Synthesis and degradation of fatty acids Martina Srbová

Fatty acids (FA) Groups of FA: mostly an even number of carbon atoms and linear chain in esterified form as component of lipids in unesterified form in plasma binding to albumin Groups of FA: according to the chain length <C6 short-chain FA (SCFA) C6 – C12 medium-chain FA (MCFA) C14 – C20 long-chain FA (LCFA) >C20 very-long-chain FA (VLCFA) according to the number of double bonds no double bond saturated FA (SAFA) one double bond monounsaturated FA (MUFA) more double bonds polyunsaturated FA (PUFA)

Overview of FA

Triacylglycerols main storage form of FA acyl-CoA and glycerol-3-phosphate synthesis of TAG in liver stored mainly in adipose tissue TAG transport from the liver to other tissues via VLDL (especially skeletal muscle, adipose tissue)

FA biosynthesis localization: enzymes: primary substrate: mainly in the liver, adipose tissue, mammary gland during lactation (always in excess calories) localization: cell cytoplasm (up to C16) endoplasmic reticulum, mitochondrion (elongation = chain extension) enzymes: acetyl-CoA-carboxylase (HCO3- - source of CO2, biotin, ATP) fatty acid synthase (NADPH + H+, pantothenic acid) primary substrate: acetyl-CoA final product: palmitate

Precursors for FA biosynthesis 1. Acetyl-CoA source: oxidative decarboxylation of pyruvate (the main source of glucose) degradation of FA, ketones, ketogenic amino acids transport across the inner mitochondrial membrane as citrate 2. NADPH source: pentose phosphate pathway (the main source) the conversion of malate to pyruvate (NADP+-dependent malate dehydrogenase - „malic enzyme”) the conversion of isocitrate to α-ketoglutarate (isocitrate dehydrogenase)

Precursors for FA biosynthesis Acetyl-CoA + HSCoA OAA - oxaloacetate

FA biosynthesis Formation of malonyl-CoA catalysed by acetyl-CoA-carboxylase (ACC) HCO3- + ATP ADP + Pi enzyme-biotin enzyme-biotin-COO- 1 carboxylation of biotin 2 transfer of carboxyl group to acetyl-CoA acetyl-CoA formation of malonyl-CoA + enzyme-biotin enzyme – acetyl-CoA-carboxylase malonyl-CoA

FA biosynthesis on the multienzyme complex – FA synthase repeated extension of FA by two carbons in each cycle to the chain length C16 (palmitate) ACP – acyl carrier protein

FA biosynthesis The course of FA biosynthesis transacylation acetyl-CoA malonyl-CoA CoASH CoASH acetyltransacylase malonyltransacylase transacylation acyl(acetyl)-malonyl- -enzyme complex

FA biosynthesis The course of FA biosynthesis condensation CO2 3-ketoacyl-synthase CO2 condensation acyl(acetyl)-malonyl-enzyme complex 3-ketoacyl-enzyme complex (acetacetyl-enzyme complex)

FA biosynthesis The course of FA biosynthesis first reduction NADPH + H+ NADP+ NADPH + H+ NADP+ H2O 3-ketoacyl-reductase 3-hydroxyacyl- dehydrase enoylreductase first reduction dehydration second reduction 3-ketoacyl-enzyme complex (acetoacetyl-enzyme complex) 3-hydroxyacyl-enzyme complex 2,3-unsaturated acyl-enzyme complex acyl-enzyme complex

FA biosynthesis Repetition of the cycle malonyl-CoA CoASH acyl-enzyme complex (palmitoyl-enzyme complex)

FA biosynthesis The release of palmitate + H2O palmitate thioesterase + H2O palmitate palmitoyl-enzyme complex

FA biosynthesis The fate of palmitate after FA biosynthesis acylglycerols cholesterol esters ATP + CoA AMP + PPi esterification palmitate palmitoyl-CoA acyl-CoA-synthetase elongation desaturation acyl-CoA

FA biosynthesis FA elongation 1. microsomal elongation system in the endoplasmic reticulum malonyl-CoA – the donor of the C2 units NADPH + H+ – the donor of the reducing equivalents extension of saturated and unsaturated FA FA > C16 elongases (chain elongation) palmitic acid (C16) fatty acid synthase 2. mitochondrial elongation system in mitochondria acetyl-CoA – the donor of the C2 unit not reverse β-oxidation

FA biosynthesis FA desaturation in the endoplasmic reticulum 4 desaturases: double bonds at position  4,5,6,9 linoleic, linolenic – essential FA enzymes: desaturase, NADH-cyt b5-reductase process requiring O2, NADH, cytochrome b5 stearoyl-CoA + NADH + H+ + O2 oleoyl-CoA + NAD+ + 2H2O

FA biosynthesis - summary Formation of malonyl-CoA Acetyl-CoA-carboxylase FA synthesis Palmitic acid FA Synthase– cytosol Saturated fatty acids(>C16) Elongation systems- mitochondria, ER Unsaturated fatty acids Desaturation system - ER -

FA degradation function: major energy source 1 2 3 4 5 (especially between meals, at night, in increased demand for energy intake – exercise) release of FA from triacylglycerols in adipose tissue into the bloodstream binding of FA to albumin in the bloodstream transport to tissues 1 entry of FA into target cells 2 activation to acyl-CoA transfer of acyl-CoA via carnitine system into mitochondria 3 4 β-oxidation 5 In the liver , acetyl CoA is converted to ketone bodies

FA degradation β-carbon -carbon -carbon -oxidation β-oxidation C10 , C12 Branched FA VLCFA http://che1.lf1.cuni.cz/html/Odbouravani_MK_3sm.pdf

FA degradation β-oxidation localization: enzymes: substrate: mainly in muscles localization: mitochondrial matrix peroxisome (VLCFA) enzymes: acyl CoA synthetase carnitine palmitoyl transferase I, II; carnitine acylcarnitine translocase dehydrogenase (FAD, NAD+), hydratase, thiolase substrate: acyl-CoA final products: acetyl-CoA propionyl-CoA

FA degradation β-oxidation PRODUCTION OF LARGE QUANTITY OF ATP repeated shortening of FA by two carbons in each cycle cleavage of two carbon atoms in the form of acetyl-CoA oxidation of acetyl-CoA to CO2 and H2O in the citric acid cycle complete oxidation of FA generation of 8 molecules of acetyl-CoA from 1 molecule of palmitoyl-CoA production of NADH, FADH2 reoxidation in the respiratory chain to form ATP PRODUCTION OF LARGE QUANTITY OF ATP

FA degradation Activation of FA fatty acid+ ATP + CoASH acyl-CoA-synthetase acyl adenylate pyrophosphate (PPi) acyl-CoA-synthetase pyrophosphatase 2Pi acyl-CoA AMP fatty acid+ ATP + CoASH acyl-CoA + AMP + PPi PPi + H2O 2Pi

FA degradation Substrate specifity for: SCFA MCFA LCFA VLCFA acyl CoA synthetase Substrate specifity for: SCFA MCFA LCFA VLCFA Localization Outer mitochondrial membrane ER SCFA, MCFA Mitochondrial matrix

FA degradation The role of carnitine in the transport of LCFA into mitochondrion FA transfer across the inner mitochondrial membrane by carnitine and three enzymes: carnitine palmitoyl transferase I (CPT I) acyl transfer to carnitine carnitine acylcarnitine translocase acylcarnitine transfer across the inner mitochondrial membrane carnitine palmitoyl transferase II (CPT II) acyl transfer from acylcarnitine back to CoA in the mitochondrial matrix

FA degradation Carnitine 3-hydroxy-4-N-trimethylaminobutyrate Sources: Exogenous: meat, dairy products Endogenous: synthesis from lysine and methionine Transported into the cell by specific transporter Deficiency: Decreased transport of acyl-CoA into mitochondria lipids accumulation myocardial damage muscle weakness Increased utilization of Glc hypoglycemia Similar symptoms are the genetically determined deficiency carnitinpalmitoyltransferase I or II

FA degradation β-oxidation Steps of cycle: acyl-CoA trans-Δ2-enoyl-CoA dehydrogenation oxidation by FAD creation of unsaturated acid acyl-CoA-dehydrogenase trans-Δ2-enoyl-CoA hydration addition of water on the β-carbon atom creation of β-hydroxyacid enoyl-CoA-hydratase L-β-hydroxyacyl-CoA L-β-hydroxyacyl-CoA- dehydrogenation oxidation by NAD+ creation of β-oxoacid -dehydrogenase β-ketoacyl-CoA cleavage at the presence of CoA formation of acetyl-CoA formation of acyl-CoA (two carbons shorter) β-ketoacyl-CoA-thiolase acyl-CoA acetyl-CoA

FA degradation Oxidation of unsaturated FA the most common unsaturated FA in the diet: β-oxidation of oleic acids oleic acid, linoleic acid degradation of unsaturated FA by β-oxidation to a double bond 3 rounds of β-oxidation Unsaturated FA are cis isomers - aren´t substrate for enoyl-coA hydratase conversion of cis-isomer of FA by specific isomerase to trans-isomer intramolecular transfer of double bond from β- to - β position continuation of β-oxidation Normal intermediates of β-oxidation http://che1.lf1.cuni.cz/html/Odbouravani_MK_3sm.pdf

FA degradation Oxidation of odd-chain FA shortening of FA to C5 propionyl-CoA shortening of FA to C5 stopping of β-oxidation HCO3- + ATP propionyl-CoA carboxylase (biotin) ADP + Pi formation of acetyl-CoA and propionyl-CoA methylmalonyl-CoA carboxylation of propionyl-CoA methylmalonyl-CoA mutase (B12) intramolecular rearrangement to form succinyl-CoA succinyl-CoA entry of succinyl-CoA into the citric acid cycle

FA degradation Peroxisomal oxidation of VLCFA Very-long-chain FA (VLCFA, > C20) transport of acyl-CoA into the peroxisome without carnitine Differences between β-oxidation in the mitochondrion and peroxisome: 1. step – dehydrogenation by FAD mitochondrion: electrons from FADH2 are delivered to the respiratory chain where they are transferred to O2 to form H2O and ATP peroxisome: electrons from FADH2 are delivered to O2 to form H2O2, which is degraded by catalase to H2O and O2 3. step – dehydrogenation by NAD+ mitochondrion: reoxidation of NADH in the respiratory chain peroxisome: reoxidation of NADH is not possible, export to the cytosol or the mitochondrion

FA degradation Peroxisomal oxidation of VLCFA Differences between β-oxidation in the mitochondrion and peroxisome: 4. step – cleavage at the presence of CoA acetyl-CoA mitochondrion: metabolization in the citric acid cycle peroxisome: export to the cytosol, to the mitochondrion (oxidation) a precursor for the synthesis of cholesterol and bile acids a precursor for the synthesis of fatty acids of phospholipids In peroxisome shortened FA bind to carnitine acylcarnitine transfer acylcarnitine into mitochondrion β-oxidation

mixed function oxidase FA degradation - oxidation Excreted in the urine mixed function oxidase Oxidation  carbon ER liver, kidney Substrates C10 a C12 FA Products: dicarboxylic acids

Comparison of FA biosynthesis and FA degradation

Ketone bodies medium strength acids - ketoacidosis Ketogenesis in the liver localization: mitochondrial matrix substrate: acetyl-CoA products: acetone acetoacetate medium strength acids - ketoacidosis D-β-hydroxybutyrate conditions: in excess of acetyl-CoA function: energy substrates for extrahepatic tissues

Ketone bodies Ketogenesis

Ketone bodies Ketogenesis acetoacetate waste product (lung, urine) spontaneous decarboxylation to acetone conversion to D-β-hydroxybutyrate by D-β-hydroxybutyrate dehydrogenase waste product (lung, urine) energy substrates for extrahepatic tissues

Ketone bodies Utilization of ketone bodies water-soluble FA equivalents energy source for extrahepatic tissues (especially heart and skeletal muscle) in starvation - the main source of energy for the brain energy citric acid cycle production

Ketone bodies Ketogenesis increased ketogenesis: lipolysis starvation prolonged exercise diabetes mellitus FA in plasma high-fat diet low-carbohydrate diet β-oxidation utilization of ketone bodies as an energy source (skeletal muscle, intestinal mucose, adipocytes, brain, heart etc.) excess of acetyl-CoA to spare of glucose and muscle proteins ketogenesis

Bibliography and sources Devlin, T. M. Textbook of biochemistry: with clinical correlations. 6th edition. Wiley-Liss, 2006. Marks, A.; Lieberman, M. Marks' basic medical biochemistry: a clinical approach. 3rd edition. Lippincott Williams & Wilkins, 2009. Matouš a kol. Základy lékařské chemie a biochemie. Galén, 2010. Meisenberg, G.; Simmons, W. H. Principles of medical biochemistry. 2nd edition. Elsevier, 2006. Murray et al. Harper's Biochemistry. 25th edition. Appleton & Lange, 2000. http://www.hindawi.com/journals/jobes/2011/482021/fig2/