Lec1 Fatty Acid and Triglycerides PDF

# OBJECTIVES - At the end of the session, students should be able to discuss: - Fatty acid synthesis enzymes - Requirement of FA Synthesis: - FA Synthase & Acetyl CoA Carboxylase - Coenzymes and cofactors: Biotin, NADPH, Mn++ - CO2: Source of CO2 is bicarbonate and AT...

# OBJECTIVES - At the end of the session, students should be able to discuss: - Fatty acid synthesis enzymes - Requirement of FA Synthesis: - FA Synthase & Acetyl CoA Carboxylase - Coenzymes and cofactors: Biotin, NADPH, Mn++ - CO2: Source of CO2 is bicarbonate and ATP as energy - Regulation of Acetyl CoA - Triacylglycerol Synthesis # FATTY ACID SYNTHESIS ## EXTRAMITOCHONDRIAL (CYTOPLASMIC) SYNTHESIS OF FATTY ACIDS: (DE NOVO SYNTHESIS) | Enzymes | Metabolites | End products | Hormones | Diet | |-------------|--------------|---------------|------------|------------------------------------| | Acetyl CoA carboxylase | Citrate | Palmitoyl CoA | Insulin | High carbohydrate diet | | | | | Glucagon | | # Lipogenesis = Fatty acid synthesis - occurs primarily in the cytoplasm of these tissues: - Liver - Adipose (fat) - Central Nervous System - Lactating mammary gland - Remember: - Glucagon and epinephrine inhibit fatty acid synthesis, and insulin stimulates it. # BIOSYNTHESIS OF FATTY ACIDS 1. Dietary carbohydrates and amino acids when consumed in excess convert to fatty acids and stored as TG. 2. Occurs predominantly in liver, kidney, adipose tissue and lactating mammary gland. 3. Biosynthesis pathway takes place in cytosol of the cell. 4. Acetyl CoA provides carbon and NADPH provides reducing equivalents. # Fatty Acid Synthesis - Occurs mainly in liver and adipocytes, in mammary glands during lactation. - Occurs in cytoplasm - FA synthesis and degradation occur by two completely separate pathways. - When glucose is plentiful, large amounts of acetyl CoA are produced by glycolysis and can be used for fatty acid synthesis. # Three stages of fatty acid synthesis: A. Transport of acetyl CoA into cytosol B. Carboxylation of acetyl CoA C. Assembly of fatty acid chain # REQUIRED FORTHE FA SYNTHESIS - Enzymes: - Fatty acid synthase, a multi-enzyme complex - Acetyl-CoA carboxylase, also a multi-enzyme - Coenzymes and cofactors: - Biotin NADPH, Mn++ - CO2: source of CO2 is bicarbonate - ATP for energy. # A. Transport of Acetyl CoA to the Cytosol - Acetyl CoA from catabolism of carbohydrates and amino acids is exported from mitochondria via the citrate transport system. - Cytosolic NADH also converted to NADPH. - Two molecules of ATP are expended for each round of this cyclic pathway. # Image Description - Page 9 & 10 The image shows a diagram displaying the export of acetyl CoA as citrate for fatty acid biosynthesis, generation of NADPH and pathway of lipogenesis. It is similar to the diagram discussed for cholesterol synthesis except this involves PDH reaction. # Sources of NADPH for Fatty Acid Synthesis 1. One molecule of NADPH is generated for each molecule of acetyl CoA that is transferred from mitochondria to the cytosol (malic enzyme). 2. NADPH molecules come from the pentose phosphate pathway. # ENZYMES (ACC & FAS) - Acetyl-CoA carboxylase: multienzyme (ACC) - Biotin, biotin carboxyl carrier protein, transcarboxylase & a regulatory allosteric site (citrate ++, palmityle CoA-ve). - Irreversible, rate limiting enzyme, HCO3 → CO2, high energy bond ATP is utilized - Fatty acid synthase (FAS): - multienzyme complex, two identical polypeptide monomeric units → each unit contains six enzyme and an ACP molecule (Acyl carrier protein) # PRODUCTION OF ACETYL COA - Produced by the oxidation of pyruvate, fatty acids, amino acids, ketone bodies. - Acetyl CoA is formed inside mitochondria but cannot come outside freely. - Comes outside the mitochondria in the form of CITRATE. # FORMATION OF MALONYL COA - Acetyl CoA carboxylase converts acetyl CoA to malonyl CoA. - This is the rate limiting step of fatty acid synthesis. # FATTY ACID SYNTHESIS 1. Production of acetyl CoA and NADPH 2. Conversion of acetyl CoA to malonyl CoA (enzyme Acetyl CoA carboxylase) 2. Reaction of fatty acid synthase complex (enzyme FAS) # REACTIONS OF FAS COMPLEX - Reaction is catalyzed by fatty acid synthase complex enzyme. - Fatty acid synthase is a dimer composed of 7 different enzymes. - Only dimer is active. - Repeated 7 times to produce palmitate (16C fatty acid), increasing fatty acid chain length by 2 carbons. - The image shows a picture of a process with Malonyl CoA, Acetyl-CoA, Acetyl-CoA, Acetyl CoA. # C. The Reactions of Fatty Acid Synthesis - Five separate stages: 1. Loading of precursors via thioester derivatives 2. Condensation of the precursors 3. Reduction 4. Dehydration 5. Reduction | Step | Reaction | Enzyme | |------|-------------------------------------------------------------------------------|------------------------------------------| | 1 | Acetyl CoA + HCO3- + ATP → malonyl CoA + ADP + Pi + H+ | Acetyl CoA carboxylase | | 2 | Acetyl CoA + ACP → acetyl ACP + CoA | Acetyl transacylase | | 3 | Malonyl CoA + ACP → malonyl ACP + CoA | Malonyl transacylase | | 4 | Acetyl ACP + malonyl ACP → acetoacetyl ACP + ACP + CO2 | Acyl-malonyl ACP condensing enzyme | | 5 | Acetoacetyl ACP + NADPH + H+ → ß-hydroxybutyryl ACP + NADP+ | ß-Ketoacyl ACP reductase | | 6 | ß-Hydroxybutyryl ACP → crotonyl ACP + H2O | 3-Hydroxyacyl ACP dehydratase | | 7 | Crotonyl ACP + NADPH + H+ → butyryl ACP + NADP+ | Enoyl ACP reductase | - Principal reaction in fatty acid synthesis in bacteria. The table describes reaction and enzyme. # Fatty Acid Synthesis - Step 1: Loading - transferring acetyl- and malonyl- groups from CoA to ACP. - Step 2: Condensation - transferring 2 carbon unit from malonyl-ACP to acetyl-ACP to form 2 carbon keto-acyl-ACP. - Step 3: Reduction - conversion of keto-acyl-ACP to hydroxyacyl-ACP (uses NADPH). - Step 4: Dehydration - Elimination of H2O to form Enoyl-ACP. - Step 5: Reduction - Reduce double bond to form 4 carbon fully saturated acyl-ACP. # Image Description - Page 20 The image shows 2 diagrams. One is a cycle with steps and enzyme, and the other is a table with step, reaction, and enzyme. # SIGNIFICANCE OF FATTY ACID SYNTHASE ENZYME - Great efficiency of fatty acid production. - No permeability barriers. - Coded by single gene so good coordination of all enzymes. # Image Description - Page 22 The image shows a structural formula of a molecule named Phosphopantetheine prosthetic group of ACP. - During the fatty acid synthesis all intermediates are linked to the protein called acyl carrier protein (ACP-SH), which is the component of fatty acyl synthase complex. - The pantothenic acid is a component of ACP. - Intermediates in the biosynthetic pathway are attached to the sulfhydryl terminus of phosphopantotheine group. # Image Description - Page 23 The image displays a chemical equation describing the conversion of Acetyl CoA and Malonyl CoA to an intermediate with Acetyl-ACP and Malonyl-ACP. - The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP. - Acetyl transacylase and malonyl transacylase catalyze these reactions. - Acetyl CoA + ACP ⇔ acetyl ACP + CoA - Malonyl CoA + ACP ⇔ malonyl ACP + CoA # Condensation reaction - Acetyl ACP and malonyl ACP react to form acetoacetyl ACP. - Enzyme - acyl-malonyl ACP condensing enzyme. # Reduction - Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP. - NADPH is the reducing agent - Enzyme: ß-ketoacyl ACP reductase # Dehydration - D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP (trans-Δ2-enoyl ACP). - Enzyme: 3-hydroxyacyl ACP dehydratase. # Reduction - The final step in the cycle reduces crotonyl ACP to butyryl ACP. - NADPH is reductant. - Enzyme - enoyl ACP reductase. - This is the end of first elongation cycle (first round). # Image Description - Page 28 The image displays a chemical equation describing the conversion of Butyryl-ACP and Malonyl ACP to an intermediate with C6-B-ketoacyl ACP, followed by reduction, dehydration and reduction. - In the second round butyryl ACP condenses with malonyl ACP to form a C6-B-ketoacyl ACP. - Reduction, dehydration, and a second reduction convert the C6-ß-ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation. # Organization of Multifunctional Enzyme Complex in Eukaryotes - The synthase is dimer with antiparallel subunits. - Each subunit has three domains. - ACP is located in domain 2. - Domain 1 contains transacylases, ketoacyl-ACP synthase (condensing enzyme) - Domain 2 contains acyl carrier protein, ß-ketoacyl reductase, dehydratase, and enoyl reductase. - Domain 3 contains thioesterase activity. # Image Description - Page 30 The image displays the complex of enzymes involved in the process of fatty acid synthesis. The structure shows the reaction taking place and the enzymes involved in each stage. # Final reaction of FA synthesis - Rounds of synthesis continue until a C16 palmitoyl group is formed. - Palmitoyl-ACP is hydrolyzed by a thioesterase. - Palmitoyl-ACP + H2O --> Palmitate + HS-ACP. - Overall reaction of palmitate synthesis from acetyl CoA and malonyl CoA. - Acetyl CoA + 7 Malonyl CoA + 14 NADPH + 14 H+ --> Palmitate + 7 CO2 + 14 NADP+ + 8 HS-CoA + 6 H2O # REGULATION OF FATTY ACID SYNTHESIS - **Covalent modification** - Insulin - Acetyl CoA carboxylase (inactive) - Acetyl CoA carboxylase (active) - Glucagon, Epinephrine - **Allosteric Regulation** - Glucose - Citrate - Acetyl-CoA - Acetyl CoA- Carboxylase - Malonyl-CoA - NADPH - G6PO - Pentose phosphate pathway - Palmitate - Palmitoyl CoA # REGULATION OF FATTY ACID SYNTHESIS - **Short term regulation:** allosteric or metabolic regulation and covalent modification of enzyme - **Long term control:** (ACC, FAS, ATP -citrate lyase) - Regulated by acetyl CoA carboxylase. - Acetyl CoA carboxylase is regulated by insulin and glucagon. - Also regulated by dietary intake, NADPH, citrate etc. # Global Regulation - is carried out by means of reversible phosphorylation. - Acetyl CoA carboxylase is switched off by phosphorylation and activated by dephosphorylation. - Insulin stimulates fatty acid synthesis causing dephosphorylation of carboxylase. - Glucagon and epinephrine have the reverse effect (keep the carboxylase in the inactive phosphorylated state). - Protein kinase is activated by AMP and inhibited by ATP. - Carboxylase is inactivated when the energy charge is low. # Local Regulation - Acetyl CoA carboxylase is allosterically stimulated by citrate. - The level of citrate is high when both acetyl CoA and ATP are abundant (isocitrate dehydrogenase is inhibited by ATP). - Palmitoyl CoA inhibits carboxylase. # Image Description - Page 36 The image shows a table and a list, describing the response of fatty acid synthesis to different diets. - **Fed state:** - Insulin level is increased. - Inhibits hydrolysis of stored TGs. - Stimulates formation of malonyl CoA, which inhibits carnitine acyltransferase I. - FA remain in cytosol (FA oxidation enzymes are in the mitochondria). - **Starvation:** - Epinephrine and glucagon are produced and stimulate adipose cell lipase and the level of free fatty acids rises. - Inactivate carboxylase, so decrease formation of malonyl CoA (lead to increased transport of FA into mitochondria and activate the b-oxidation pathway) # THE CONTROL OF FATTY ACID METABOLISM - Acetyl CoA carboxylase plays an essential in regulating fatty acid synthesis and degradation. - The carboxylase is controlled by hormones: - glucagon - epinephrine - insulin - Another regulatory factors: - citrate - palmitoyl CoA - AMP # Synthesis of Triglycerides - The major building block for the synthesis of triacylglycerols, in tissues other than adipose tissue, is glycerol-3-phosphate. - Adipocytes lack glycerol kinase, therefore, the 3C dihydroxyacetone phosphate, produced during glycolysis, is the precursor for triacylglycerol synthesis in adipose tissue. - This means that adipocytes must have glucose to oxidize in order to store fatty acids in the form of triacylglycerols. # SYNTHESIS OFTAG 1. There are two pathways for synthesis of Glycerol 3-P. - Liver is primary site for TAG synthesis. - **First pathway**: Liver and Adipose tissue:- - Glucose (glycolysis)→DHAP → reduced by Glycerol 3-P dehydrogenase Glycerol-3-Phosp. - **Second Pathway**: Liver but not in Adipose tissues (no enzyme). - Glycerol Kinase converts Free Glycerol Glycerol 3-Phosphate. - When plasma glucose and therefore Insulin levels are low -- Glucose cannot enter adipocyte (GLUT+ is Insulin dependent) -- Adipocytes→↓ glycerol-3-P synthesis →↓ TAG synthesis. # Breakdown of Triacylglycerols - The diagram shows a cell with triacylglycerol and another cell with glycolysis, gluconeogenesis, fatty acid oxidation, and CAC. - Adipocytes do not have Glycerol Kinase. # Image Description - Page 41 The diagram shows the synthesis of triacylglycerol with various intermediates including glucose, dihydroxyacetone-P, glycerol-3-P, and FA. # FATTY ACIDS ARE STORED AS TRIACYLGLYCEROLS (TAG) - Triglycerides are a highly concentrated store of energy. - Major energy reserve of body gives 9 kcal/g vs 4 kcal/g for carbohydrates or proteins. # TRIACYLGLYCEROL SYNTHESIS - Addition of 3 Acyl groups from Acyl-CoA to Glycerol-3-phosphate - Glycerol-3-phosphate + 2 Acyl-CoA --> Phosphatidate + CoA --> Triacylglycerol + CoA + Pi. # Image Description - Page 44 The image displays a diagram of a cell with all significant molecules and pathways for triacylglycerol synthesis. # Image Description - Page 45 The image displays a diagram of the pathway, describing the synthesis of triacylglycerol. The pathway show the reactions and enzymes involved. # Image Description - Page 46 The image shows a list of questions related to the lecture of Fatty Acid Synthesis and Triacylglycerol Synthesis. # FILL IN THE BLANKS - When glucose is plentiful, large amounts of acetyl CoA are produced by glycolysis and can be used for **fatty acid synthesis.** - Biosynthesis pathway takes place in **the cytosol** of the cell. - Acetyl CoA from catabolism of CHO and Amino acids is exported from mitochondria via **the citrate transport** system. - Acetyl CoA provides carbon and **NADPH** provides reducing equivalents. - NADPH molecules come from the **pentose phosphate** pathway. - **Acetyl CoA carboxylase** enzyme is rate limiting, irreversible and activate with the help of biotin. - **Acetyl CoA** is formed inside mitochondria but cannot come outside freely. - Acetyl CoA comes outside the mitochondria in the form of **citrate.** - Fatty acid synthesis occurs with loading of precursors via **thioester** derivatives. - During the fatty acid synthesis all intermediate are linked to the protein called **acyl carrier protein (ACP-SH).** - The elongation phase of FA synthesis starts with the formation of **acetyl ACP and malonyl ACP.** - Acetyl CoA carboxylase is regulated by **insulin and glucagon.** - Acetyl CoA also regulated by dietary intake, NADPH, and **citrate.** - The major building block for the synthesis of triacylglycerols, in tissues other than adipose tissue is **glycerol-3-phosphate.**