Medical Biochemistry Lecture Notes PDF

Medical Biochemistry Semester II Week 2 LIPID BIOSYNTHESIS FATTY ACIDS TAGs PHOSPHOLIPIDS Assistant prof. DACE REIHMANE Medicine 2.0, Medical Biochemistry 1 (CFUBK_072), A...

Medical Biochemistry Semester II Week 2 LIPID BIOSYNTHESIS FATTY ACIDS TAGs PHOSPHOLIPIDS Assistant prof. DACE REIHMANE Medicine 2.0, Medical Biochemistry 1 (CFUBK_072), Assist. Prof. Dace Reihmane  Lipid Biosynthesis Learning goals: Describe the pathway of fatty acid synthesis, particularly the roles of acetyl-CoA carboxylase and the multifunctional enzyme fatty acid synthase Explain the concepts of elongation and desaturation of the fatty acid chain Explain regulation of fatty acid synthesis Describe the synthesis of triacylglycerides Describe the synthesis of phospholipids Medicine 2.0, Medical Biochemistry 2 (CFUBK_072), Assist. Prof. Dace Reihmane Corresponding literature: 21| Lipid biosynthesis © 2017 W. H. Freeman and Company Medicine 2.0, Medical Biochemistry 3 (CFUBK_072), Assist. Prof. Dace Reihmane Additional literature Medical Biochemistry Principles of Medical Biochemistry HERE HERE Medicine 2.0, Medical Biochemistry 4 (CFUBK_072), Assist. Prof. Dace Reihmane  Catabolism and Anabolism of Fatty Acids Proceed via Different Pathways Catabolism of fatty acids produces acetyl-CoA produces reducing power (NADH+H+, FADH2) takes place in the mitochondria Anabolism of fatty acids requires acetyl-CoA and malonyl-CoA requires reducing power from NADPH+H+ takes place in cytosol in animals, chloroplast in plants Medicine 2.0, Medical Biochemistry 5 (CFUBK_072), Assist. Prof. Dace Reihmane  Fatty acid biosynthesis - place of action Every cell with mitochondria, but with different rate Cytosol Medicine 2.0, Medical Biochemistry 6 (CFUBK_072), Assist. Prof. Dace Reihmane  Fatty Acid Synthesis Occurs in Cell Compartments Where NADPH+H+ Levels Are High Cytosol for animals, yeast Sources of NADPH+H+: in adipocytes: pentose phosphate pathway and malic enzyme NADPH+H +made as malate converts to pyruvate + CO2 in hepatocytes and mammary gland: pentose phosphate pathway NADPH+H + made as glucose 6-phosphate converts to ribulose 5-phosphate Medicine 2.0, Medical Biochemistry 7 (CFUBK_072), Assist. Prof. Dace Reihmane  Acetyl-CoA Is Transported into the Cytosol for Fatty Acid Synthesis In nonphotosynthetic eukaryotes… Acetyl-CoA is made in the mitochondria But fatty acids are made in the cytosol So acetyl-CoA is transported into the cytosol with a cost of 2 ATPs Therefore, cost of FA synthesis is 3 ATPs per 2C ~50% of NADPH+H+ unit necessary for FA biosynthesis Medicine 2.0, Medical Biochemistry 8 (CFUBK_072), Assist. Prof. Dace Reihmane  Overview of Fatty Acid Synthesis Fatty acids are built in several passes, processing one acetate unit at a time The acetate is coming from activated malonate in the form of malonyl-CoA Each pass involves reduction of a carbonyl carbon to a methylene carbon Medicine 2.0, Medical Biochemistry 9 (CFUBK_072), Assist. Prof. Dace Reihmane  The Acetyl-CoA Carboxylase Reaction: Commited step Medicine 2.0, Medical Biochemistry 10 (CFUBK_072), Assist. Prof. Dace Reihmane  Synthesis of Fatty Acids Is Catalyzed by Fatty Acid Synthase (FAS) FAS I: Single polypeptide chain with 7 active sites in vertebrates Leads to single product: palmitate 16:0 FAS II: Seperate enzymes in plants and bacteria create variety of FA products – saturated, unsaturated, branched etc. All of the active sites in the mammalian system are located in different domains within a single large polypeptide chain. The different enzymatic activites are: β-ketoacyl-ACP synthase (KS), malonyl/acetyl-CoA–ACP transferase (MAT), β-hydroxyacyl-ACP dehydratase (DH), enoyl-ACP reductase (ER), and β-ketoacyl-ACP reductase (KR). ACP is the acyl carrier protein. Medicine 2.0, Medical Biochemistry 11 (CFUBK_072), Assist. Prof. Dace Reihmane  Fatty Acid Synthesis Overall goal: to attach acetate unit (2C) from malonyl-CoA to a growing chain and then reduce it Reaction involves cycles of four enzyme-catalyzed steps: condensation of the growing chain with activated acetate reduction of carbonyl to hydroxyl dehydration of alcohol to trans-alkene reduction of alkene to alkane The growing chain is initially attached to the enzyme via a thioester linkage Medicine 2.0, Medical Biochemistry 12 (CFUBK_072), Assist. Prof. Dace Reihmane  Acyl Carrier Protein (ACP) Serves as a Shuttle in Fatty Acid Synthesis Contains a covalently attached prosthetic group 4’-phosphopantetheine flexible arm to tether acyl chain while carrying intermediates from one enzyme subunit to the next Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase Medicine 2.0, Medical Biochemistry 13 (CFUBK_072), Assist. Prof. Dace Reihmane Charging: addition of Acetyl-CoA to ACP and further transfer to –SH of KS in synthesis Denovo Medicine 2.0, Medical Biochemistry 14 (CFUBK_072), Assist. Prof. Dace Reihmane  Charging: addition of Malonyl-CoA to ACP By using activated malonyl groups in the synthesis of fatty acids and activated acetate in their degradation, the cell makes both processes energetically favorable, although one is effectively the reversal of the other Medicine 2.0, Medical Biochemistry 15 (CFUBK_072), Assist. Prof. Dace Reihmane  Condensation by β-ketoacyl-ACP synthase Coupling condensation to decarboxylation of malonyl- CoA makes the reaction energetically favorable /β-ketoacyl-ACP Medicine 2.0, Medical Biochemistry 16 (CFUBK_072), Assist. Prof. Dace Reihmane Reduction by β-ketoacyl-ACP reductase Stereoisomer for intermediate in β-oxidation /β-ketoacyl-ACP D- /D-β-Hydroxyacyl-ACP Medicine 2.0, Medical Biochemistry 17 (CFUBK_072), Assist. Prof. Dace Reihmane Dehydration by -hydroxyacyl-ACP dehydratase D- /D-β-Hydroxyacyl-ACP /trans-Δ2-Enoyl-ACP Medicine 2.0, Medical Biochemistry 18 (CFUBK_072), Assist. Prof. Dace Reihmane Reduction by Enoyl-ACP reductase /trans-Δ2-Enoyl-ACP /Acyl-ACP (C=N+2) Medicine 2.0, Medical Biochemistry 19 (CFUBK_072), Assist. Prof. Dace Reihmane Translocation of extended chain to thiol group of Ketoacyl-ACP synthase domain /Acyl-ACP (C=N+2) Medicine 2.0, Medical Biochemistry 20 (CFUBK_072), Assist. Prof. Dace Reihmane Recharging ACP for another round of synthesis by malonyl/acetyl-CoA ACP transferase Medicine 2.0, Medical Biochemistry 21 (CFUBK_072), Assist. Prof. Dace Reihmane Overall Palmitate (16C) Synthesis Medicine 2.0, Medical Biochemistry 22 (CFUBK_072), Assist. Prof. Dace Reihmane  Fatty Acid Synthesis Is Tightly Regulated via Acetyl-CoA Carboxylase Acetyl CoA carboxylase (ACC) catalyzes the rate-limiting step ACC is feedback-inhibited by palmitoyl-CoA ACC is activated by citrate Citrate is made from acetyl-CoA in mitochondria (acetyl-CoAmt) Citrate signals excess energy to be converted to fat while inhibits glycolysis (PFK-1) Medicine 2.0, Medical Biochemistry 23 (CFUBK_072), Assist. Prof. Dace Reihmane Acetyl-CoA Carboxylase Is Also Regulated by Covalent Modification Inhibited when energy is needed Glucagon and epinephrine: reduce sensitivity of citrate activation lead to phosphorylation and inactivation of ACC ACC is inactive as phosphorylated monomers When dephosphorylated, ACC polymerizes into long active filaments Phosphorylation reverses the polymerization Medicine 2.0, Medical Biochemistry 24 (CFUBK_072), Assist. Prof. Dace Reihmane  Reciprocal regulation by Malonyl-CoA Benefit for segregating synthetic and degradative pathways in different cellular compartments Medicine 2.0, Medical Biochemistry 25 (CFUBK_072), Assist. Prof. Dace Reihmane Palmitate Can Be Lengthened to Longer-Chain Fatty Acids acyl-CoA desaturase Elongation systems in the endoplasmic reticulum and mitochondria create longer fatty acids (CoASH used, otherwise identical) Stearate (18:0) is the most common product Humans have 4, 5, 6, and 9 desaturases but cannot desaturate beyond 9, thus cannot produce Polyunsaturated fatty acids (PUFAs), such as omega-3 and omega-6 families, which help control membrane fluidity and are precursors to eicosanoids Linoleate and α-Linolenate are essential fatty acids for mammals In plants and bacteria PUFAs ensure membrane fluidity at reduced temperature Medicine 2.0, Medical Biochemistry 26 (CFUBK_072), Assist. Prof. Dace Reihmane  Summarize your knowledge about Fatty Acid Biosynthesis HOMEWORK TASK 4: Take a time to fill it out! Medicine 2.0, Medical Biochemistry 27 (CFUBK_072), Assist. Prof. Dace Reihmane  Synthesis of Fat (Triacylglycerides) and Phospholipids Animals and plants store fat for fuel plants: in seeds, nuts and fruits typical 70-kg human has ~15 kg fat enough to last 12 weeks compare with 12 hours worth of glycogen in liver and muscle Animals and plants and bacteria make phospholipids for cell membranes Both molecules contain glycerol backbone and 2 (e.g., phospholipids) or 3 (e.g., triacylglycerides) fatty acids Medicine 2.0, Medical Biochemistry 28 (CFUBK_072), Assist. Prof. Dace Reihmane Synthesis of Fat (Triacylglycerides) and Phospholipids Medicine 2.0, Medical Biochemistry 29 (CFUBK_072), Assist. Prof. Dace Reihmane  Synthesis of Backbone of TAGs and Phospholipids Most glycerol 3-phosphate comes from siphoning off dihydroxyacetone phosphate (DHAP) from glycolysis via glycerol 3-phosphate dehydrogenase Some glycerol 3-phosphate is made from glycerol via glycerol kinase minor pathway in liver and kidney glycerol kinase absent in adipocytes Medicine 2.0, Medical Biochemistry 30 (CFUBK_072), Assist. Prof. Dace Reihmane Synthesis of Phosphatidic Acid Occurs Before Synthesis TAGs Phosphatidic acid is the precursor for TAGs and phospholipids fatty acids attached by acyl transferases, after the activation by acyl-CoA synthetase releases CoASH Advantage of making phosphatidic acid It can then be made into triacylglycerol OR phospholipid /diacylglycerol 3-phosphate Medicine 2.0, Medical Biochemistry 31 (CFUBK_072), Assist. Prof. Dace Reihmane  Phosphatidic Acid Can Be Modified to Form Phospholipids or TAGs Phosphatidic acid phosphatase (lipin) removes the 3-phosphate from the phosphatidic acid yields 1,2-diacylglycerol The third carbon is then acylated with a third fatty acid yields triacylglycerol Medicine 2.0, Medical Biochemistry 32 (CFUBK_072), Assist. Prof. Dace Reihmane Regulation of Triacylglycerol Synthesis by Insulin Insulin results in stimulation of triacylglycerol synthesis Lack of insulin results in: Increased lipolysis Increased fatty acid oxidation sometimes to ketones if citric acid cycle intermediates (oxaloacetate) that react with acetyl CoA are depleted Failure to synthesize fatty acids from carbohydrates and proteins In case of untreated diabetes – increased rates of fat oxidation, therefore loss of weight Medicine 2.0, Medical Biochemistry 33 (CFUBK_072), Assist. Prof. Dace Reihmane  The Triacylglycerol Cycle Seventy-five percent (75%) of free fatty acids (FFAs) released by lipolysis are reesterified to form TAGs, rather than be used for fuel under all metabolic conditions (even in case of starvation) Some recycling occurs in adipose tissue Some FFAs from adipose cells are transported to the liver, remade into TAG, and redeposited in adipose cells Futile cycle? Where does the glycerol 3-phosphate come from? Medicine 2.0, Medical Biochemistry 34 (CFUBK_072), Assist. Prof. Dace Reihmane Glyceroneogenesis Makes DHAP for Glycerol 3- Phosphate Generation During TAG Cycle During lipolysis (stimulated by glucagon or epinephrine), glycolysis in adipocytes is inhibited So DHAP is not readily available to make glycerol 3-phosphate And adipose cells do not have glycerol kinase to make glycerol 3-phosphate on site Glyceroneogenesis contains some of the same steps of gluconeogenesis Converts pyruvate  DHAP Basically, an abbreviated version of gluconeogenesis in the liver and adipose tissue Uses pyruvate, alanine, glutamine or any substances from the Citric acid cycle as precursors for glycerol 3- phosphate Medicine 2.0, Medical Biochemistry 35 (CFUBK_072), Assist. Prof. Dace Reihmane  Thiazolidinediones Treat Type 2 Diabetes by Increasing Glyceroneogenesis High levels of free fatty acids in blood interfere with glucose utilization in muscle and promote insulin resistance that lead to type 2 diabetes Thiazolidinediones promote the synthesis of PEP carboxykinase Increase the rate of resynthesis of TAGs Medicine 2.0, Medical Biochemistry 36 (CFUBK_072), Assist. Prof. Dace Reihmane  Biosynthesis of Membrane Phospholipids All the biosynthetic pathways of different phospholipid species follow a few basic patterns: Synthesis of the backbone molecule (glycerol or sphingosine) Attachment of fatty acid(s) to the backbone through an ester or amide linkage Addition of a hydrophilic head group to the backbone through a phosphodiester linkage In some cases, alteration or exchange of the head group to yield the final phospholipid product Medicine 2.0, Medical Biochemistry 37 (CFUBK_072), Assist. Prof. Dace Reihmane  Phospholipid biosynthesis - place of action Every cell Primarily surface of the smooth ER and the inner mitochondrial membrane Transport to other cellular locations not fully understood Medicine 2.0, Medical Biochemistry 38 (CFUBK_072), Assist. Prof. Dace Reihmane  Biosynthesis of Membrane Phospholipids Begins with phosphatidic acid (diacylglycerol) Attach head group to C-3 OH group C-3 has OH; head group has OH new phospho-head group created when phosphoric acid condenses with these two alcohols eliminates two H2O Medicine 2.0, Medical Biochemistry 39 (CFUBK_072), Assist. Prof. Dace Reihmane  Attaching Phospholipid Head Group Requires Activation by Cytidine diphosphate Either one of the alcohols is activated by attaching to CDP (cytidine diphosphate) The free (not bound to CDP) alcohol then does a nucleophilic attack on the CDP- activated phosphate Medicine 2.0, Medical Biochemistry 40 (CFUBK_072), Assist. Prof. Dace Reihmane Example: Phospholipid Synthesis in E. coli Two main pathways: Phosphatidylserine is synthesized and can be decarboxylated to phosphatidylethanolamine Phosphatidylglycerol is synthesized by addition of a Glycerol-3-phosphate Further modification to cardiolipin can be achieved by replacement of the glycerol head group with another phospholipid Medicine 2.0, Medical Biochemistry 41 (CFUBK_072), Assist. Prof. Dace Reihmane  Synthesis of Phosphatidylserine and Phosphatidylcholine in Mammals Constitutes a Salvage Pathway Phosphatidylserine is made “backwards” from phosphatidyl-ethanolamine or phosphatidylcholine via head- group exchange reactions catalyzed by specific synthases Medicine 2.0, Medical Biochemistry 42 (CFUBK_072), Assist. Prof. Dace Reihmane  Summary of Phospholipid Biosynthesis Pathways in Eukaryotes Use Similar Pathways Used for synthesis of: Ether Lipids Plasmolagens Sphingolipids Central role in signal transduction Anti-inflammatory properties of fosfolipids Role of Phospholipids in Endocytosis, Phagocytosis, and Macropinocytosis Medicine 2.0, Medical Biochemistry 43 (CFUBK_072), Assist. Prof. Dace Reihmane Subcellular Localization of Lipid metabolism Medicine 2.0, Medical Biochemistry 44 (CFUBK_072), Assist. Prof. Dace Reihmane  Summarize your knowledge about TAG and phospholipid biosynthesis HOMEWORK TASK 5: Take a time to fill it out! Medicine 2.0, Medical Biochemistry 45 (CFUBK_072), Assist. Prof. Dace Reihmane  Summary of lipid anabolism Fatty acid synthesis and storage are essential components of body energy homeostasis Fatty acid synthesis takes place in the cytosol; its committed step is the reaction catalyzed by acetyl-CoA carboxylase Elongation of the fatty acid chain (up to the length of 16 carbon atoms) is carried out by the dimeric fatty acid synthase, which possesses 7 enzyme activities; both acetyl-CoA carboxylase and fatty acid synthase are subject to a complex regulation The citrate facilitates the transfer of two-carbon units from the mitochondria to cytoplasm for use in fatty acid synthesis The reducing power for fatty acid synthesis in the form of NADPH+H+ is supplied by the pentose phosphate pathway and also by the malic enzyme The essential unsaturated fatty acids are linoleic and linolenic acid; linoleic acid is converted to arachidonic acid, which in turn serves as the precursor of prostaglandins Medicine 2.0, Medical Biochemistry 46 (CFUBK_072), Assist. Prof. Dace Reihmane  Summary of lipid anabolism The synthesis of triacylglycerides and phospholipids starts after the formation of phosphatidic acid Further synthesis of triacylglycerides involves dephosphorylation and acylation and is associated with energy storage promoted by insulin Glyceroneogenesis in adipocytes is required to ensure the TAG cycle; activation of glyceroneogenesis is used in the treatment of type II diabetes Further synthesis of phospholipids involves the addition of a polar «head» group and is associated with growth processes in cells/body promoted by insulin Medicine 2.0, Medical Biochemistry 47 (CFUBK_072), Assist. Prof. Dace Reihmane Thank you!  Medicine 2.0, Medical Biochemistry 48 (CFUBK_072), Assist. Prof. Dace Reihmane