP (three oxidations yielding NADH, one yielding FADH2 , and a substrate level phosphorylation). The ATP count for 16:0 is, noting that each round of beta oxidation yields 5 ATP (three per NADH + two per FADH2) # ATP = 7 rounds of beta oxidation x 5 ATP per round + 8 acetyl CoA x 12 ATP per acetyl CoA 2 = 129 Oxidation of unsaturated fatty acids requires two auxiliary enzymes (an isomerase and a reductase . Actually, there are two separate isomerases for a total of three). in addition to those of beta oxidation. Although double bonds are formed in beta oxidation they are trans as opposed to cis in naturally occurring unsaturated fatty acids. Additionally they are typically in the wrong location. See Figure 19-15 for a demonstration for a doubly unsaturated fatty acid. The first auxiliary enzyme, an isomerase, is required for mono unsaturated fatty acids, and is straightforward. Mono unsaturated fatty acids typically have their double bond between carbons 9 and 10. Thus, oleate is 18:1 “del”9. Three rounds of beta oxidation, each round removing two carbons, place the double bond at the number 3 position (9 6 = 3). The hydratase of beta oxidation requires the double bond to be in the trans configuration and at carbon 2. The first isomerase catalyzes the reconfiguring the cis, 3 double bond to a trans, 2 and the enzymes of beta oxidation do the rest. Note that the ATP count for an monounsaturated fatty acid is two less than the fully saturated analog (for 18:0, there are 8 x 5 + 9 x 12 2 = 146 ATP; for 18:1 there would be 144 ATP, two less than for 18:0 because one less FADH2 is generated. To process the second double bond in 18:2 “del”9, 12 , for example, the reductase and isomerase 2 are required. The action of the second auxiliary enzyme, a reductase, is a little trickier to understand. Refer to Figure 19-15. Since the double bonds are in the 9 and 12 positions for 18:2, for example, three rounds of beta oxidation can proceed as normal, resulting in the double bonds being at the 3 and 6 positions. Isomerase 1 can reconfigure the cis 3 double bond to a trans 2 double bond, and the enzymes of beta oxidation will yield a product 2 carbons shorter with a cis double bond at the 4 position (= 6 2). Beta oxidation will then proceed to form a trans-2, cis-4 product. The mammalian version of the second auxiliary enzyme, the reductase, reduces this to a singly unsaturated product with a trans double bond in the 3 position. Isomerase 2 then converts this to a trans, 2 double bond which can be processed via beta oxidation. Note that isomerase 1 converts a cis, 3 double bond to a trans, 2 double bond, whereas isomerase 2 converts a trans, 3 double bond to a trans,2 double bond. The ATP count is a little more complicated than it is for only one double bond. Anytime the reductase is involved, the ATP count will be three fewer than for a fully saturated version because, in addition to “losing” the FADH2 required to make a double bond, when the reductase is involved one double bond is reduced at the expense of an NADPH, then recreated yielding an FADH2. Thus we trade 3 ATP for 2. The ATP count for 18:2, then would be 148 2 2 3 = 141 (don’t forget the two ATP for activation). Oxidation of fatty acids with an odd number of carbons, produced by some plants and marine organisms, will eventually yield a product with an odd number of carbons since beta oxidation shortens the chain by an even number (2) of carbons. Thus 15:0 will yield a three carbon product, propionyl CoA after 6 rounds of beta oxidation (15 6x2 = 3). This 3-carbon product enters the citric acid cycle by acquiring an additional carbon to yield succinyl CoA: O H3C CH2 C S Propionyl CoA CoA ATP + CO2 Carboxylase ADP + Pi O O C HC O C CoA S (S)-Methylmalonyl CoA CH3 (R)-Methylmalonyl CoA Mutase O O C H2C CH2 C S CoA Succinyl CoA O NOTE: The carboxylase requires biotin as a cofactor. Recall the similarity between this reaction and the anaplerotic reaction of the citric acid cycle in which pyruvate was carboxylated in a biotin-assisted reaction to form oxaloacetate. In this case the product of the reaction is not succinyl CoA, the desired product, because direct formation of succinyl CoA from propionyl CoA would require carboxylation at the beta carbon (relative to the carbonyl carbon); however, as we know carboxylation occurs at the alpha carbon because of the acidity of the alpha carbon proton, making the alpha carbon a better nucleophile to attack the carboxylated biotin. NOTE: The mutase requires coenzyme B12 (Figure 19-17). This coenzyme has a heme-like tetrapyrrole ring containing a Co atom, rather than Fe+3, Fe+2/+3 for heme or Mg++ for chlorophylls. The Co C bond at the number 5 coordinated position is unique to biology, and the weakness of this bond leads to its dissociation, thereby facilitating the subsequent repositioning of a proton and acyl CoA group on the substrate to form succinyl CoA via a free radical intermediate (see Figure 19-19): H H O H C C C H C OO S CoA H H H C C C O C H O O S CoA O H H O CoAS C C C C O H H Succinyl CoA In general coenzyme B12 facilitates the “swap” of a proton and R group on adjacent carbons as shown in the first structure on the left above. Because of the catalytic nature of the citric acid cycle, succinyl CoA can only regenerate the catalyst, oxaloacetate. In order for succinyl CoA to be catabolized, it is first converted to pyruvate by an oxidative decarboxylation (malic enzyme) which can then be converted to acetyl CoA which is then catabolized to CO2 via the citric acid cycle. S CoA O NADP+ NADPH O O C CH2 H C OH C O CH2 CH2 CH2 C O O C O C O O C O C O O O O C Citric Acid Cycle Acetyl CoA C O CH3 NOTE: The bracketed intermediate, oxaloacetate, is converted to pyruvate by an enzymeassisted beta decarboxylation. Beta oxidation occurs in peroxisomes as well as mitochondria. These are specialized organelles in which reactions that generate potentially harmful free radicals and hydrogen peroxide (H2O2) are segregated. The hydrogen peroxide is degraded by catalase, which converts it to H2O and O2. The FADH2 generated during double bond formation in peroxisomes transfers its electrons directly to O2, thereby bypassing the electron transport chain and sacrificing the 2 ATP that are possible via transfer to system III in the electron transport chain. Thus, each round of beta oxidation in peroxisomes yields 3, rather than 5 ATP. Ketone Bodies Catabolism of fatty acids requires the citric acid cycle to process the acetyl CoA produced by beta oxidation. Since this is a catalytic cycle, the anaplerotic reaction that replenishes the supply of catalyst is critical to operation of the cycle. This reaction requires pyruvate, which in turn is the end product of gluconeogenesis. The meaning of the phrase, “fat burns in the flame of carbohydrate,” is thus clear. Without a well-balanced diet containing carbohydrate, citric acid cycle activity can be compromised. This is the premise of the Atkins diet. Under such conditions, or during prolonged starvation or uncontrolled diabetes, the acetyl CoA produced during beta oxidation accumulates due reduced citric acid cycle activity. Under such conditions the thiolase reaction can be reversed to form the penultimate product of beta oxidation, acetoacetyl CoA, which then is converted to ketone bodies: O O O H3C C SCoA + H3C C SCoA Thiolase O H3C C H2C C SCoA O + H3C C SCoA O O OH O O H3C C H2C C SCoA H3C C SCoA + H3C C H2C C Acetoacetate O CH2 C O O HMG CoA Acetoacetate is unstable because it can undergo spontaneous beta decarboxylation to form acetone. Acetoacetate is thus converted to a stable form by reducing the carbonyl group (which provides the electron sink in the decarboxylation) to a hydroxyl group. The resulting product, beta-hydroxy butyrate is stable and can be used as a transportable fuel: O O H3C C H2C C O NADH NAD+ O H3C C CH3 Acetone OH O H3C C H2C C O H Beta-OH butyrate It was once thought that ketone body formation was the result of metabolism gone astray, but it is now recognized that the liver forms ketone bodies for export to other tissues, as it does glucose. In fact, muscle cells’ major fuels are glucose, fatty acids and ketone bodies (see chapter 21). Beta-OH butyrate is converted to acetyl CoA in the target cells: OH O H3C C CH2 C H O NAD+ O NADH O H3C C CH2 C O Succinyl CoA Succinate O O H3C C CH2 C S CoA Thiolase O 2 H3C C SCoA Citric acid cycle Fatty Acid Biosynthesis Fatty acid biosynthesis is chemically very similar to the reverse of beta oxidation. However, beta oxidation and fatty acid biosynthesis utilize different enzymes and different cellular compartments; biosynthesis occurs in the cytoplasm. In eukaryotes the multiple enzyme functions associated with fatty acid synthesis are contained on a single polypeptide chain, fatty acid synthase, which is present as a homo-dimer. This is a variation on a theme of the kinase/phosphatase functions of the tandem enzyme involved in the formation/hydrolysis of fructose 2,6-bisphosphate in glycolysis/gluconeogenesis. Biosynthesis must also be exergonic overall to proceed spontaneously. Although acetyl CoA is the starting point, as it is the end point of beta oxidation, the actual donor of C-2 groups is malonyl CoA, an activated form of acetyl CoA. Malonyl CoA formation is catalyzed by acetyl CoA carboxylase in a biotindependent reaction very similar to the anaplerotic reaction of the citric acid cycle we studied (pyruvate to oxaloacetate), as well that catalyzed by propionyl CoA carboxylase in the catabolism of odd-number carbon fatty acids: O O C C S CoA S CoA CH2 CH3 ATP HCO3- ADP + Pi C O 2Malonyl CoA The different functions performed by fatty acid synthase are summarized in Figure 19-26: Note that reactions 4, 5 and 6 are the counterparts of beta oxidation. Reactions 1 3 involve the enzyme activities of the acyl carrier protein (ACP) and condensing enzyme (CE). The incoming malonyl CoA groups are transferred to ACP, which contains a phosphopanteine group identical to that of CoA (see Figure 19-25). The growing fatty acid is bound to CE via a thioester linkage. The condensation reaction consists of the CH2 group of the ACP-bound malonyl CoA displacing the S of the nascent fatty acyl moiety bound to CE (reaction 3). Notice that the nascent fatty acyl group, now increased in length by two carbons, is bound to ACP. The reduction steps (4 6) are carried out with substrate bound to ACP. Since the next incoming malonyl CoA must be bound to ACP, the next step in elongation necessitates a transfer of the nascent fatty group to CE. Finally, Finally, after seven rounds of elongation, palmitate (16:0) is released via the action of palmitoyl thioesterase (reaction 7). Since formation of each malonyl CoA required ATP, and since each round consumed two NADPH, the overall energy requirement for the synthesis of palmitate from eight acetyl CoA is 7 ATP 14 NADPH. Further elongation of saturated fatty acids, as well as the formation of unsaturated fatty acids, are carried out by different enzymes (elongases and desaturases) in the ER. Linoleic acid (18:2 )9, 12), however, cannot by synthesized by animals and is an essential nutrient. Linoleic acid is a precursor to prostaglandins and is an important constituent of the skins water permeability barrier. Regulation of fatty acid metabolism: (See Figure 19-27) Malonyl CoA and its formation provide the essence of this regulatory process. Recall that malonyl CoA inhibits the transferase I that denies access of malonyl CoA into the mitochondria where beta oxidation occurs. Acetyl CoA carboxylase, which catalyzes the formation of malonyl CoA is subject to modulation via phosphorylation/dephosphorylation. Phosphorylation via c-AMP-dependent protein kinase inhibits this enzyme. Thus glucacon inhibits fatty acid biosynthesis in the liver. This makes sense because glucagon is released during times of energy need, hence stimulates catabolic pathways and inhibits biosynthetic pathways. We already know that insulin stimulates an end to glycogen catabolism, so it should be logical that insulin also acts similarly in fatty acid metabolism by dephosphorylating acetyl CoA carboxylase, thus stimulating this enzyme and also fatty acid biosynthesis. Cholesterol Biosynthesis Cholesterol biosynthesis proceeds from acetyl CoA and involves formation of HMG CoA, thus having these steps in common with ketone body formation. Note that cholesterol biosynthesis occurs in the cytoplasm, whereas ketone body takes place in the mitochondria of a liver cell. The reduction of HMG CoA to form mevalonate is the next step in the biosynthesis of cholesterol and constitutes the key regulatory step in cholesterol biosynthesis. Treatment of patients with competitive inhibitors of HMG CoA reductase, namely fungal products compactin and lovastatin (See p. 607), which resemble mevalonate, can decrease endogenous cholesterol synthesis. This, together with ingestion of resins that bind bile salts constitutes a two-pronged drug therapy that can lower serum cholesterol levels, a risk factor in the development of atherosclerosis. See p. 606 for a more complete discussion. Mevalonate is then converted to the isoprene-containing isopentenyl pyrophosphate, six of which are converted to squalene, which is then cyclized to form the steroid nucleus of cholesterol in a nifty little reaction involving epoxide formation (Figure 19-40). Problems: 1, 2, 3, 6