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Lipid Metabolism_In_Biochemistry

Lipid Metabolism_In_Biochemistry.
Agricultural University of Plovdiv, Bulgaria
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Lipid Metabolism_In_Biochemistry

  1. 1. Lipid Metabolism Plant Chemistry In Biochemistry Agricultural University - Plovdiv Plovdiv; 2017 1
  2. 2. Lipids are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents, e.g., chloroform, ether, or benzene. Lipids 2
  3. 3. 3
  5. 5. Summary of events that must occur before triacyglycerols (TAGs) can reach the bloodstream through the digestive process. 5
  6. 6. A gram of nearly anhydrous fat stores more than six times as much energy as a gram of hydrated glycogen, which is likely the reason that triacylglycerol's rather than glycogen were selected in evolution as the major energy reservoir. The glycogen and glucose stores provide enough energy to sustain biological function for about 24 hours, whereas the Triacylglycerol stores allow survival for several weeks. TRIGLYCERIDES V/S GLYCOGEN 6
  7. 7. Lipolysis - hydrolysis of triacylglycerol's by lipases. A hormone-sensitive lipase converts TGs to free fatty acids and monoacylglycerol. Monoacylglycerol is hydrolyzed to fatty acid and glycerol or by a hormone- sensitive lipase or by more specific and more active monoacylglycerol lipase L I P O L Y S I S 7
  8. 8. Hydrolysis of stored triacylglycerols in adipose tissue is triggered by hormones that stimulate cAMP production within adipose cells. 8
  9. 9. Isomers  Steps: phosphorylation, oxidation and isomerisation.  Glyceraldehyde 3- phosphate is an intermediate in: • Glycolytic pathway • Gluconeogenic pathways Oxidation of Glycerol 9
  10. 10.  Glycerol – glycerol 3-phosphate - 1 ATP  Glycerol 3-phosphate - dihydroxyaceton phosphate 2.5ATP (1 NADH)  Glyceraldehyde 3-phosphate – pyruvate 4,5 ATP (1NADH + 2 ATP)  Pyruvate – acetyl CoA 2.5 ATP (1 NADH)  Acetyl CoA in Krebs cycle 10 ATP (3NADH + 1 FADH2 + 1GTP) Total 19,5-1 = 18,5 ATP ATP Generation from Glycerol Oxidation. 10
  11. 11. TYPES OF FATTY ACID OXIDATION Fatty acids can be oxidized by: 11
  12. 12. Overview of beta oxidation: A saturated acyl Co A is degraded by a recurring sequence of four reactions: 1) Oxidation 2) Hydration 3) Oxidation 4) Thiolysis • Acyl CoA undergoes dehydrogenation by an FAD-dependent flavoenzyme, acyl CoA Dehydrogenase. • A double bond is formed between α and β carbons (i.e., 2 and 3 carbons) Enoyl CoA hydrates brings. About the hydration of the double bond to form β - hydroxyacyl CoA.  β-Hydroxyacyl CoA dehydrogenase catalyzes the second oxidation and generates NADH.  The product formed is β-ketoacyl CoA. The final reaction in β -oxidation is the liberation of a 2 carbon fragment, acetyl CoA from acyl CoA. This occurs by a thiolytic cleavage catalysed by β- ketoacyl CoA thiolase (or thiolase). The new acyl CoA, containing two carbons less than the original, reenters the β-oxidation cycle. The process continues till the fatty acid is completely oxidized. 12
  13. 13. 1. Activation of fatty acids in the cytosol 2. Transport of activated fatty acids into mitochondria (carnitine shuttle) 3. Beta oxidation proper in the mitochondrial matrix Fatty acids to be oxidized must be entered the following steps: 1) Activation of FA: This proceeds by FA thiokinase (acyl COA synthetize) present in endoplasmic reticulum and in the outer mitochondrial membrane. Thiokinase requires ATP, COA SH, Mg++. The product of this reaction is acyl COA and water. Fatty acids must first be converted to an active intermediate before they can be catabolized. This is the only step in the complete degradation of a fatty acid that requires energy from ATP. The activation of a fatty acid is accomplished in two steps: The beta oxidation of fatty acids involve three stages: 13
  14. 14. Impermeable to ions and most other compounds In inner membrane knobs Mitochondrion The mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation. 14
  15. 15. • ATP is converted to AMP + P~P, the energy released is utilized for formation of high energy bond (thioester bond) in acyl COA (RCO ~ S COA). • The high energy of P~P is lost by pyrophosphatase thus two high energy phosphates are lost during activation. 15
  16. 16. 2-Transport of fatty acyl CoA from cytosol into mitochondria: Long chain acyl CoA cannot readily traverse the inner mitochondria membrane and so a special transport mechanism called carnitine shuttle is needed.  It is synthesized in liver and kidney from lysine.  It is essential for oxidation of long chain fatty acids.  Carnitine is not required for the permeation of medium chain acyl CoA into the mitochondrial matrix.  Carnitine (β-hydroxy-y-trimethyl- ammonium butyrate) is a carrier.  Acyl groups from acyl COA is transferred to hydroxyl group of carnitine to form acyl carnitine, catalyzed by carnitine acyltransferase I, located in the outer mitochondrial membrane. 16
  17. 17. Acylcarnitine is then shuttled across the inner mitochondrial membrane by a translocase enzyme. The acyl group is transferred back to CoA on the inner border of the matrix side of the inner mitochondrial membrane by carnitine acyl transferase II. Finally, carnitine is returned to the cytosolic side by translocase, in exchange for an incoming acyl carnitine. 17
  18. 18. STEPS OF BETA OXIDATION СН2 СН2 СН3 С О ОН α α β 18
  19. 19. STEPS OF BETA OXIDATION Electrons from the FADH2 prosthetic group of the reduced acyl CoA dehydrogenase are transferred to electron- transferring flavoprotein (ETF).  ETF donates electrons to ETF: ubiquinone reductase, an iron-sulfur protein.  Ubiquinone is thereby reduced to ubiquinol, which delivers its high- potential electrons to the second proton-pumping site of the respiratory chain. 19
  20. 20. Step-1 Dehydrogenation- The first step is the removal of two hydrogen atoms from the 2(α)- and 3(β)- carbon atoms, catalyzed by acyl-CoA dehydrogenase and requiring FAD. This results in the formation of Δ2-trans- enoyl-CoA and FADH2. Step-2- Hydration Water is added to saturate the double bond and form 3- hydroxyacyl- CoA, catalyzed by Δ 2-enoyl- CoA hydrates. Step-3- dehydrogenation- The 3-hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed by L(+)-3-hydroxyacyl- CoA dehydrogenase to form the corresponding 3-ketoacyl-CoA compound. In this case, NAD+ is the coenzyme involved. Step-4- Thiolysis- 3-ketoacyl-CoA is split at the 2,3- position by thiolase (3- ketoacyl-CoA- thiolase), forming acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoA molecule. 20
  21. 21. STEPS OF BETA OXIDATION The acyl-CoA formed in the cleavage reaction reenters the oxidative pathway at reaction 2.  Since acetyl-CoA can be oxidized to CO2 and water via the citric acid cycle the complete oxidation of fatty acids is achieved. 21
  22. 22. BETA OXIDATION- ENERGY YIELD Total ATP per turn of the fatty acid spiral is:  Step 1 - FAD into e.t.c. = 2 ATP  Step 3 - NAD+ into e.t.c. = 3 ATP Total ATP per turn of spiral = 5 ATP NET ATP from Fatty Acid Spiral = 35 - 1 = 34 ATP  One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain.  Example with Palmitic Acid = 16 carbons = 8 acetyl groups.  Number of turns of fatty acid spiral = 8-1 = 7 turns  ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP. 22
  23. 23. 23
  24. 24. Cycles of Beta Oxidation Beta-oxidation is the process by which fatty acids, in the form of Acyl-CoA molecules, are broken down in mitochondria and/or in peroxisomes to generate Acetyl-CoA, the entry molecule for the Citric Acid cycle 24
  25. 25. β- OXIDATION OF ODD CHAIN FATTY ACIDS  The propionyl residue from an odd-chain fatty acid is the only part of a fatty acid that is glucogenic. Acetyl CoA cannot be converted into pyruvate or Oxaloacetate in animals. Fatty acids with an odd number of carbon atoms are oxidized by the pathway of β-oxidation, producing acetyl-CoA, until a three-carbon (propionyl-CoA) residue remains. This compound is converted to Succinyl-CoA, a constituent of the citric acid cycle 25
  26. 26. BETA OXIDATION OF UNSATURATED FATTY ACIDS  In the oxidation of unsaturated fatty acids, most of the reactions are the same as those for saturated fatty acids, only two additional enzymes an isomerase and a reductase are needed to degrade a wide range of unsaturated fatty acids.  Energy yield is less by the oxidation of unsaturated fatty acids since they are less reduced.  Per double bonds 2 ATP are less formed, since the first step of dehydrogenation to introduce double bond is not required, as the double already exists. 26
  27. 27. BETA OXIDATION OF UNSATURATED FATTY ACIDS Palmitoleoyl Co A undergoes three cycles of degradation, which are carried out by the same enzymes as in the oxidation of saturated fatty acids.  The cis- Δ 3-enoyl CoA formed in the third round is not a substrate for acyl CoA dehydrogenase.  An isomerase converts this double bond into a trans- Δ 2 double bond.  The subsequent reactions are those of the saturated fatty acid oxidation pathway, in which the trans- Δ 2-enoyl CoA is a regular substrate . 27
  29. 29. A different set of enzymes is required for the oxidation of Linoleic acid, a C18 polyunsaturated fatty acid with cis-Δ 9 and cis-Δ12 double bonds. The cis- Δ 3 double bond formed after three rounds of β oxidation is converted into a trans- Δ 2 double bond by isomerase. The acyl CoA produced by another round of β oxidation contains a cis- Δ 4 double bond. Dehydrogenation of this species by acyl CoA dehydrogenase yields a 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the β -oxidation pathway. This impasse is circumvented by 2,4-dienoyl CoA reductase, an enzyme that uses NADPH to reduce the 2,4-dienoyl intermediate to trans-D 3-enoyl CoA. 29
  30. 30. MINOR PATHWAYS OF FATTY ACID OXIDATION 3) Peroxisomal fatty acid oxidation- Occurs for the chain shortening of very long chain fatty acids. 1) α- Oxidation- Oxidation occurs at C-2 instead of C-3 , as in β oxidation 2) ω- Oxidation – Oxidation occurs at the methyl end of the fatty acid molecule. 30
  31. 31. 31
  32. 32.  Unsaturated fatty acids: - mono, ∆9 (odd) - poly, ∆9, ∆12 (odd, even) -> isomerization, reduction  Odd chain length fatty acids: -> propionyl-CoA in the last cycle  Very long-chain fatty acids (> C22 atoms): -> first β-oxidation in peroxisomes  Branched chain fatty acids: - chlorophyll’s phytanic acid -> α-oxidation, formyl-CoA + propionyl-CoA Special cases of β-oxidation 32
  33. 33. BIOLOGICAL SIGNIFICANCE OF ALPHA OXIDATION 1) α- Oxidation is most suited for the oxidation of phytanic acid, produced from dietary phytol, a constituent of chlorophyll of plants. 2) The hydroxy fatty acids produced as intermediates of this pathway like Cerebronic acid can be used for the synthesis of cerebrosides and sulfatides. 3) Odd chain fatty acids produced upon decarboxylation in this pathway, can be used for the synthesis of sphingolipids and can also undergo beta oxidation to form propionyl co A and Acetyl co A. The number of acetyl co A depend upon the chain length. Propionyl co A is converted to Succinyl co A to gain entry in to TCA cycle for further oxidation.  Phytanic acid is a significant constituent of milk lipids and animal fats.  Normally it is metabolized by an initial α- hydroxylation followed by dehydrogenation and decarboxylation.  Beta oxidation can not occur initially because of the presence of 3- methyl groups, but it can proceed after decarboxylation.  The whole reaction produces three molecules of propionyl co A, three molecules of Acetyl co A, and one molecule of iso butyryl co A . 33
  34. 34. Phytanic acid is oxidized by Phytanic acid α oxidase (α- hydroxylase enzyme) to yield CO2 and odd chain fatty acid Pristanic acid that can be subsequently oxidized by beta oxidation. 34
  35. 35. OMEGA OXIDATION OF FATTY ACIDS  Involves hydroxylation and occurs in the endoplasmic reticulum of many tissues.  Hydroxylation takes place on the methyl carbon at the other end of the molecule from the carboxyl group or on the carbon next to the methyl end.  It uses the “mixed function oxidase” type of reaction requiring Cytochrome P450, O2 and NADPH, as well as the necessary enzymes.  Hydroxy fatty acids can be further oxidized to a dicarboxylic acid via sequential reactions of Alcohol dehydrogenase and aldehyde dehydrogenases.  The process occurs primarily with medium chain fatty acids. Dicarboxylic acids so formed can undergo beta oxidation to produce shorter chain dicarboxylic acids such as Adipic acids(C6) and succinic acid (C4). 35
  36. 36. PEROXISOMAL OXIDATION OF VERY LONG CHAIN FATTY ACIDS  In peroxisomes, a flavoprotein dehydrogenase transfers electrons to O2 to yield H2O2 instead of capturing the high-energy electrons as FADH2, as occurs in mitochondrial beta oxidation.  Catalase is needed to convert the hydrogen peroxide produced in the initial reaction into water and oxygen.  Subsequent steps are identical with their mitochondrial counterparts,  They are carried out by different isoform of the enzymes.  The specificity of the peroxisomal enzymes is for longer chain fatty acids. Thus peroxisomal enzymes function to shorten the chain length of relatively long chain fatty acids to a point at which beta oxidation can be completed in mitochondria. 36
  37. 37. 37
  38. 38. 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 Three stages of fatty acid synthesis: A. Transport of acetyl CoA into 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 B. Carboxylation of acetyl CoA. C. Assembly of fatty acid chain 38
  39. 39. Glucose Pyruvate Malate Oxaloacetate Citrate Mitochondrial matrix Pyruvate Acetyl CoA Citrate Oxaloacetate Amino acids FA Pyruvate carboxylase PDH Complex Citrate synthaseATP ADP+Pi CoASH Acetyl CoA Citrate lyase MDH NADH + H+ NAD+ NADPH + H+ NADP+ HMP Shunt CO2 Fatty acid Cytosol Transfer of acetyl CoA from mitochondria to cytosol Malic enzyme 39
  40. 40. B. Carboxylation of Acetyl CoA Enzyme: acetyl CoA carboxylase Prosthetic group - biotin  A carboxybiotin intermediate is formed.  ATP is hydrolyzed.  The CO2 group in carboxybiotin is transferred to acetyl CoA to form malonyl CoA.  Acetyl CoA carboxylase is the regulatory enzyme. Five separate stages: 1. Loading of precursors via thioester derivatives. 2. Condensation of the precursors. 3. Reduction. 4. Dehydration. 5. Reduction C. The Reactions of Fatty Acid Synthesis 40
  41. 41. 41
  42. 42. 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 42
  43. 43. Condensation reaction. Acetyl ACP and malonyl ACP react to form acetoacetyl ACP. Enzyme - acyl-malonyl ACP condensing enzyme. 43
  44. 44. Reduction. Acetoacetyl ACP is reduced to D-3- hydroxybutyryl ACP. NADPH is the reducing agent Enzyme: - ketoacyl ACP reductase 44
  45. 45. Dehydration. D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP Enzyme: 3-hydroxyacyl ACP dehydratase 45
  46. 46. 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).  In the second round butyryl ACP condenses with malonyl ACP to form a C6--ketoacyl ACP.  Reduction, dehydration, and a second reduction convert the C6--ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation. 46
  47. 47.  Rounds of synthesis continue until a C16 palmitoyl group is formed  Palmitoyl-ACP is hydrolyzed by a thioesterase. Final reaction of FA synthesis 47
  48. 48. 48
  49. 49. 1. Phosphorilation of glycerol through the action of glycerol kinase: 2. Reduction of dihydroxyacetone phosphate which is the product of the aldolase reaction of glycolysis. Dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the NAD-linked glycerol-3- phosphate dehydrogenase of the cytosol: ATP + glycerol  glycerol 3-phosphate + ADP Dihydroxyacetone phosphate + NADH + H+  glycerol 3-phosphate + NAD The ways of formation of active form of glycerol. There are two ways of formation of active form of glycerol. 49
  50. 50. Biosynthesis of Triacylglycerol The first stage in triacylglycerol formation is the acylation of the free hydroxyl groups of glycerol phosphate by two molecules of fatty acyl-CoA to yield first a lysophosphotidic acid and then a phosphatidic acid: H2C HC OH OH H2C O P R1 - COSKoA KoA - SH H2C HC O OH H2C O P C O R1 H2C HC O OH H2C O P R2 - COSKoA KoA - SH H2C HC O O H2C O P C O R1C O R1 C R2 O 50
  51. 51. 51
  52. 52. 52 ACP: acyl carrier protein ADP: adenosine diphosphate AMP: adenosine monophosphate cAMP: cyclic AMP ATP: adenosine triphosphate ATPase: adenosine triphosphatase CDP: cytidine diphosphate CMP: cytidine monophosphate CoA: coenzyme A CoQ: coenzyme Q (ubiquinone) CTP: cytidine triphosphate cAMP: adenosine 3′,5′-cyclic monophosphate cGMP: guanosine 3′,5′-cyclic monophosphate DNA: deoxyribonucleic acid cDNA: complementary DNA DNAse: deoxyribonuclease EcoRI: EcoRI restriction endonuclease FAD: flavin adenine dinucleotide (oxidized form) FADH2: flavin adenine dinucleotide (reduced form) fMet: formylmethionine FMN: flavin mononucleotide (oxidized form) FMNH2: flavin mononucleotide (reduced form) GDP: guanosine diphosphate GMP: guanosine monophosphate cGMP: cyclic GMP GSH: reduced glutathione GSSG: oxidized glutathione GTP: guanosine triphosphate Hb: hemoglobin HDL: high-density lipoprotein ITP: inosine triphosphate LDL: low-density lipoprotein NAD+: nicotinamide adenine dinucleotide (oxidized form) NADH: nicotinamide adenine dinucleotide (reduced form) NADP+: nicotinamide adenine dinucleotide phosphate (oxidized form) NADPH: nicotinamide adenine dinucleotide phosphate (reduced form) PFK: phosphofructokinase Pi: inorganic orthophosphate PLP: pyridoxal phosphate PPi: inorganic pyrophosphate PRPP: 5-phosphoribosyl-1-pyrophosphate Q: ubiquinone (or plastoquinone) QH2: ubiquinol (or plastoquinol) RNA: ribonucleic acid mRNA: messenger RNA rRNA: ribosomal RNA tRNA: transfer RNA RNAse: ribonuclease TPP: thiamine pyrophosphate TTP: thymidine triphosphate VLDL: very low density lipoprotein
  53. 53. Electronic References. Bibliographic References.  KOLEVA, L. (2017) – The Notes or Lectures of Subject of Plant Chemistry in Biochemistry: Biochemistry. Faculty of Agronomy. Plovdiv: Agricultural University of Plovdiv; Bulgaria.  MICHAEL, S. & GOLDSTEIN, J. (1996). Synthesis and Degradation of Lipids. Department Of Molecular Genetics. Texas: University Of Texas Health Science Center, & Southwestern Medical School: Dallas and Texas; United State Of America (U.S.A). [In line]. [Consultation on 08/12/2017]. Available at <URL: https://www.unifr.ch/biochem/assets/files/schneiter/cours/Voet_Pratt/Voet_chap_20_ new.pdf >  KUMAR, R (2016).Beta Oxidation of Fatty Acids. Department Of Health and Medicine. [In line]. [Consultation on 06/12/2017]. Available at <URL: https://www.slideshare.net/RajanKumar16/beta-oxidation-of-fatty-acids-6084897 >  ARA BEGUM, I (2014). Lipolysis and Fatty Acid Oxidation. Department Of Chemistry. Dhaka: Medical College of Dakha, India. [In line]. [Consultation on 08/12/2017]. Available at <URL: https://www.slideshare.net/enamifat/lipolysis-fatty-acid-oxidation >  ALVIOALA, G (2016). Lipid Metabolism. Chemical Physiology: Department of Biology, Davao; Philippine. [In line]. [Consultation on 06/12/2017]. Available at <URL: https://www.slideshare.net/RajanKumar16/beta-oxidation-of-fatty-acids-6084897 > 53