Fatty Acid Metabolism Part II PDF

1 Metabolism of Fatty acids Dr. Haroon Habib, Ph.D. Assistant Professor Dept of Biochemistry Alatoo International University  2 FATTY ACID METABOLISM BREAKDOWN OF FATTY ACID OR FATTY ACID F...

1 Metabolism of Fatty acids Dr. Haroon Habib, Ph.D. Assistant Professor Dept of Biochemistry Alatoo International University  2 FATTY ACID METABOLISM BREAKDOWN OF FATTY ACID OR FATTY ACID FATTY ACID BIOSYNTHESIS CATABOLISM  3 FATTY ACID BIOSYNTHESIS  4 Overview of Fatty Acid Synthesis Fatty acids are synthesized by In most mammals, glucose is an extramitochondrial the primary substrate for system, which is responsible lipogenesis, but in ruminants for the complete synthesis of it is acetate, the main fuel palmitate from acetyl-CoA molecule they obtain from the in the cytosol. diet. Fatty acids are synthesized by Fatty acid synthesis the condensation of two-carbon involves a separate series of units. However, in terms of the reactions to build up long- enzymic steps involved, the process is not the reverse of chain hydrocarbons from Beta- oxidation. acetyl CoA units.  THE NUTRITIONAL STATE 5 REGULATES LIPOGENESIS  Excess carbohydrate is stored as fat in many animals in anticipation of periods of caloric deficiency such as starvation, hibernation, etc, and to provide energy for use between meals.  Lipogenesis converts surplus glucose and intermediates such as pyruvate, lactate, and acetyl-CoA to fat, assisting the anabolic phase of this feeding cycle.  The nutritional state of the organism is the main factor regulating the rate of lipogenesis.  6 This system is present in many tissues, including liver, kidney, brain, lung, mammary gland, THE MAIN and adipose tissue. PATHWAY FOR Its cofactor requirements SYNTHESIS OF include NADPH, ATP, Mn2+, FATTY ACIDS biotin, and HCO3 − (as a source (LIPOGENESIS) of CO2 ). OCCURS IN THE CYTOSOL Acetyl-CoA is the immediate substrate, and free palmitate is the end product  Transport into the 7 cytosol  Since fatty acid synthesis takes place in the cytosol, the acetyl CoA produced from pyruvate has to be transported out of the mitochondria.  However,the inner mitochondrial membrane is not permeable to this compound, so it is first combined with oxaloacetate to form citrate which readily crosses the membrane.  In the cytosol the citrate is cleaved to regenerate the acetyl CoA.  8 Shuttle for Transfer of Acetyl Groups from Mitochondria to Cytosol  Acetyl-CoA, generated in the mitochondria, is shuttled to the cytosol as citrate 9  In most eukaryotes, the acetyl- CoA for lipid synthesis is made in the mitochondria  – But lipid synthesis occurs in the cytosol And there is no way for acetyl-CoA to cross  mitochondrial inner membrane to the cytosol  So acetyl-CoA is converted to citrate  – Acetyl-CoA + oxaloacetate------------------ citrate  Same rxn as occurs in CAC Catalyzed by citrate synthase Citrate passes through citrate transporter  10 Citrate is cleaved to regenerate acetyl-CoA  Citrate (now in cytosol) is cleaved by citrate lyase  – Regenerates acetyl-CoA and oxaloacetate – Rxn requires ATP – Acetyl-CoA can now be used for lipid synthesis  What happens to the oxaloacetate because there is no oxaloacetate transporter either? Oxaloacetate is converted to malate 11  Malate dehydrogenase in cytosol reduces  oxaloacetate to malate Two potential fates for malate:  – Can be converted to NADPHcyt and pyruvatecyt via the malic enzyme  NADPH used for lipid synthesis  Pyruvatecyt sent back to mito via pyruvate transporter  Converted back to oxaloacetatemito by pyruvate carboxylase, requires ATP  – Can be transported back to mito via malate - α- ketoglutarate transporter  Malatemito is reoxidized to oxaloacetatemito  12 Pathways for NADPH Productio n  13 REACTIONS OF THE FATTY ACID BIOSYNTHESIS PATHWAY  Production of Malonyl-COA Is the Initial & 14 Controlling Step in Fatty acid Synthesis  Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to malonyl-CoA in the presence of ATP and acetyl-CoA carboxylase.  This enzyme has a major role in the regulation of fatty acid synthesis.  Acetyl-CoA carboxylase has a requirement for the B vitamin biotin and is a multienzyme protein containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and a carboxyl transferase, as well as a regulatory allosteric site.  15 Production of Malonyl-COA Is the Initial & Controlling Step in Fatty acid Synthesis  16 Malonyl-CoA is formed from acetyl- CoA and bicarbonate  The reaction carboxylates acetyl CoA Catalyzed by acetyl-CoA carboxylase (ACC) Enzyme has three subunits: One unit has Biotin covalently linked to Lys  Biotin carries CO2 In animals, all three subunits are on one polypepeptide chain  HCO3 − (bicarbonate) is the source of CO2  The Acetyl-CoA Carboxylase 17 (ACC) Reaction  Two-step rxn similar to carboxylations catalyzed by pyruvate carboxylase (gluconeogenesis) and propionyl-CoA carboxylase (odd f.a. metabolism)  CO2 binds to biotin  Regulation of fatty acid 18 biosynthesis  The synthesis of fatty acids takes place when carbohydrate and energy are plentiful and when fatty acids are scarce.  The key enzyme in the regulation of fatty acid synthesis is acetyl CoA carboxylase which synthesizes malonyl CoA.  Acetyl CoA carboxylase (ACC) catalyzes the rate- limiting step  – ACC is feedback-inhibited by palmitoyl-CoA  – ACC is activated by citrate  Remember citrate is made from acetyl-CoAmito Citrate signals excess energy to be converted to fat  – When [acetyl-CoA]mito ↑, converted to citrate...citrate exported to cytosol  Importance of Citrate to 19 Regulation of Fatty Acid Synthesis In animals, citrate stimulates fatty acid synthesis! – Precursor for acetyl-CoA Sent to cytosol and cleaved to become AcCoA when AcCoA and ATP ↑ (energy excess) – Allosteric activator of ACC  Acetyl CoA carboxylase is also 20 subject to hormonal and allosteric regulation  When energy is required, glucagon and epinephrine inhibit protein phosphatase 2A, thus keeping acetyl CoA carboxylase in the inactive form and blocking fatty acid synthesis.  Acetyl CoA carboxylase is also allosterically regulated. The citric acid cycle intermediate citrate, the level of which is high when both acetyl CoA and ATP are abundant, allosterically stimulates acetyl CoA carboxylase  21 Synthesis of fatty acids is catalyzed by fatty acid synthase (FAS)  FASsystem: – Catalyzes a repeating four-step sequence that elongates the fatty acyl chain by two carbons at each step – Uses NADPH as as the electron donor – Uses two enzyme-bound -SH groups as activating groups  22 Fatty acid synthesis  Overall goal: attach two-C acetate unit from malonyl-CoA to a growing chain and then reduce it  Reaction involves cycles of four enzyme-catalyzed steps a) Condensation of the growing chain with activated acetate b) Reduction of carbonyl to hydroxyl c) Dehydration of alcohol to trans-alkene d) Reduction of alkene to alkane  The growing chain is initially attached to the enzyme via a thioester linkage  During condensation, the growing chain is transferred to the acyl carrier protein (ACP)  After the second reduction step, the elongated chain is transferred back to fatty acid synthase  23 Fatty acid biosynthesis : Reactions Condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP, releasing free ACP and CO2 (catalyzed by Reduction of acetoacetyl-ACP to form D-3- hydroxybutyryl-ACP, using NADPH as reductant (catalyzed by beta-ketoacyl-ACP reductase). Dehydration of D-3-hydroxybutyryl-ACP to produce crotonyl-ACP (catalyzed by 3-hydroxyacyl- ACP dehydratase).  Reduction of crotonyl-ACP by a second NADPH molecule to give butyryl- ACP (catalyzed by enoyl- ACP reductase).  Fatty acid biosynthesis : 24 Reactions  This first round of elongation produces the four-carbon butyryl-ACP.  The cycle now repeats with malonyl-ACP adding two-carbon units in each cycle to the lengthening acyl-ACP chain.  This continues until the 16-carbon palmitoyl- ACP is formed.  This molecule is not accepted by the acyl-malonyl-ACP condensing enzyme, and so cannot be elongated further by this process.  Instead it is hydrolyzed by a thio-esterase to give palmitate and ACP.  For each of the seven rounds of fatty acid elongation, one ATP is used in the synthesis of malonyl-CoA and two NADPH are used in the reduction reactions.  Fatty acid biosynthesis : 25 Reactions  In eukaryotes the elongation of fatty acids beyond C16 palmitate is carried out by enzymes located on the cytosolic surface of the smooth endoplasmic reticulum (SER).  Malonyl CoA is used as the two-carbon donor, and the fatty acid is elongated as its CoA derivative rather than its ACP derivative.  In prokaryotes, each of the reactions of fatty acid synthesis is catalyzed by a separate enzyme. However, in eukaryotes, the enzymes of the fatty acid synthesis elongation cycle are present in a single polypeptide chain, multifunctional enzyme complex, called fatty acid synthase.  26 Enzymes in Fatty Acid Synthase Condensation with acetate – β-ketoacyl-ACP synthase (KS) Reduction of carbonyl to hydroxyl – β-ketoacyl-ACP reductase (KR) Dehydration of alcohol to alkene – β-hydroxyacyl-ACP dehydratase (DH) Reduction of alkene to alkane – enoyl-ACP reductase (ER) Chain transfer/charging – Malonyl/acetyl-CoA ACP transferase  Stoichiometry of Synthesis of 27 Palmitate (16:0) 1) 7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs... using ATP 7AcCoA+7CO2 +7ATP 7malCoA+7ADP+7Pi 2) Seven cycles of condensation, reduction, dehydration and reduction...using NADPH to reduce the β-keto group and trans-double bond AcCoA + 7 malCoA + 14 NADPH + 14 H+ Palmitate + 7 CO2 + 8CoA+14NADP+ +6H2O  28 Formation of double bond  In eukaryotes the Smooth Endoplasmic reticulum has enzymes able to introduce double bonds into fatty acyl CoA molecules in an oxidation reaction that uses molecular oxygen.  This reaction is catalyzed by a membrane-bound complex of three enzymes:  NADH-cytochrome b5 reductase,  cytochrome b5  desaturase.  The overall reaction is:  29 Formation of double bond  Mammals lack the enzymes to insert double bonds at carbon atoms beyond C-9 in the fatty acid chain.  They cannot synthesize linoleate and linolenate, both of which have double bonds later in the chain than C-9.  Hence, in mammals linoleate and linolenate are called essential fatty acids since they have to be supplied in the diet.  These two unsaturated fatty acids are also the starting points for the synthesis of other unsaturated fatty acids, such as arachidonate.  This C20:4 fatty acid is the precursor of several biologically important molecules, including the prostaglandins, thromboxanes and leukotrienes  Catabolism and anabolism of fatty 30 acids proceed via different pathways Catabolism of fatty acids (excergonic and oxidative) – produces acetyl-CoA – produces reducing power (NADH and FADH2) – activation of fatty acids by CoA – takes place in the mitochondria Anabolism of fatty acids (endergonic and reductive) – requires acetyl-CoA and malonyl-CoA – requires reducing power from NADPH – activation of fatty acids by 2 different –SH groups on protein – takes place in cytosol in animals, chloroplast in plants Thank You 32