Lipid Metabolism Lecture 8 PDF

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 email: [email protected]  Energy expenditure during exercise http://highered.mheducation.com/sites/00725...

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 email: [email protected]  Energy expenditure during exercise http://highered.mheducation.com/sites/0072507470/student_view0/ch apter25/animation__energy_sources_for_prolonged_exercise.html Mathews, van Holde, Ahern: Biochemistry 3rd edition Scanning electron micrograph of an adipose cell Garett & Grisham: Biochemistry 4th edition  Stored metabolic fuel in a 70-kg person Advantages for storing energy in the form of fatty acids: 1. The carbon in fatty acids is almost completely reduced compared to the carbon in sugars or amino acids. Therefore, oxidation of fatty acids will yield more energy in form of ATP than any other form of carbon. 1-Palmitoyl-2,3-dioleoyl-glycerol 2. Fatty acids are not generally as hydrated as monosaccharides are, and thus they can pack more closely in storage tissues. Voet, Voet: BIOCHEMISTRY 3rd edition Garett & Grisham: Biochemistry 4th edition Primary sources of TAGs Diet De novo biosynthesis in the liver Storage depots in adipocytes or adipose cells. Because of their insolubility, fats are usually emulsified with bile salts or complexed with proteins as lipoproteins © 2016 Pearson Education, Ltd.  Synthesis of TAG and its deposition in adipose cells is unlimited Mobilization of stored fat (lipolysis) is hormonally controlled via cAMP Mediated by epinephrine during stress situations and by glucagon during fasting Other hormones regulate the process under different conditions (parathyroid hormone etc.) Garett & Grisham: Biochemistry 4th edition  Alkaline pancreatic juice secreted into the duodenum raises the pH of the digestive mixture, allowing hydrolysis of the triacylglycerols by pancreatic lipase and by nonspecific esterases. These processes depend upon the presence of bile salts. These agents act as detergents to emulsify the triglycerols and facilitate the hydrolytic activity of the lipases and esterases. The fatty acids pass into the epithelial cells, where they are condensed with glycerol to form new triacylglycerols, which aggregate with lipoproteins to form particles called chylomicrons. Garett & Grisham: Biochemistry 4th edition  Bile salts emulsify TAGs in the intestine Conjugation site © 2016 Pearson Education, Ltd. Bile acids are derivative of cholesterol Synthesized in the hepatocytes Cholesterol is converted into the bile acid cholic and chenodeoxycholoic acid These bile acids are conjugated to glycine or taurine to yield anions called bile salts © 2016 Pearson Education, Ltd.  Generalized plasma lipoprotein structure The spherical particle, part of which is shown, has a hydrophobic inner core (yellow) composed of cholesterol esters and triacylglycerols surrounded by a hydrophilic surface formed by the polar head groups of phospholipids and free cholesterol. © 2016 Pearson Education, Ltd.  Classification of lipoproteins ©Function 2016 Pearson Education, Delivery of Delivery of Delivery of Delivery of Picking up Ltd. dietary dietary and dietary and cholesterol excess fatty acids other other cholesterol from fatty acids fatty acids cells for delivery back to the liver  Fate of chylomicrons LPL- lipoprotein lipase; ApoE, ApoCII - lipoproteins Mark’s Basic Medical Biochemistry: A clinical Approach,, 4th Edition  Binding of a chylomicron to lipoprotein lipase on the inner capillary surface Breakdown of chylomicrons (and VLDL) at capillaries for hydrolysis of TAG to glycerol and free fatty acids for uptake into cells Lipoprotein lipase, like other lipases, is a member of the serine esterase family (like trypsin) with active site Ser, His, and Asp © 2016 Pearson Education, Ltd. Once a fatty acid enters the cytosol of a cell that need the energy, three successive processes must occur: 1. Activation The fatty acid must be activated by conversion to fatty acyl-CoA. 2. Transport The fatty acyl-CoA, which can not cross the mitochondrial membrane by diffusion, must be transported from the cytosol into the mitochondrial matrix. Carnitine, an amino-oxy acid, undergoes an ester-formation exchange reaction with the fatty acyl-CoA, resulting in a fatty acyl-carnitine ester that moves across the membrane into the mitochondria by facilitated diffusion. 3. The β-oxidation pathway Franz Knoop - fatty acids must be degraded by oxidation at the b-carbon, followed by cleavage of the Cα-Cβ bond. Albert Lehninger - this degradative process took place in the mitochondria. F. Lynen and E. Reichart - the two carbon unit released is acetyl-CoA. In mammalian cells, b-oxidation take place primarily in mitochondria, but a similar pathway occurs in peroxisomen. Acetyl-CoA Mitochondrial b-oxidation provides energy to the organism, whereas peroxisomal b-oxidation is responsible for shortening long-chain fatty acids that are poor substrates for mitochondrial b-oxidation.  For long-chain fatty acids, this reaction occurs at the outer membrane in higher eukaryotes before entry of the fatty acid into the mitochondria. The overall reaction has a net DGO’ of about - 0.8 kJ/mol, so the reaction is favorable but easily reversible. However, the pyrophosphate produced in this reaction is rapidly hydrolyzed by inorganic pyrophosphatase to two molecules of phosphate, with a net DGO’ of about -33.6 kJ/mol. Thus, pyrophosphate is maintained at a low concentration in the cell, and the synthetase reaction is strongly promoted. Garett & Grisham: Biochemistry 4th edition  Long-chain fatty acyl-CoA derivatives must be converted to acylcarnitine derivatives. Carnitine acyltransferase I, associated with the outer mitochondrial membrane, catalyzes the formation of the O- acylcarnitine, which is then transported across the inner membrane by a translocase. At this point, the acylcarnitine is passed to carnitine acyltransferase II on the matrix side of the inner membrane, which transfers the fatty acyl group back to CoA to re-form the fatty acyl-CoA, leaving free carnitine, which can return across the membrane via the translocase. Mary K. Campbell, Shawn O. Farrell: Biochemistry, 6th Edition  The total energy output from fatty acid catabolism, like that from glucose catabolism, is measured by the total number of ATP molecules produced. Each round of oxidation produces one NADH, one FADH2 and one Acetyl-CoA. Oxidation of acetyl-CoA via the citric acid cycle generates additional FADH2 and NADH which are reoxidized through oxidative phosphorylation to form ATP.  Oxidation of fatty acids The route of metabolism for a fatty acid depends somewhat on its chain length. Fatty acids are generally classified as: very long-chain fatty acids (> C20), long-chain fatty acids (C12 to C20), medium-chain fatty acids (C6 to C12), and short-chain fatty acids (C4). 1. Saturated fatty acids 2. Unsaturated fatty acids 3. Fatty acids with odd numbered carbon chain COT, carnitine octanoyltransferase; CAT, carnitine acetyltransferase; CAC, carnitine acylcarnitine carrier; CPTI, carnitine palmitoyltransferase I; CPTII, carnitine palmityltransferase II  Palmitoyl-CoA + 7 CoA-SH + 7 FAD + 7 NAD + + 7 H2O 8 Acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+ Reaction ATP Yield Activation of palmitate to palmitoyl-CoA -2 Oxidation of 8 acetyl-CoA 8 x 10 = 80 Oxidation of 7 FADH2 7 x 1.5= 10.5 Oxidation of 7 NADH 7 x 2.5= 17.5 Net: Palmitate → CO2 + H2O 106 Mathews, van Holde, Appling, Anthony-Cahill: BIOCHEMISTRY 4rd edition  Energy yield for one cycle of β-oxidation of palmitic acid 17 ATP http://www.wiley.com/college/pratt/0471393878/student/exercises/chapter_14.html  Energy yield for the complete β-oxidation of palmitic acid 1. 2. 3. 4. 5. 6. 7. http://www.wiley.com/college/pratt/0471393878/student/exercises/chapter_14.html  Very-long-chain fatty acids begin β- oxidation in the peroxisomes. This process is almost identical to β- oxidation in the mitochondria, with one key difference. Instead of reducing ubiquinone in the first step, the peroxisomes produce hydrogen peroxide. ω-oxidation is an alternative pathway in some animal species. 15ATP minor catabolic pathway for medium chain fatty acid become more important when β-oxidation is defect. http://www.wiley.com/college/pratt/0471393878/student/exercises/chapter_14.html  Oxidation of fatty acids with odd numbered carbon chains The final product of b-oxidation of odd-carbon fatty acids is the three-carbon propionyl-CoA. The pathway involves an initial carboxylation at the a- carbon of propionly-CoA to produces D-methylmalonyl- CoA. The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxylation of biotin, followed by nucleophilic attack by the a-carbanion of propionyl-CoA in a stereospecific manner. Conversion of propionyl-CoA to succinyl-CoA is carried out by a trio of enzymes. Succinyl-CoA can enter the TCA cycle. Leninger Principles of BIOCHEMISTRY 4rd edition Voet, Voet: BIOCHEMISTRY 3rd edition Unsaturated fatty acids are also catabolized by b-oxidation, but two additional mitochondrial enzymes: – an isomerase and – a novel reductase are required to handle the cis double bonds. As shown for linoleic acid, b-oxidation proceeds three cycles, and enoyl-CoA isomerase converts the cis-D3 double bond to trans-D2 double bond to permit one more round of b-oxidation. What results is a cis-D4 enoyl-CoA, which is oxidized to the trans-D2, cis-D4 species by acyl-CoA dehydrogenase. The subsequent action of 2,4-dienoyl-CoA reductase yields the trans-D3 product, which is converted by enoyl-CoA ismerase to the trans-D2 form. Garett & Grisham: Biochemistry 4th edition  The process in which acetyl-CoA is converted to three important metabolites: acetone, acetoacetate and b-hydroxybutyrate is known as ketogenesis, and the metabolites are known as ketone bodies. Ketone bodies are synthesized primarily in the liver (mitochondrial matrix) but are important sources of fuel and energy for many tissues, including brain, heart, and skeletal muscle. Ketone bodies are easily transportable forms of fatty acids that move through the circulatory system without the need for complex formation with serum albumin and other fatty acid-binding proteins. Garett & Grisham: Biochemistry 4th edition  Acetoacetyl-CoA b-Hydroxy-b-methyl- glytaryl CoA Acetone Acetoacetate D-b-Hydroxybutyrate The Lynen cycle describes the formation of acetoacetate from two molecules of Acetyl-CoA. The cycle results in the formation of 2 HSCoA molecules and 1 acetoacetate. Reconversion of ketone bodies to acetyl-CoA in the mitochondria of many tissues (other than liver) provides significant metabolic energy. Karlson, P.: Biochemistry for medicine and science 13th edition Voet, Voet: BIOCHEMISTRY 3rd edition Acetyl-CoA is a key intermediate between fat and carbohydrate metabolism Arrows identify major routes of formation or utilization of acetyl-CoA. Citrate serves as a carrier to transport acetyl units from the mitochondrion to the cytosol for fatty acid synthesis. Note that acetyl-CoA is readily converted into fatty acids, but acetyl- CoA cannot undergo net conversion to carbohydrate.  A comparison of fatty acid Voet, Voet: BIOCHEMISTRY 3rd edition  Amino acids CO2 NAD+ NAD+ Glycolysis NADH + H Pyruvate NADH + H dehydrogenase Glucose © 2016 Pearson Education, Ltd.  acetyl-CoA carboxylase The carboxylation of acetyl-CoA to form Malonyl-CoA malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids. The reaction is catalyzed by acetyl-CoA carboxylase (ACC), which contains a biotin prosthetic group and is regulated by phosphorylation, allosteric modification and induction/repression if its synthesis. Citrate allosterically activates acetyl CoA carboxylase by causing the individual enzyme molecules to polymerize. Palmitoyl-CoA, the final product of fatty acid biosynthesis, inhibits acetyl CoA carboxylase. Garett & Grisham: Biochemistry 4th edition  The fatty acid synthase complex has seven different active sites In E. coli and some plants, the seven active sites for fatty acid synthesis (six enzymes and ACP) reside in seven separate polypeptides. In these complexes, each enzyme is positioned with its active site near that of the preceding and succeeding enzymes of the sequence. The flexible pantetheine arm of ACP can reach all the active sites, and it carries the growing fatty acyl chain from one site to the next; intermediates are not released from the enzyme complex until it has formed the finished product. © 2016 Pearson Education, Ltd.  The b-ketoacyl-ACP synthase catalyzes the decarboxylative condensation of the acyl group with malonyl-ACP to produce an acetoacetyl-ACP. There are two crucial differences between fatty acid biosynthesis and fatty acid oxidation: 1. the alcohol formed in biosynthesis has the D-configuration rather than the L-form; 2. the reducing coenzyme is NADPH, whereas NAD+ and FAD are the oxidants in the catabolic pathway. Lehninger: Principles of Biochemistry, 4th edition  Shorter chains are easily made if the chain is released before reaching 16 carbons in length. Longer chains are made through special elongation reactions, which occur both in the mitochondria and at the surface of the endoplasmatic reticulum (ER). Elongation in the ER involves the successive condensations of malonyl-CoA with acyl-CoA and the NADPH-associated reduction. The mitochondrial reactions involve addition (and subsequent reduction) of acetyl units. These reactions are essentially a reversal of fatty acid oxidation, with the exception that NADPH is utilized in the saturation of the double bond, instead of FADH2. Voet, Voet: BIOCHEMISTRY 3rd edition Both pro-and eukaryotes are capable of introducing a single cis double bound in a newly synthesized fatty acid. Bacteria and eukaryotes carry it out in an O2-independent and O2-dependent pathway, respectively. In eukaryotes this reaction is catalyzed by a desaturase. NADH, O2 and the two proteins cytochrome b5 reductase and cytochrome b5 are required. All these proteins are associated with the ER membrane. Cytochrome b5 reductase transfers a pair of e- from NADH through FAD to cytochrome b5. Oxidation of reduced cytochrome b5 is coupled to reduction of nonheme Fe3+ to Fe2+ in the desaturase. Thus Fe3+ accepts a pair of e- from cytochrome b5 and creates a cis double bond in the fatty acid. O2 is the terminal e- acceptor in the fatty acyl desaturation cycle. Note that two H2O molecule are made, which means that four e- are transferred overall. Two of these come through the reaction sequence from NADH, and two come from the fatty acyl substrate that is being dehydrogenated. Mathews, van Holde, Ahern: Biochemistry 3rd edition Voet, Voet: BIOCHEMISTRY 3rd edition  Control of fatty acid synthesis Insulin promotes glucose uptake, promotes dephosphorylation (activation) of pyruvate dehydrogenase, citrate lyase, acetyl-CoA carboxylase (ACC) AMP-activated protein kinase and protein kinase A promote phosphorylation of acetyl-CoA carboxylase (inhibition) Citrate (activation) and long-chain fatty acids (inhibition) are also allosteric modulators of ACC Malonyl CoA inhibits carnitine acyl transferase of the outer mitochondrial membrane inhibiting the transport of fatty acyl CoA into mitochondrial matrix. © 2016 Pearson Education, Ltd.