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Fatty Acid Oxidation 4 b 3 2 O C 1 O fatty acid with a cis-9 double bond A 16-C fatty acid with numbering conventions is shown. Most naturally occurring fatty acids have an even number of carbon atoms & unsaturated fatty acids are in the cis configuration The pathway for catabolism of fatty acids is referred to as the b-oxidation pathway, because oxidation occurs at the b-carbon (C-3). O H2C OH HC OH O HO H2C C OH glycerol fatty acid H2C O C O R HC O C O R H2C O C R R triacylglycerol Triacylglycerols (triglycerides) are the most abundant dietary lipids. Each triacylglycerol has a glycerol backbone to which are esterified 3 fatty acids Most triacylglycerols are “mixed.” The 3 fatty acids differ in chain length & number of double bonds. O H2C OH HC OH O HO H2C C OH glycerol fatty acid H2C O C O R HC O C O R H2C O C R R triacylglycerol Lipases hydrolyze triacylglycerols, yielding glycerol and three fatty acids 4 b 3 2 O C 1 O fatty acid with a cis-9 double bond Free fatty acids are transported in the blood bound to albumin, a plasma protein produced by the liver. Several proteins have been identified that facilitate transport of long chain fatty acids into cells Fatty acid activation: Acyl-CoA Synthases (Thiokinases) of ER & outer mitochondrial membranes catalyze activation of long chain fatty acids, esterifying them to coenzyme A. This process is ATP-dependent, & occurs in 2 steps. There are different Acyl-CoA Synthases for fatty acids of different chain lengths. Acyl-CoA Synthases Exergonic PPi (P~P) hydrolysis, is catalyzed by Pyrophosphatase 2 ~P bonds of ATP are cleaved. The acyl-CoA product includes one "~" thioester linkage. NH2 Fatty acid activation O fatty acid O O O P O R O O O N P O O CH2 H 2 Pi R O C H OH H OH P N N O CH2 O CoA SH H O H H OH H OH N acyladenylate AMP O R NH2 N O O N ATP H PPi O N O O P C N C S CoA acyl-CoA Summary of fatty aid activation: fatty acid + ATP + HS-CoA acyl-CoA + AMP + 2 Pi Mitochondrion Fatty acid b-oxidation is considered to occur in the mitochondrial matrix. Fatty acids must enter the matrix to be oxidized. b-Oxidation pathway in matrix Fatty acyl-CoA formed in cytosol by enzymes of outer mitochondrial membrane & ER Fatty acyl-CoA formed outside can pass through the outer mitochondrial membrane, but cannot penetrate the inner membrane. CH3 H3C Transfer of the fatty acid across the inner mitochondrial membrane involves carnitine. + N CH3 CH2 OH R CH CH2 COO + C carnitine O SCoA Carnitine Palmitoyl Transferase R C CH3 H3C + N CH3 O O CH2 CH CH2 COO + HSCoA fatty acyl carnitine Carnitine Palmitoyl Transferases catalyze transfer of a fatty acid between the thiol of Coenzyme A and the hydroxyl on carnitine. cytosol mitochondrial matrix O O R-C-SCoA HO-carnitine 1 HO-carnitine R-C-SCoA 3 2 HSCoA R-C-O-carnitine O R-C-O-carnitine HSCoA O Carnitine-mediated transfer of the fatty acyl into the mitochondrial matrix is a 3-step process: 1. Carnitine Palmitoyl Transferase I, an enzyme on the cytosolic surface of the outer mitochondrial membrane, transfers a fatty acid from CoA to the OH on carnitine. 2. An antiporter in the inner mitochondrial membrane mediates exchange of carnitine for acylcarnitine. cytosol mitochondrial matrix O O R-C-SCoA HO-carnitine 1 HO-carnitine R-C-SCoA 3 2 HSCoA R-C-O-carnitine O R-C-O-carnitine HSCoA O 3. Carnitine Palmitoyl Transferase II, an enzyme within the matrix, transfers the fatty acid from carnitine to CoA. (Carnitine exits the matrix in step 2.) The fatty acid is now esterified to CoA in the matrix. O H3C C SCoA acetyl-CoA O OOC CH2 C SCoA malonyl-CoA Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria. Malonyl-CoA (which is also a precursor for fatty acid synthesis) inhibits Carnitine Palmitoyl Transferase I. Malonyl-CoA thus inhibits fatty acid oxidation by preventing its transport into mitochondria. H H O b-Oxidation 3 2 1 Pathway: H3C (CH2)n C C C SCoA b fatty acyl-CoA Step 1. Acyl-CoA H H FAD Dehydrogenase Acyl-CoA Dehydrogenase FADH2 catalyzes oxidation H O of the fatty acid of H3C (CH2)n C C C SCoA acyl-CoA to trans-2-enoyl-CoA H produce a double bond between carbon atomsH22O& 3. H O There are different Acyl-CoA Dehydrogenases for short (4-6 C), medium (6-10H3C), long and very long (12-18 C) C (CH SCoA 2)n C CH2 C chain fatty acids. OH H+ + NADH NAD + O O H 3 H3C (CH2)n C b H FAD H O 2 C H C 1 SCoA fatty acyl-CoA Acyl-CoA Dehydrogenase FADH2 H O H3C (CH2)n C C C H SCoA trans-2-enoyl-CoA FAD His2Othe prosthetic group that functions as e acceptor H Dehydrogenase. O for Acyl-CoA H SCoA (CH2)n C isCH The stereospecific, 3C reaction 2 C bond in enoyl-CoA. OH H+ + NADH NAD + O O yielding a trans double H Step 2. Enoyl-CoA Hydratase catalyzes stereospecific hydration of the trans double bond produced in the 1st step, yielding L-hydroxyacylCoenzyme A. 3 H3C (CH2)n C b H FAD H O 2 C H C 1 fatty acyl-CoA Acyl-CoA Dehydrogenase FADH2 H O H3C (CH2)n C C C H H2O SCoA trans-2-enoyl-CoA Enoyl-CoA Hydratase H O H3C (CH2)n C CH2 C OH H+ + NADH SCoA SCoA 3-L-hydroxyacyl-CoA H H2O H O H3C (CH2)n C CH2 C Step 3. Hydroxyacyl-CoA Dehydrogenase catalyzes oxidation of the hydroxyl in the b position (C3) to a ketone. NAD+ is the electron acceptor. NAD + H+ + NADH OH SCoA 3-L-hydroxyacyl-CoA Hydroxyacyl-CoA Dehydrogenase O O H3C (CH2)n C CH2 C SCoA b-ketoacyl-CoA b-Ketothiolase HSCoA O O H3C (CH2)n C SCoA + CH3 C fatty acyl-CoA (2 C shorter) SCoA acetyl-CoA O Step 4. b-Ketothiolase catalyzes thiolytic cleavage. Thiol sulfur of CoA attacks the bketo carbon O H3C (CH2)n C CH2 C SCoA b-ketoacyl-CoA HSCoA O O H3C (CH2)n C SCoA + CH3 C fatty acyl-CoA (2 C shorter) SCoA acetyl-CoA b-Ketothiolase Acetyl-CoA is released, leaving the fatty acyl in thioester linkage to the CoA -fatty acyl-CoA (2 C less). Summary of one round of the b-oxidation pathway: fatty acyl-CoA + FAD + NAD+ + HS-CoA fatty acyl-CoA (2 C less) + FADH2 + NADH + H+ + acetyl-CoA The b-oxidation pathway is cyclic. The product, 2 carbons shorter, is the input to another round of the pathway. If, as is usually the case, the fatty acid contains an even number of C atoms, in the final reaction cycle butyryl-CoA is converted to 2 molecules of acetyl-CoA. FADH2 & NADH produced during fatty acid oxidation are reoxidized by transfer of electrons to respiratory chain.Transfer of electrons in the respiratory chain leads to production of ATP Acetyl-CoA can enter Krebs cycle, yielding additional NADH, FADH2, and ATP. Fatty acid oxidation is a major source of cell ATP. The reactions presented accomplish catabolism of a fatty acid with an even number of C atoms & no double bonds. Additional enzymes deal with catabolism of fatty acids with an odd number of C atoms or with double bonds. The final round of b-oxidation of a fatty acid with an odd number of C atoms yields acetyl-CoA & propionyl-CoA. Propionyl-CoA is converted to the Krebs cycle intermediate succinyl-CoA, by a pathway involving vitamin B12. Most double bonds of naturally occurring fatty acids have the cis configuration. They are not correct substrates for Enoyl-CoA hydratase, which acts only on trans compounds. Additional enzymes, isomerase and reductase , are required for oxidation of unsaturated fatty acids. b-Oxidation of very long-chain fatty acids also occurs within peroxisomes. Within the peroxisome, FADH2 generated by fatty acid oxidation is reoxidized producing hydrogen peroxide: FADH2 + O2 FAD + H2O2 The peroxisomal enzyme Catalase degrades H2O2: 2 H2O2 2 H2O + O2 These reactions produce no ATP. Once fatty acids are reduced in length within the peroxisomes they may shift to the mitochondria to be catabolized to acetylCoA. Glucose-6-phosphatase glucose-6-P glucose Gluconeogenesis Glycolysis pyruvate fatty acids During fasting acetyl CoA ketone bodies or carbohydrate cholesterol starvation, oxaloacetate citrate oxaloacetate is depleted in Krebs Cycle liver due to gluconeogenesis. This impedes entry of acetyl-CoA into Krebs cycle. Acetyl-CoA in liver mitochondria is converted then to ketone bodies, acetoacetate & b-hydroxybutyrate. Ketone body synthesis: b-Ketothiolase. The final step of the boxidation pathway runs backward. HMG-CoA Synthase catalyzes condensation with a 3rd acetate (from acetyl-CoA). HMG-CoA Lyase cleaves HMG-CoA to yield acetoacetate & acetyl-CoA. O H3C O C acetyl-CoA SCoA + H3C HSCoA H3C O H3C C SCoA O O Thiolase O O C H2 C C SCoA acetyl-CoA SCoA acetoacetyl-CoA acetyl-CoA HSCoA C C HMG-CoA Synthase OH H2 C C O H2 C CH3 C SCoA HMG-CoA HMG-CoA Lyase O O C O H2 C acetoacetate C O CH3 + H3C C SCoA acetyl-CoA b-Hydroxybutyrate Dehydrogenase b-Hydroxybutyrate CH3 + H Dehydrogenase C O NADH catalyzes reversible interconversion of CH2 the ketone bodies COO acetoacetate & acetoacetate b-hydroxybutyrate. CH3 + NAD HO CH CH2 COO D-b-hydroxybutyrate Ketone bodies are transported in the blood to other cells, where they are converted back to acetyl-CoA for catabolism in Krebs cycle, to generate ATP. Ketone bodies thus function as an alternative fuel. Ketoacidosis is caused by excess of ketone bodies. Fatty Acid Synthesis O H3C C SCoA acetyl-CoA The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to malonyl-CoA. O OOC CH2 C SCoA malonyl-CoA ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid. Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA. Enzyme-biotin HCO3 + ATP 1 ADP + Pi Enzyme-biotin-CO2 O ll CH3-C-SCoA acetyl-CoA 2 Enzyme-biotin O - ll O2C-CH2-C-SCoA malonyl-CoA As with other carboxylation reactions, the enzyme prosthetic group is biotin. ATP-dependent carboxylation of the biotin, carried out at one active site 1 , is followed by transfer of the carboxyl group to acetyl-CoA at a second active site 2 . Enzyme-biotin HCO3 + ATP 1 ADP + Pi Enzyme-biotin-CO2 O ll CH3-C-SCoA acetyl-CoA 2 Enzyme-biotin O - ll O2C-CH2-C-SCoA malonyl-CoA The overall reaction may be summarized as: HCO3 + ATP + acetyl-CoA ADP + Pi + malonyl-CoA O O C C O N NH CH CH CH H2C S Carboxybiotin O O (CH2)4 C NH C (CH2)4 CH lysine NH residue Biotin is linked to the enzyme by an amide bond between the terminal carboxyl of the biotin side chain and the e-amino group of a lysine residue. Fatty acid synthesis from acetyl-CoA & malonyl-CoA occurs by a series of reactions that are: catalyzed by individual domains of a very large polypeptide that includes an ACP domain. This multienzyme complex is called Fatty Acid Synthase NADPH serves as electron donor in the two reactions involving substrate reduction. The NADPH is produced mainly by the Pentose Phosphate Pathway. H H3N+ C SH COO CH2 CH2 CH2 SH NH Fatty Acid cysteine Synthase prosthetic groups: the thiol of the sidechain of a cysteine residue. the thiol of phosphopantetheine, equivalent in structure to part of coenzyme A. Coenzyme A C b-mercaptoethylamine O CH2 CH2 pantothenate NH C NH2 O ADP-3'phosphate HO C H H3C C CH3 O H2C O P N N O O O P N N O CH2 O O H H O H OH H phosphopantetheine O P O O SH phosphopantetheine of acyl carrier protein CH2 Phosphopantetheine (Pant) is covalently linked via a phosphate ester to a serine OH of the acyl carrier protein (ACP) of Fatty Acid Synthase. CH2 b-mercaptoethylamine NH C O CH2 CH2 pantothenate NH C O HO C H H3C C CH3 O H2C O P NH O O phosphate CH2 CH C serine residue O acetyl-S-CoA HS-CoA Pant SH Cys SH Pant 1 SH CO2 malonyl-S-CoA HS-CoA Cys Pant 2 S C CH3 O Cys S S C O C CH2 1 Malonyl/acetyl-CoA-ACP Transacylase COO Acetyl-CoA-ACP Transacylase 2 Malonyl/acetyl-CoA-ACP Transacylase Malonyl-CoA-ACP Transacylase 3 Condensing Enzyme (b-Ketoacyl Synthase) CH3 3 O Pant Cys S SH C O CH2 C O CH3 The condensation reaction (step 3) involves decarboxylation of the malonyl, followed by attack of the acetyl (or acyl). NADPH NADP+ Pant Cys S SH C O O CH3 Pant Cys S SH C O HC 5 Pant Cys S SH C CH CH2 CH2 C 4 NADPH NADP+ H2O OH CH3 HC CH3 O 6 Pant Cys S SH C O CH2 CH2 CH3 4 b-Ketoacyl-ACP Reductase 5 b-Hydroxyacyl-ACP Dehydratase 6 Enoyl-ACP Reductase 4. The b-ketone is reduced to an alcohol by e transfer from NADPH. 5. Dehydration yields a trans double bond. 6. Reduction by NADPH yields a saturated chain. Malonyl-S-CoA HS-CoA Pant Cys S SH C O 7 Pant Cys SH S C 2 O Pant Cys S S C O C CH2 CH2 CH2 CH2 CH2 CH2 COO CH2 CH3 CH3 O CH3 7 Condensing Enzyme Malonyl-CoA-ACP Transacylase (repeat)(repeat). 2 Malonyl/acetyl-CoA-ACP Transacylase Following transfer of the growing fatty acid from phosphopantetheine to cysteine sulfhydryl, the cycle begins again, with another malonyl-CoA. Product release: When the fatty acid is 16 carbon atoms long, a Thioesterase catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine. The 16-C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex. Palmitate, a 16-C saturated fatty acid, is the final product of the Fatty Acid Synthase reactions. Summary (ignoring H+ & water): acetyl-CoA + 7 malonyl-CoA + 14 NADPH palmitate + 7 CO2 + 14 NADP+ + 8 CoA Accounting for ATP-dependent synthesis of malonate: 8 acetyl-CoA + 14 NADPH + 7 ATP palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi Fatty acid synthesis occurs in the cytosol. Acetyl-CoA generated in mitochondria is transported to the cytosol via a shuttle mechanism involving citrate. b-Oxidation & Fatty Acid Synthesis Compared b Oxidation Pathway Fatty Acid Synthesis mitochondrial matrix cytosol acyl carriers (thiols) Coenzyme-A phosphopantetheine (ACP) & cysteine e acceptors/donor FAD & NAD+ NADPH -OH intermediate L D 2-C product/donor acetyl-CoA malonyl-CoA (& acetyl-CoA) pathway location Elongation beyond the 16-C length of the palmitate product of Fatty Acid Synthase occurs in mitochondria and endoplasmic reticulum (ER). Fatty acid elongation within mitochondria involves the b-oxidation pathway running in reverse, but NADPH serves as electron donor for the final reduction step. Fatty acids esterified to CoA are substrates for the ER elongation machinery, which uses malonyl-CoA as donor of 2-carbon units. The reaction sequence is similar to Fatty Acid Synthase but individual steps are catalyzed by separate proteins. A family of enzymes designated Fatty Acid Elongases catalyze the initial condensation step for elongation of saturated or polyunsaturated fatty acids. 10 9 O C OH oleate 18:1 cis 9 Desaturases introduce double bonds at specific positions in a fatty acid chain. Mammalian cells are unable to produce double bonds at certain locations, e.g., 12. Thus some polyunsaturated fatty acids are dietary essentials, e.g., linoleic acid, 18:2 cis 9,12 (18 C atoms long, with cis double bonds at carbons 9-10 & 12-13). 10 9 O C OH oleate 18:1 cis 9 Formation of a double bond in a fatty acid involves the following endoplasmic reticulum membrane proteins in mammalian cells: NADH-cyt b5 Reductase, a flavoprotein with FAD as prosthetic group. Cytochrome b5 Desaturase The overall reaction for desaturation of stearate (18:0) to form oleate (18:1 cis 9) is: stearate + NADH + H+ + O2 oleate + NAD+ + 2H2O There is a 4-electron reduction of O2 2 H2O as a fatty acid is oxidized to form a double bond. 2e pass from NADH to the desaturase via the FAD-containing reductase & cytochrome b5, the order of electron transfer being: NADH FAD cyt b5 desaturase 2e are extracted from the fatty acid as the double bond is formed. Control of fatty acid synthesis is exerted mainly at acetyl-CoA carboxylase step • Citrate & Insulin activate the enzyme • Acyl-CoA & Glucagon & Epinephrine inhibite the enzyme Thank you for your attention