* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Fatty Acid Biosynthesis
Catalytic triad wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Genetic code wikipedia , lookup
Metalloprotein wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Oligonucleotide synthesis wikipedia , lookup
Proteolysis wikipedia , lookup
Lipid signaling wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
15-Hydroxyeicosatetraenoic acid wikipedia , lookup
Peptide synthesis wikipedia , lookup
Glyceroneogenesis wikipedia , lookup
Specialized pro-resolving mediators wikipedia , lookup
Butyric acid wikipedia , lookup
Citric acid cycle wikipedia , lookup
Biochemistry wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biosynthesis wikipedia , lookup
LIPID MAPS Lipid Metabolomics Tutorial Fatty Acid Biosynthesis Professor Edward A. Dennis Department of Chemistry and Biochemistry Department of Pharmacology, School of Medicine University of California, San Diego Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org E.A. DENNIS 2010 © Metabolism and Energy Overview • Proteins Carbohydrates Lipids • • Amino Acids Simple Sugars Fatty Acids • Many biomolecules are degraded to Acetyl CoA Acetyl CoA provides biologic energy Excess acetyl CoA is stored as Fatty Acids (FA’s) FA’s are assembled into more complex lipids like triglycerides (TG’s) Pyruvate Acetyl CoA Energy (CO2, H2O) E.A. DENNIS 2010 © What is a “Fatty Acid”? Palmitic acid Fatty acid: a carboxylic acid with a long hydrocarbon chain. Usually, they have an even number of carbons. Reactive and toxic. Ester group Fatty acid ester: a fatty acid in which the carboxylic acid group has reacted with the alcohol group of another molecule (often glycerol) to form a stable, less reactive ester bond. E.A. DENNIS 2010 © What is a “Triglyceride”? Glycerol: common name for 1,2,3-trihydroxy-propane. Glycerol Triglyceride: a glycerol molecule with three esterfied fatty acid side chains. Also known more correctly as a “triacylglycerol”. Stable, nonpolar, hydrophobic. Triacylglycerol E.A. DENNIS 2010 © Common Saturated Fatty Acids Saturated FA’s have no double bonds 16 Carbons = Palmitic Acid (Palmitate) 18 Carbons = Stearic Acid (Stearate) E.A. DENNIS 2010 © Common Unsaturated Fatty Acids Unsaturated FA’s have at least one double bond, usually in the Z (cis) conformation 18 Carbons, 1 double bond at c9 = Oleic Acid (Oleate) 18 9 1 18 Carbons, 2 double bonds at c9 and c12 = Linoleic Acid (Linoleate) 18 12 9 1 E.A. DENNIS 2010 © More Unsaturated Fatty Acids 18 Carbons, 3 cis double bonds at 9, 12 & 15 = a-Linolenic Acid (a-Linolenate) 18 15 12 9 1 20 Carbons, 4 cis double bonds at 5,8,11 & 14 Arachidonic Acid (Arachidonate) (5Z,8Z,11Z,14Z-Eicosatetraenoic Acid) E.A. DENNIS 2010 © What are Essential Fatty Acids? • Two “Essential” FA’s cannot be synthesized by humans Diet Linoleic acid Linolenic acid Arachidonic acid EPA – Linoleic acid – Linolenic acid • Used in the biosynthesis of polyunsaturated fatty acid • Must come from diet E.A. DENNIS 2010 © Key Enzyme: Acetyl-CoA Carboxylase • Acetyl-CoA Carboxylase is a key enzyme • Converts acetyl-CoA into malonyl-CoA – The CO2 is released later – Biotin is a cofactor • It is the “committed step” in FA synthesis • It is the regulated, ratelimiting enzyme in FA synthesis “E” above is the enzyme acetyl-CoA carboxylase, which is conjugated to biotin. E.A. DENNIS 2010 © FA Synthesis: Step 1 Step 1: Set up acetyl-ACP Acetyl-CoA-ACP Transacylase ACP is “acyl carrier protein” and is a part of a large enzyme complex. It holds the reactants in place while other enzymes catalyze the subsequent reaction steps. E.A. DENNIS 2010 © FA Synthesis: Step 2 Step 2: Set up malonyl-ACP Acetyl-CoA-ACP Transacylase HCO3- Acetyl-CoA Carboxylase Malonyl-CoA-ACP Transacylase This step will iterate many times, adding carbons to the growing FA backbone. E.A. DENNIS 2010 © FA Synthesis: Step 3 Step 3: Condense them, giving Acetoacetyl-ACP and CO2 condensing enzyme CO2 ACP The condensing enzyme is also known as b-ketoacyl-ACP synthase. It is part of the FA synthase complex E.A. DENNIS 2010 © FA Synthesis: Step 4 Step 4: Use NADPH to reduce the b-carbonyl to a hydroxyl group H+ + NADPH b-ketoacyl-ACP reductase NADP+ E.A. DENNIS 2010 © FA Synthesis: Step 5 Step 5: Remove the hydroxyl group as H 2O leaving a double bond b-hydroxylacyl-ACP dehydratase E.A. DENNIS 2010 © FA Synthesis: Step 6 Step 6: Use another NADPH to reduce the double bond H+ + NADPH Enoyl-ACP reductase NADP + E.A. DENNIS 2010 © FA Synthesis: Repeat Cycle Repeat from step 2 using the new 4-carbon butyryl-ACP in place of acetyl-ACP recycle reactions 2-6 six more times thioesterase • 6 iterations makes Palmitoyl-ACP. • Finally, the enzyme thioesterase cleaves the ACP from palmitoylACP • Palmitate is released. E.A. DENNIS 2010 © Fatty Acid Synthesis Summary One iteration: Step 1: Set up acetyl-ACP Step 2: Set up malonyl-ACP Step 3: Condense them, giving Acetoacetyl ACP Step 4: Use NADPH to reduce distal carbonyl to a hydroxyl group Step 5: Remove the hydroxyl group as H 2O leaving a double bond Step 6: Use another NADPH to reduce the double bond REPEAT: From step 2 using the new 4-carbon butyryl-ACP in place of acetyl-ACP in step 3 E.A. DENNIS 2010 © [2a] Acetyl-CoA Carboxylase Acetyl-CoA enters cycle Acetyl-CoA [1] Malonyl-CoA Acetyl-CoA-ACP transacylase Malonyl-CoA-ACP transacylase [2b] Initiation Acetyl-ACP Malonyl-ACP Fatty acid synthase cycle [3] b-ketoacylACP synthase Release from FA synthase complex Acetoacetyl-ACP [4] b-ketoacyl-ACP reductase Elongation Palmitoyl-ACP b-hydroxybutyrylACP Butyryl-ACP thioesterase [6] Palmitate enoyl-ACP reductase b-hydroxyacyl-ACP dehydratase 2-trans-butenoyl-ACP [5] E.A. DENNIS 2010 © The FA Synthase Enzyme • FA synthase is an enzyme complex • It includes all the FA synthesis enzymes except for acetyl-CoA carboxylase • Actually exists as a dimer of two complete, anti-parallel complexes -- like Ying and Yang • Cytosolic Figure: Voet, D, Voet JG, Pratt CW (2002), Fundamentals of Biochemistry: Lif e at the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc. Figures: Nelson DL, Cox MM (2005), Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman & Co. E.A. DENNIS 2010 © Tuberculosis Tuberculosis cases (thousands) Reported Tuberculosis in the US 1982-2008 AIDS increase 50% decrease • Incidence: one of the leading causes of death due to infectious diseases – Often follows HIV infection • Symptoms: pulmonary infection – cough, sputum, pleural effusions • see picture below left – urogenital & brain affects also seen Year • Mechanism: Mycobacterium tuberculosis infection • Treatments: – First-line combination: • Pyrazinamide and Isoniazid – stop mycobacterial FA synthase! • Rifampin (RNA transcription inhibitor) – Various second-line agents – If needed, HIV treatment Normal lung Source: CDC Tuberculosis infection Treating Tuberculosis • Mycobacteria – make their outer membrane with mycolic acids using FA synthases • FAS-1 is a single, eukaryote-like enzyme with multiple actions • FAS-2 is a multi-unit, prokaryote-like enzyme with multiple actions • Pyrazinamide Figure: Draper, Nat. Med. 6, 977-8 (2000). Mycolic acid; R1 and R2 are long-chain aliphatic hydrocarbons (“peer-ah-ZIN-a-mide”) – Inhibits FAS-I – Relatively specific for M. tuberculosis – Arrests synthesis of both fatty acids and mycolic acids • Isoniazid (“eye-so-NYE-a-zid”) – Inhibits FAS-II – Stops synthesis of mycolic acids E.A. DENNIS 2010 © Bacteria vs. Mammals FA Synthesis in Bacteria FA Synthesis in Mammals • Acyl carrier protein (ACP) is a separate molecule, as are each of the six enzymes. • ACP is part of the FA synthase complex, which is one large protein present as a dimer. • “Acetyl CoA carboxylase” is two separate enzymes, plus a biotin cofactor joined to a third enzyme. • Acetyl-CoA carboxylase is one enzyme conjugated to a biotin cofactor. E.A. DENNIS 2010 © Regulation of FA Synthesis Regulation occurs primarily at acetylCoA carboxylase, the rate limiting step Feedback Mechanisms Hormonal Mechanisms • Citrate, which builds up when acetyl-CoA is plentiful, accelerates FA synthesis. • Insulin, which signals a resting, energy rich state, dephosphorylates and accelerates the enzyme. • Palmitoyl-CoA weakly inhibits FA synthesis. • Glucagon, epinephrine and norepinephrine, which signal immediate energy needs, phosphorylate and slow the enzyme [via AMP -dependent protein kinase and also via CMPdependent PKA]. E.A. DENNIS 2010 © Acknowledgement This tutorial is based on an evolving subset of lectures and accompanying slides presented to medical students in the Cell Biology and Biochemistry course at the School of Medicine of the University of California, San Diego. I wish to thank Dr. Bridget Quinn and Dr. Keith Cross for aid in developing many of the original slides, Dr. Eoin Fahy for advice in applying the LIPID MAPS nomenclature and structural drawing conventions [Fahy et al (2005) J Lipid Res, 46, 839-61; Fahy et al (2009) J Lipid Res, 50, S9-14] and Masada Disenhouse for help in adopting to the tutorial format. Edward A. Dennis September, 2010 La Jolla, California E.A. DENNIS 2010 ©