Exam 5 Notes PDF

Lecture 24 → Fatty Acid Metabolism Part 1 1. Which one is more energy dense- carbohydrates or fats? a. Fats are more energy dense than carbohydrates i. Fats > Alcohol > Carbohydrates = Proteins 1. Lipids undergo lipolysis to yield fatty acids...

Lecture 24 → Fatty Acid Metabolism Part 1 1. Which one is more energy dense- carbohydrates or fats? a. Fats are more energy dense than carbohydrates i. Fats > Alcohol > Carbohydrates = Proteins 1. Lipids undergo lipolysis to yield fatty acids that are metabolized to Acetyl-CoA that enters the TCA Cycle 2. Carbohydrates are metabolized to yield Acetyl-CoA which forms Citrate which can be used for fatty acid synthesis 3. Proteins are broken down to amino acides to enter the TCA cycle as one of the TCA cycle intermediates, and procude Acetyl-CoA or Citrate which are then used in fatty acid synthesis b. Fats are long hydrocarbons that are energy dense and lightweight i. Have a lot of hydrocarbons, can make a lot of Acetyl-CoA ii. ~15kgs per human and can sustain 80 days of fasting 1. During prolonged fasting, the fuel for most tissues/organs/cells is fatty acids that are released from fat tissue aftr lipolysis a. Fatty acids cannot be used by the brain (cannot cross blood brain barrier) c. Oher fuel sources i. Muscle protein (not intended as fuel storage) 1. 6 kg, ~ 12 days ii. Muscle glycogen (for muscle use only) 1. < 500 g a. Cannot contribute to blood glucose homeostasis i. Muscles lack glucose 6 phosphatatse so they cannot turn G6P to glucose iii. Liver glycogen (for extrahepatic tissues) 1. 80-100g, < 8 hours a. Brain consumes ~120g per day i. Brain primarily uses glucose ii. Red blood cells also only use glucose iv. Glucose in circulating blood 1. < 5g, < 20 min d. Liver glycogen depletes (8-12 hours) durings starvation. Meanswhile lipolysis (fat breakdown) and ketogenesis increase → glycogen used up first i. Gluconeogenesis also increases but then decreases and stabilized after prolonged starvation 1. Non carbohydrate glucose synthesis ii. During fasting and starvation, the brain uses glucose and ketone bodies (but more ketone bodies than glucose) 2. A triglyceride (TG) is made of what? a. Three fatty acids esterified to a glycerol backbone i. Triglycerides are syntehsized by esterifiation of fatty acids to Glycerol in liver and adipose tissues 1. In liver they are packed to VLDLs and secreted in blood to peripheral tissues 2. In adipocytes they are stored as triglycerides 3. Exogenous lipids uptake process (exogenous = diet) a. Exogenous lipids are dietary triglycerides that are broken down by intestinal lipases into fatty acids and monoglycerides b. In the intestinal cells they are converted back to triglycerides and packaged into chylomicrons for tissue distribution c. Chylomicrons i. Synthesized by intestinal cells ii. Secreted into the circulation via lymphatics iii. Major lipid content are dietary triglycerides (98%) iv. Apoprotein is Apo-B48 4. Endogenous fatty acids are derived from Acetyl CoA. Where do we get Acetyl-CoA from? (endogenous = liver and adipose tissue) a. Acetyl-CoA comes from Carbohydrate metabolism (PDC after glycolysis), Beta Oxidation (fatty acids broken down in mitochondria, especially in fasting), breakdown of certain amino acids, ketone bodies, etc b. Endogenous lipids are synthesized in the cytosol, mainly in the liver and adipose tissues. c. They are transported by the Very Low Density Lipoproteins (VLDLs) into the circulation d. This biosynthesis is ‘reductive’, where it starts as Acetyl-CoA and NADPH is used as a reducing agent e. VLDL i. Synthesized by liver cells ii. Secreted directly into the circulation iii. Major lipid content are endogenous triglycerides (60%) iv. Apoprotein is Apo-B100 f. Endogenous triglyceride synthesis releases 3H2Os (3 fatty acids per triglyceride) 5. How are Triglycerides mobilized from Adipose tissue. Know the Process a. White Adipocytes → energy storage b. Brown Adipocytes → heat generation (more mitochondria) c. During fasting & starvation the body needs triglycerides from adipose tissue i. Breakdown activated by glucagon 1. Also activated glycogenolysis in the liver  2. Epinephrine/Norepinephrine activated glycogenolysis in Muscle d. Glucagon Receptor (GPCR) will activate Adenylyl Cyclase i. Converts ATP to cAMP 1. Activated Protein Kinase A a. Activated Hormone Sensitive Lipase i. The rate limiting enzyme of triglyceride breakdown b. HSL converts Triglycerides to Fatty Acids i. Which will then go to beta-oxidation 6. Rate limiting enzyme in Triglyceride Breakdown a. Hormone Sensitive Lipase (HSL) is the rate limiting enzyme in lipolysis (triglyceride breakdown) b. Triacylglycerol –adipose triglyceride lipase→ Diacylglycerol c. Diacylglycerol –HSL→Monoacylglycerol d. Monoacylglycerol –monoglyceride lipase→Glycerol 7. What is the function of phospholipases? a. Phospholipases act on phospholipids to release fatty acids b. Different lipases can act on different sites of the phospholipid to release different fragments. i. Release important molecules that can be used in inflammatory processes and cellular signaling 8. Fate of glycerol? And Fate of fatty acids? a. Glycerol is transported to the liver where it participates in gluconeogenesis i. Glycerol (in liver to do gluconeogenesis) –glycerol kinase→ G3P ii. G3P –Glycerol phosphate dehydrogenase → DHAP b. DHAP can also come fromglycerol and glycolysis c. Glycerol kinase would be present in the liver and kidneys (where gluconeogenesis is) i. No need in adipocytes → glycerol won’t be released if there is glycerol kinase 1. Adipocytes need to release glycerol d. Fatty Acids travel to different tissues where they undergo beta oxidation to release Acetyl-CoA which is ultimately used to generate energu 9. What is beta oxidation? What is the process? a. Beta oxidation is when fatty acids are broken down in the mitochondria (or peroxisomes) to generate Acetyl-CoA, NADH, and FADH2 b. It involves breaking the bond between the alpha and bate carbons i. Short Chain = 2-4C ii. Medium Chain = 5-11C iii. Long Chain = 12-22C iv. Very Long Chain > 22C c. It is a key step in the production of energy from fat, especially during fasting, exercise, or low carbohydrate intake d. Each cycle of beta-oxidation shortens a fatty acid by two carbons, generating Acetyl-CoA, NADH, and FADH2 e. Process of Fatty Acid Oxidation i. Long Chain Fatty Acid Activation ii. Enter the Mitochondrial Matrix iii. Beta-Oxidation of Fatty Acids f. Triglyceride broken down into free fatty acids and glycerol in adipose tissue i. Go to plasma LCFAin the blood vessels → can also come from diet ii. LCFA go into the cell, where there are also MCFA and SCFA 1. MCFA and SCFA do not need to be activated 2. LCFA gets activated by adding a CoA to become LCFA-CoA a. These then go into the mitochondria where they undergo beta oxidation and then ETC g. Step 1: Fatty Acid Activation i. Entry of long-chain fatty acids (LCFA) into the mitochondria requires their activation. ii. Activation occurs in the cytosol by attaching the fatty acid to a helper molecule CoA iii. It used 2 ATP → 2AMP + 2PPi 1. Fatty Acid → Fatty Acyl CoA via Thiokinase h. Step 2: Fatty Acid entry into the Mitochondria i. Acyl CoA- Synthetase → Activates LCFA in the cytoplasm ii. LC-Acyl-CoA reacts with Carnitine to remove the CoA to form LC-Acyl-Carnitine so that it can pass through the outer mitochondrial membrane via CPT-1 (carnitine palmitoyl transferase 1) iii. LC-Acyl-Carnitine then passed through CACT (carnitine-acylcarnitine translocase) to cross the inner membrane to enter the mitochondrial matrix iv. LC-Actyl-Carnitne then reacts with CoA via CPT-2 (carnitine palmitoyl transferase 2) to remake LC-Acyl-CoA and Carnitine v. This LC-Acyl-CoA then can undergo beta oxidation to Acetyl-CoA to then enter the TCA cycle vi. The Carnitine then travels back to the cytoplasm (outside mitochondria) via CACT i. Stap 3: Beta Oxidation i. Fatty Acyl-Coa undergoes oxidation via Acyl-CoA dehydrogenase to produce trans-2-enoyl-CoA, while using FAD to create FADH2 1. Acyl-CoA dehydrogenase makes a double bond between beta and alpha carbon  2. FAD → riboflavin precursor a. FAD deficiency impacts beta oxidation the most (first needed cofactor) ii. trans-2-enoyl-CoA undergoes hydration (addition of H2O) via Enoyl-CoA hydratase to make 3Hydroxyacyl-CoA 1. Hydration used H2O 2. Enoyl-CoA hydratase adds an OH to the double bond iii. This then undergoes oxidation via 3-Hydrocyacyl-CoA dehydrogenase to create beta-ketoacyl-CoA using NAD to create NADH+H 1. 3-Hydrocyacyl-CoA dehydrogenase will turn the OH into a carbonyl (c = o) 2. NAD → niacin precursor iv. This then undergoes thiolysis via beta-ketoacyl-CoA thiolase to create a Fatty Acyl-CoA that is now 2 carbons shorter. This process uses CoASH which gets converted into Acetyl-CoA (from the 2 carbons from FA) 1. Thiolysis will cleave the bond between the alpha and beta carbons v. Overall = 2 oxidations, 1 hydration, 1 thiolysis 10. Why Fatty Acids need to be activated? a. To help keep the fatty acid from leaving the cell b. To prevent from emulsifying the membrane 11. Know the Carnitine Shuttle, and its enzymes, what will happen if this shuttle does not function? a. Step 2: Fatty Acid entry into the Mitochondria i. Acyl CoA- Synthetase → Activates LCFA in the cytoplasm ii. LC-Acyl-CoA reacts with Carnitine to remove the CoA to form LC-Acyl-Carnitine so that it can pass through the outer mitochondrial membrane via CPT-1 (carnitine palmitoyl transferase 1) iii. LC-Acyl-Carnitine then passed through CACT (carnitine-acylcarnitine translocase) to cross the inner membrane to enter the mitochondrial matrix iv. LC-Actyl-Carnitne then reacts with CoA via CPT-2 (carnitine palmitoyl transferase 2) to remake LC-Acyl-CoA and Carnitine v. This LC-Acyl-CoA then can undergo beta oxidation to Acetyl-CoA to then enter the TCA cycle vi. The Carnitine then travels back to the cytoplasm (outside mitochondria) via CACT b. If this shuttle is disrupted, it will decrease the energy being produced by energy metabolism → stops producing source for Acetyl-CoA 12. What kind of Fatty Acid metabolism will be affected?  a. This will effect the Long Chain Fatty Acid Metabolism → require carnitine shuttle to be transported to mitochondria to be broken down → cannot be broken down i. Acculumatin of fatty acids b. Will also decrease ATP production 13. What does beta oxidation yield? What does it consume? What is required in final thiolysis? a. Yields: Acetyl-CoA, FADH2, NADH, Shortened Fatty Acyl-CoA i. About 14 ATP is produced (not directly) for each round of beta oxidation b. Consumes: Fatty Acyl-CoA, CoA, H2O, FAD, NAD+ c. Thiolysis requires a CoA and the enzyme beta-ketoacyl-CoA thiolase 14. Stage to make Acetyl-CoA? a. Long-chain fatty acids are transported into the mitochondria via the carnitine shuttle and broken down into Acetyl-CoA through beta-oxidation b. Key Steps: i. Oxidation (FADH₂ generation). ii. Hydration iii. Oxidation (NADH generation). iv. Thiolysis (release of Acetyl-CoA) 15. What will accumulate with each of LCAD, MCAD, and SCAD deficiency? a. LCF Acyl-CoA Dehydrogenase deficiency will accumulate excess Long Chain Fatty Acyl-CoAs because they cannot be broken down. This will inhibit teh ability to extract energy from these long chain fatty acids b. MCF Acyl-CoA Dehydrogenase deficiency will accumulate excess Medium Chain fatty acyl-CoAs because they will not be able to be brken down further to short chain fatty acyl-coas. This will still allow energy to be extracted because longer chains can be broken down to medium, just not shorter c. SCF Acyl-CoA Dehydrogenase deficiency will accumulate Short Chain Fatty Acyl-CoAs because these cannot be fully broken down. There is still a lot of energy that can be broken down and hearvested from this deficiency because long chains can be broken down to short chains, just not fully broken down. 16. What is SIDS? a. Sudden Infant Death Syndrome b. Results from a Medium Chain Acyl CoA dehydrogenase deficiency c. Mothers milk has mainly medium chain fatty acids i. When napping, teh body needs to make energy fron stores but it cannot break down these medium chains 17. How is Fatty Acid oxidation regulated? a. The availability of free fatty acids regulateqs how much utilization occurs by beta oxidation → regulated by Insulin : Glucagon ratio  b. During low energy state: high glucagon (and epinephrine → meaning the cell needs sugar/energy) promotes fatty acid breakdown by synthesizing cAMP → PKA activation which then activated the Hormone Sensitive Lipase which is the rate limiting enzyme of beta oxidation c. Regulation i. Substrate Availiabilty ii. Activation of HSL (to convert Triglycerides to Fatty Acids) 18. What is the fate of Acetyl-CoA? a. Used to Make i. Triglygerides ii. Phospholipid iii. Eicosanoidss iv. CO2 + H2O + ATP v. Cholesterol 1. Steroid hormones 2. Bile salts vi. Ketone bodies vii. Fatty acids b. Made From i. Ketone bodies ii. Fatty acids iii. Amino acids iv. Pyruvate (from glycolysis) Lesson 25 → Fatty Acid Metabolism Part 2 1. What are the products of Oxidation of odd chain fatty acids? What is the difference between beta oxidation of even chain vs odd chain fatty acids? a. Products: Propionyl-CoA (3 carbons), Acetyl-CoA, NADH, FADH2 b. The difference between even and odd chains is that even chains are fully broken down to Acetyl-CoA (2 carbons per Acetyl-CoA), while the last 3 carbon of the odd chain fatty acid will be made into Propionyl-CoA (which later will be need to be converted to Succinyl-CoA) 2. What 3 vitamins are required for this process? a. Riboflavin (FAD), Niacin (NAD), Pantothenic Acid (component of CoA, B5) 3. Which 2 vitamins are required for converting odd chain and branched chain fatty acids to Succinyl CoA? a. Biotin (B7) i. Coenzyme for propionyl-CoA carboxylase 1. converts propionyl-CoA into D-methylmalonyl-CoA 2. addition of a carboxyl group using bicarbonate (HCO₃⁻) and ATP  b. B12 (Cobalamin) i. Coenzyme for methylmalonyl-CoA mutase ii. converts L-methylmalonyl-CoA to Succinyl-CoA via a carbon rearrangement reaction → can go into TCA 4. How is beta-oxidation different in saturated and unsaturated fatty acids? a. Monounsaturated Fatty Acids (one double bond) i. Regular beta oxidation will occur until it reaches the double bond (where the alpha or beta carbon are involved in c=c) ii. An Isomerase will covert the cis double bond to a trans double bond between the alpha and beta carbons so that it can undergo hydration 1. Acyl-CoA dehydrogenase (creation of double bond) is not used like it in in saturated fatty acids (there is already a doubel bond, it will just be relocated), therefore it does not need FAD and does not create FADH2 iii. After the isomerase, a hydratease will react to allow the molecule to continue to undergo beta oxidation b. Polyunsaturated Fatty Acids (multiple double bonds) i. The Isomerase will convert a cis double bond to a trsans double bond between the alpha and beta carbons 1. No FADH produced because no dehydrogenase used c. In Saturated fatty acids, a dehydrogenase is required in the initial reaction to create a double bond. For unsaturated fatty acids, since double bond already exists, dehydrogenase is not used. An isomerase is used to reposition the double bond at C2-C3 position for action of hydratase 5. What are 3 main functions of peroxisomes? a. Beta Oxidation of very Long Chain Fatty Acids b. Plasmalogen (Ether lipid) Synthesis c. Alpha Oxidatyion of Phytanic Acid d. Peroxisomes are single membrane enclosed organelles taht carry out oxidation reactions and produce hydrogen peroxide i. Very Long Chain Fatty Acids (VLCFA) undergo prelimiary beta oxidation in the peroxisomes to shorted the VLC to MC which is then transferred to the mitochondria for further beta oxidation ii. Any Acetyl-CoA produced in peroxisomes are used for biosynthesis or transferred to mitochondria 6. What metabolic changes occur during fasting/starvation? Why? What substrates are broken down to produce which energy molecules? a. Energy Sources i. Fed State  1. Glucose is the primary energy source for brain, RBCs, Kidneys, Lens epithelial cells 2. Fatty Acids are the primary energy source for heart, lung, liver, intestines, and skeletal muscle ii. Starvation State (more than 3 days usually) 1. Out of preferred energy source → no more glucose (glycogen stores depleted) a. The body adapts i. To maintain adequate blodo glucose levels ii. Brian must adapt to use ketone bodies iii. Prevent cellular protein degradation b. Starts synthesis of ketone bodies i. Beta hydroxybutyrate ii. Acetoacetate 2. Activates lipolysis and ketogenesis to produce fatty acids and ketone bodies a. Fatty Acids are used in beta oxidation to produce ATP b. Fatty Acids are used in ketogenesis to produce ketone bodies 7. Where are ketone bodies synthesized and where are they broken down? Which organelle (specify where) and the organ? a. Ketone bodies are synthesized only in the liver, in the liver mitochondria. Ketogenesis and ketolysis occur in the mitochondrial matrix b. The liver converts excess acetyl-CoA (from beta-oxidation of fatty acids) into ketone bodies i. Mainly make Acetoacetate and Beta-hydroxybutyrate 1. Very small amounts of acetone 8. Rate limiting enzyme in ketone body synthesis? a. HMG-CoA Synthase is the rate limiting enzyme b. Catalyzes condensation of acetyl-CoA and acetoacetyl-CoA to form HMG-CoA 9. What is the significance of ketone body synthesis? a. Ketone bodies are alternative to glucose for energy provision to cells. The brain normally uses glucose, but in starvation (and diabetes) when glucose is scare, the brain adapts to use ketone bodeies in addition to glucose for energy b. Regeneration of CoA for sustaining ketone body synthesis since CoA is required for beta oxidation of fatty acids 10. What are the 3 ketone bodies? a. Acetoacetate i. Primary ketone body produced in the liver during fasting or prolonged starvation  ii. Formed from HMG-CoA via the action of HMG-CoA lyase iii. Can be used to make beta-hydroxybutyrate and acetone iv. Used directly for energy b. Beta-Hydroxybutyrate i. Most abundant ketone body during starvation or fasting. ii. Formed by the reduction of acetoacetate (via beta-hydroxybutyrate dehydrogenase) in the liver. iii. More stable and efficient for energy transport than acetoacetate iv. Utilized by peripheral tissues (e.g., brain, muscle) for ATP production after being converted back to acetoacetate. c. Acetone i. A minor, non-metabolizable ketone body. ii. Formed by the spontaneous decarboxylation of acetoacetate iii. Exhaled in breath, no energy use 11. What determines the prevalence of one ketone body over another? Which enzyme is used in interconversion of the 2 ketone bodies? How is acetone produced? a. The ratio of beta-hydroxybutyrate and acetoacetate in circulation depends on the amount of NADH in the liver mitochondria i. High NADH (low NAD+)= High Beta-Hydroxybutyrate ii. High NAD+ (low NADH) = Acetoacetate iii. Typically the ratio of beta-hydroxybutyrate to acetoacetate is 3:1 b. Beta-Hydroxybutyrate Dehydrogenase is used to convert Acetoacetate to Beta-Hydroxybutyrate and the reverse i. The forward reaction required NADH (released NAD+) to make beta-hydroxybutyrate ii. To make acetoacetate back it required NAD+ (to make NADH) c. Acetone is created via Acetoacetate dehydrogenase which converts acetoacetate to acetone while releasing CO2 12. Name the enzyme responsible for synthesis of Acetyl-CoA from ketone bodies? a. Thiophorase i. Will be upregulated by the brain during fasting and starvation ii. Uses Succinyl CoA to convert Acetoacetate to Acetoacetyl-CoA (releasing Succinate which will feed back into TCA) iii. Acetyl CoA will then process through the TCA cycle and ETC to generate ATP iv. NOT IN LIVER 1. In peripheral tissues 13. What regulates ketone body synthesis? a. Substrate Availability i. More availability of fatty acids means more ketogenesis  b. Regulation of Beta Oxidation i. Increase in the Glucagon/Insulin ratio inhibits acetyl-CoA carboxylase which results in reduced Malonyl-CoA (an intermediate in fatty acid synthesis) ii. Malonyl CoA is an inhibitor of CPT-1 1. Less Malonyl-CoA means CPT-1 will be more active and will cause an increase in beta oxidation a. CPT-1 helps move fatty acids into the mitochondrial matrix for beta oxidation iii. HMG-CoA Synthase (rate limiting enzyme in ketogenesis) is stimulated by fasting, high fat intake, and untreated diabetes 1. Fatty acids are strong incuders of HMG-CoA Syntahse 14. What is Rothera’s test? Which ketone bodies are detected by this test and which one is not? a. Rothera’s test is a urine analysis for ketone bodies i. It detects Acetone and Acetoacetate but it does not detect beta-hydroxybutyrate 1. This is because beta-hydroxybutyrate does not have a ketone in its structure (has OH and carboxylic acid) while Acetone and Acetoacetatae both have ketone groups b. A blood test for beta-hydroxybutyrate will be the most accurate Lecture 26 → Fatty Acid Metabolism Part 3: Lipid Biosynthesis 1. Functining of citrate shuttle, its componenets, and what is produced? a. Fatty Acid Syntehsis occurs in the cytoplasm b. Acetyl-CoA for Fatty Acid Synthesis comes from the mitochondira i. The 2C of Acetyl-CoA move out to the cytoplams in the form of citrate by the Citrate Shuttle System  ii. This Citrate is then broken down in the cytoplasm by Citrate Lypase to extract the Acetyl-CoA iii. NADPH is also a product of the citrate shuttle that is used in reductive biosynthesis c. In the Mitochondria i. Pyruvate is turned into Oxaloacetate ii. Citrate Synthase combines Oxaloacetate and Acetyl-CoA to create Citrate iii. Citrate is transported across the membrane to the cytoplams/cytosol d. In the Cytosol/Cytoplasm i. Citrate Lyase splits Citrate into Acetyl-CoA and Oxaloacetate ii. Oxaloacetate combines with NADH to create Malate iii. Malate breaks into NADPH and Pyruvate 1. NADPH (like from hmp shunt and glycolysis) is used in biosynthesis iv. The pyruvate travels back into the mitochondria 2. What is the required starting material for fatty acid synthesis? How are they transported? a. Fatty Acid Synthesis occurs in the liver and adipose tissue, but the storage is only in adipose tissue. But abnormal production or lack of transport can cause accumulation in the liver b. The starting materials are Acetyl-CoA and NADPH c. FA Synthesis produces 16C Palmitic Acid d. FA is produced in the liver and is packaged into lipoproteins and transported via circulation e. Lipoporteins (VLDL) are used to transport triglycerides and cholesterol from the liver to the peripheral tissues 3. Rate limiting enyme in fatty acid sythesis? a. Acetyl-CoA Carboxylase (ACC) is the rate limiting enzyme that catalyzes the carboxylation of Acetyl-CoA to Malonyl-CoA 4. What does ACC need in order to function (hint: ABC). What other enzymes need ABC? a. The carboxylation of Acetyl-CoA to Malonyl CoA requires ATP, Biotin, and CO2 b. ACC is activated by Citrate and inhibited by Palmitic Acid (end product of fatty acid synthesis) c. Malonyl-CoA inhibits CPT-1 (FA crossing outer mitochondrial membrane to enter mitochondria) to help prevent beta-oxidation 5. Where are carbons for fatty acids derived from? What role does Malonyl-CoA have in this process? a. Carbon atoms for fatty acid synthesis are primarily derived from acetyl-CoA i. Which is produced from the breakdown of carbohydrates (via glycolysis) and fatty acids (via beta-oxidation)  b. The carbon atoms are incorporated into malonyl-CoA (a key intermediate in fatty acid biosynthesis), which then contributes to the elongation of the fatty acid chain i. In the cytoplasm, acetyl-CoA is converted into malonyl-CoA through the action of acetyl-CoA carboxylase (ACC), which adds a carbon dioxide (CO₂) group to acetyl-CoA (consumes an ATP) ii. Malonyl-CoA provides the 2 Carbons that are used to elongate the fatty acid chain 6. Different processes that occur in fatty acid synthesis a. Synthesis of Malonyl-CoA from Acetyl-CoA (via ACC) i. Uses ATP, Biotin, and CO2 ii. Enzyme activated by Citrate and inhibited by Palmitic Acid (end product) b. Binding of Acyl Carrier Protein i. Acetyl CoA – Acyl Transferase → Acetyl-ACP (Primer) 1. Uses ACP (acyl carrier protein) 2. Releases CoA-SH which replaces CoA ii. Malonyl-CoA –Acyl Transferase → Malonlyl-ACP (Chain Extender) 1. Uses ACP (acyl carrier protein) 2. Releases CoA-SH which replaces CoA 3. Malonyl-ACP has 3 carbons, but only 2 are used to extend the chain c. Extension of Fatty Acid Chain and Synthesis of Palmitate i. Acetyl-ACP (primer) + Malonyl-ACP (extender) to make Acetoacetyl-ACP 1. Enzyme: beta-ketoacyl-ACP synthetase 2. Condensation 3. Releases CO2 and ACP-SH a. The decarboxylation brings down deltaG b. Carbon in CO2 is the 3rd carbon from Malonyl-ACP ii. Acetoaceytl-ACP to make 3-hydroxybutyryl-ACP 1. Enzyme: beta-ketoacyl-ACP reductase 2. Reduction 3. Uses NADPH + H+ to release NADP+ iii. 3-hydroxybutyryl-ACP to make Crotonyl-ACP 1. Enzyme: beta-hydroxyacyl-ACP dehydrase 2. Dehydration 3. Released H2O iv. Crotonyl-ACP to make Butyryl-ACP 1. Enzyme: Enoyl-ACP reductase 2. Reduction 3. Uses NADPH + H+ to release NADP+  a. Now 4 carbons long v. Conversion to Palmitate 1. Thioesterase adds back the CoA to create the long fatty acid chain 2. Swaps the ACP on Palmitoyl-ACP with CoA d. For UNSATURATED fatty acids i. Saturated fatty acids undergo desaturation by Desaturases in the ER to add double bonds 7. Fatty Acid Synthase - how many enzymes are there? (no need to memorize the names of enzymes in FAS complex), which is the key/central component of FAS a. b. Fatty acid synthase (FAS) complex is a multi-enzyme system that synthesizes palmitate (C16:0), a saturated fatty acid, using acetyl-CoA, malonyl-CoA, and NADPH. c. There are 7 enzymes in the multi-enzyme complex of Fatty Acid Synthase i. Also the Acyl carrier protein (ACP) which is the central and key component of the FAS complex 1. It holds the growing fatty acid chain as it undergoes sequential enzymatic reactions 2. It facilitates the transfer of intermediates between the enzymatic domains within the FAS complex d. Fatty Acid Synthase is a homodimer → not the rate limiting enzyme i. Fatty acid synthase is a single polypeptide chain with seven enzyme activities 1. All 7 enzymes are encoded by a single gene ii. ACP is the center of the enzyme complex 1. KS, MAT, and DH are kind of separated from the rest 8. What is the role of Thioesterase? a. Convert the 16C Palmitoyl-ACP to Palmitoyl-CoA (Palmitic Acid) which is the end product b. It removes the ACP and replaces it with CoA 9. ACP - Which amino acid links ACP to the phosphopantethene arm? a. ACP has a Serine residue i. The OH interacts with the phosphopantetheine 10. Sources of NADPH a. The primary source of NADPH is the HMP shunt (pentose phosphate pathway) in the cytosol b. Malate to Pyruvate conversion reaction in catalyzed by Malic enzyme in cytosol to produce NADPH c. Isocitrate dehydrogenase carries out oxidative decarboxylation of isocitrate to form alpha-ketoglutarate and produces NADPH in the cytosol i. This Isocitrate dehydrogenase is not in th emitochondria like it usually is in TCA d. NADPH is used in reductive biosynthesis i. For fatty acid synthesis, cholesterol, and steroid hormone synthesis 11. Positive and negative regulators of fatty acid synthesis (or ACC) a. Acetyl COA Carboxylase (ACC) is the rate limiting enzyme b. Allosteric Control i. Citrate allosterically activates (+) ACC through feed forward mechanism 1. Feed forward → goes ahead to activate the enzyme to pull the pathway forward a. other feed forward regulation occurs in Glycolysis. Fructose-1,6- bisphosphate acts on Pyruvate kinase (at the last step in Glycolysis) to help pull all the reactions forward from the bottle neck at the split part of glycolysis (DHAP and Glyceraldehyde 3P) 2. Citrate is abundant during the well fed state and indicated plentiful supply of Acetyl-CoA ii. Palmitoyl CoA is the end product of the pathway and is a negative (-) feedback inhibitor for ACC 1. Long chain fatty acids (besides Palmitoyl CoA) also inhibit ACC iii. Malonyl-CoA is an allosteric inhibitor (-) of CPT-1 1. Prevents newly synthesized fatty acids from going to mitochondrial matrix for beta oxidation c. Hormonal Cycle i. High Insulin:Glucagon ratio activates fatty acid synthesis 1. Insulin triggers activation of citrate lyase to make Acetyl-CoA  2. Insulin triggers dephosphorylation of ACC to activate it ii. Low ratio inhibits ACC activity by triggering ACC Phosphorylation 1. Phosphorylation of ACC = inactivation a. Triggered by glucagon & epinephrine (GPCR activates the adenylyl cyclase to make cAMP which activates PKA which will phosphorylate iii. Insulin 1. Active phosphatase a. Activate Enzyme i. Too much present, need to pack and store iv. Glucagon 1. Activate Kinase a. Deactivate Enzyme i. No need to store, need to break down for energy/use 12. What covalent modification activates/inactivates ACC? How does Insulin and glucagon regulated this covalent modification? a. Hormonal control (covalent modification) → phosphorylation/dephosphorylation b. Insulin i. Active phosphatase 1. Activate Enzyme a. Too much present, need to pack and store c. Glucagon i. Activate Kinase 1. Deactivate Enzyme a. No need to store, need to break down for energy/use ii. Phosphorylation of ACC = inactivation 1. Triggered by glucagon & epinephrine (GPCR activates the adenylyl cyclase to make cAMP which activates PKA which will phosphorylate 13. Difference between fatty acid synthesis and degradation? a. Fatty Acid Degradation (Beta Oxidation) i. Location: Mitochondrial Matrix ii. Carrier: CoA iii. Enzymes: Enzymes are independent, not linked iv. Oxidant/Reductant: NAD+ used for Oxidation v. Isomeric Form: Hydroxy acyl intermediate is L-form b. Fatty Acid Synthesis i. Locatiom: Cytosol ii. Carrier: ACP (Acyl Carrier Protein)  iii. Enzymes: Fatty acid Synthase is a complex with various enzymes are linked covalently in a single polypeptide chain iv. Oxidant/Reductant: NADPH used as Reductant (reductive biosynthesis) v. Isomeric Form: Hydroxy acyl intermediate is D-form 14. Characteristic feature of cholesterol? What is cholesterol ester? Why is it made? a. The Signature is the Sterol Nucleus (rings) → signature in things synthesized via cholesterol b. Hydroxyl group (water soluble) & Hydrocarbon tail (fat soluble) c. Cholesterol ester is the stored form of cholesterol d. VLDL and Chylomicrons will circulate things e. Cholesterol i. All nucleated cells can make cholesterol ii. Lipoproteins transport cholesterol in circulation iii. Sources 1. 75% from De novio synthesis (like in liver and intestines) 2. 25% from diet 3. Stored as cholesterol esters iv. Functions 1. Structural component of the cell membrane a. Lipid bilayer, provides flexibility and transitioning between states w/changing temperature 2. Building block for steroid hormones (cortisol, aldosterone, estrogens, etc) 3. Precursor for Bile Acids 4. Synthesis of prenylated proteins, heme A, CoQ, and Dolichol 5. Important for production of vitamin D 15. What is synthesized in cholesterol synthesis pathway besides cholesterol? Rate limiting enzyme? Its regulation? Mechanism of action of Statins, what type of inhibitors are they? a. Cholesterol is synthesized by Acetyl-CoA b. Synthesis of Mevalonate is the committed, irreversible step in the pathway c. HMG-CoA Reductase is the rate limiting enzyme i. HMG-CoA is reduced to Mevalonate ii. It is in the cytosol iii. Regulation of HMG-CoA Reductase 1. Feedback Inhibition (cholesterol) 2. Transciptional Control of Gene Expression 3. Rate of enzyme synthesis/degradation 4. Hormonal Regulation d. Squalene intermediate is specific to making cholesterol e. Other possible products from Mevalonate are heme A (ETC), Ubiquinone, Dolichol, Vitamin D, etc f. Cholesterol can be used to make Bile Salts, Steroids, Sex Hormones, Cell membranes, etc g. Statins inhibit HMG-CoA Reductase i. They are structual analogs to HMG-CoA which is the usual substrate for the enzyme. ii. Statins are competitive inhibitors that bind to the active site of HMG-CoA Reductase with much higher affinity than HMG-CoA iii. They are used to treat High Cholesterol (not make more)