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Lipid Metabolism II

Lipid Metabolism II. Andy Howard Biochemistry Lectures, Fall 2010 10 November 2010. Lipid anabolism & catabolism. We’ll discuss the anabolic pathways associated with lipids Then we’ll turn it around and talk about lipid catabolism. Lipid Anabolism Isoprenoid and steroid synthesis

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Lipid Metabolism II

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  1. Lipid Metabolism II Andy HowardBiochemistry Lectures, Fall 201010 November 2010 Lipid Anabolism & Catabolism

  2. Lipid anabolism & catabolism • We’ll discuss the anabolic pathways associated with lipids • Then we’ll turn it around and talk about lipid catabolism Lipid Anabolism & Catabolism

  3. Lipid Anabolism Isoprenoid and steroid synthesis Medical aspects Fatty acid oxidation Steps and enzymes Energetics Special cases Control Other topics in lipid catabolism Triacylglycerol breakdown Lipid transport Phospholipid breakdown Lipoproteins Serum albumin Ketone bodies What we’ll discuss Lipid Anabolism & Catabolism

  4. Steroid synthesis: overview • Cholesterol is important on its own & as a precursor of steroid hormones, bile salts • Derived formally from isoprene • Isoprenoid synthesis based on mevalonate &isopentenyldiphosphate Lipid Anabolism & Catabolism

  5. Making HMG-CoA • Condense 3 molecules of acetyl CoA: • 2 acetyl CoA  acetoacetyl CoA + CoASH;catalyzed by acetoacetyl CoA synthase • Acetoacetyl CoA + acetyl CoA + H2O 3-hydroxy-3-methylglutaryl CoA + CoASH + H+catalyzed by HMG CoA synthase • These are important intermediates: precursor to steroids and ketone bodies • Not the committed step toward isoprenoids because we can also make ketone bodies from HMG-CoA Lipid Anabolism & Catabolism

  6. HMG CoA synthase • Acetyl CoA + acetoacetyl CoA  HMG CoA + CoASH • Cytosolic and mitochondrial forms are different • Cytosolic forms are primarily used for isoprenoid synthesis • Mitochondrial form is primarily used in making ketone bodies Human HMG CoA synthase IPDB 2P8U108 kDa dimer EC 2.3.3.10, 2Å Lipid Anabolism & Catabolism

  7. iClicker quiz question 1 • Creation of new C-C bonds requires energy. Where is it coming from in these condensations? • (a) hydrolysis of thioester bonds • (b) enzymatic catalysis • (c) hydrolysis of thioether bonds • (d) hydrolysis of ATP • (e) none of the above Lipid Anabolism & Catabolism

  8. Answer: (a) • (b) no. Enzymatic catalysis doesn’t change thermodynamics: it changes kinetics • (c) no. These acyl CoA molecules contain thioester linkages,not thioether linkages • (d) no. There’s no ATP explicitly involved. • (a) yes. Hydrolysis of thioester linkages yields substantial amounts of free energy Lipid Anabolism & Catabolism

  9. PDB 1DQA205 kDa tetramerhumanEC 1.1.1.342Å PDB 1DQA205 kDa tetramer Human HMGCoA tomevalonate • HMGCoA reductase is the first committed step on pathway toward isoprenoids • HMGCoA + 2NADPH + 2H+ mevalonate + 2NADP+ + CoASH • Many drug-discovery projects involve inhibition of this enzyme Lipid Anabolism & Catabolism

  10. Atorvastatin (lipitor) • Most prominent statin,i.e. inhibitor of HMGCoA reductase • Currently the biggest-selling prescription drug still on its original patent • Other classes of statins exist • Competitive inhibitor of enzyme:Km = 6 µM (low!); but KI = 5nM Lipid Anabolism & Catabolism

  11. Mevalonate to isopentenyl diphosphate • Two successive ATP-dependent kinase steps convert mevalonate to mevalonate 5-diphosphate • ATP-dependent decarboxylation yields isopentenyl diphosphate • This is an isoprene-donating group involved in making non-steroidal isoprenoid compounds as well as steroids Lipid Anabolism & Catabolism

  12. Mevalonate kinase • Converts mevalonate tomevalonate 5-phosphate • Secondary control point inisoprenoid synthetic pathway • Human diseases associated with abnormalities • Mevalonic aciduria • Hyperimmunoglobulinemia (Periodic fever syndrome) PDB 2HFS 73 kDa dimer;monomer shownLeishmania majorEC 2.7.1.36 1.75Å Lipid Anabolism & Catabolism

  13. Isopentenyl diphosphate to squalene • Isomerized to dimethylallyl diphosphate • That condenses with another molecule of IPDP to make geranyl diphosphate (C10) • Another condensation with IPDP (with the same enzyme) makes farnesyl diphosphate (C15) • Two farnesyl diP fuse head-to-head to make squalene (C30, no heteroatoms) Lipid Anabolism & Catabolism

  14. Geranyl & farnesyl diphosphate Geranyl diphosphate:C10 Farnesyl diphosphate:C15 Lipid Anabolism & Catabolism

  15. Squalene • Made via head-to-head synthesis from 2 molecules of farnesyl diphosphate Squalene: C30 Lipid Anabolism & Catabolism

  16. Squalene to cholesterol lanosterol • Several messy steps move the double bonds around • replace double bonds with ring closures  lanosterol • Eliminate 3 methyls, move one double bond, remove another double bond, and voila: cholesterol lanosterol synthase; converts 2,3-oxidosqualene to lanosterolPDB 1W6K81 kDa monomerhumanEC 5.4.99.7, 2.1Å Lipid Anabolism & Catabolism

  17. What happens to cholesterol? • Inserted into membranes • Assembled into lipoproteins • Derivatized to make bile salts • Modified into hormones Lipid Anabolism & Catabolism

  18. Other isoprenoids • Generally made from isopentenyl pyrophosphate • Pathways to isopentenyl pyroP are ancient: used in bacteria • Pathways to steroids comparatively recent • Cholesterol essential in animal membranes; plants have other sterols like campesterol (24-methyl-cholesterol) Lipid Anabolism & Catabolism

  19. Fatty acid oxidation • Degradation proceeds 2 C at a time • somewhat like synthesis • Called -oxidation because in each round the form that gets shortened is a -ketoacyl CoA • Activated form is acyl CoA, not acyl ACP • Product is n molecules of acetyl CoA from a 2n-carbon fatty acid • Yields n-1 NADH and n-1 QH2 • Occurs in the mitochondrion or peroxisome, whereas synthesis occurs in the cytosol Lipid Anabolism & Catabolism

  20. Reactions in -oxidation • Proceeds through cycle n times for a 2n-carbon fatty acid Diagram courtesy Richard Paselk, Humboldt State U. Lipid Anabolism & Catabolism

  21. Acyl-CoA dehydrogenase • Converts fatty acid saturated at C2,3 to trans-2-Enoyl CoA:—CH2—CH2—COSCoA • Several isozymes for various sizes of FAs • FAD-dependent enzyme PDB 2UXWHuman very long chain isozymeEC 1.3.99.—, 1.45Å135kDa dimermonomer shown Lipid Anabolism & Catabolism

  22. Electron-Transferring Flavoprotein (ETF) • Here, it converts FADH2 created by acyl-CoA dehydrogenase back to FAD via Fe-S protein • Plays role in other redox reactions • Ultimate acceptor is Q, which can be re-oxidized in the ETS Human ETFPDB 1EFV62 kDaheterodimer, 2.1Å Lipid Anabolism & Catabolism

  23. Hydration step • Enzyme is 2-enoyl CoA dehydratase • Converts enoyl CoA to L-3-hydroxyacyl CoA • Remember this is the opposite stereochemistry relative to synthetic intermediate PDB 1PN2 Candida dehydratase domainEC 4.2.1.—, 1.95Å127 kDa tetramer Lipid Anabolism & Catabolism

  24. Second oxidative step • Enzyme is L-3-hydroxyacyl-CoA dehydrogenase • NADH is reduced product • NADH can be used in biosynthesis (via shuttles) or oxidized in the ETS PDB 1E6Wdehydrogenase domain, rat brain111 kDa tetramerEC 1.1.1.35, 1.7Å Lipid Anabolism & Catabolism

  25. Thiolysis • HS-CoA attacks C3-carbonyl and cleaves off acetyl CoA, resulting in shortening by two carbons • Enzyme is 3-ketoacyl-CoA thiolase • Similar to acetoacyl-CoA thiolase found in isopentenyl diP pathway • Substrate can go through another round PDB 3GOASalmonellaEC 2.3.1.16, 1.7Å84 kDa homodimer Lipid Anabolism & Catabolism

  26. Formal similarity • … between FA oxidation steps 1-3 and middle reactions of TCA cycle: • –CH2CH2– oxidized to trans-CH=CH—:like succinate to fumarate • Trans-ene hydrated to L-CHOH-CH2—:like fumarate to L-malate • Alcohol oxidized to ketone:like L-malate to oxalacetate Lipid Anabolism & Catabolism

  27. Peroxisomal -oxidation • Very common • in many non-mammalianeukaryotes it’s the only kind • In mammals this handles odd cases;mitochondria are the primary oxidizers • Initial reaction doesn’t produce QH2:it produces hydrogen peroxide as the other product besides trans-D2-enoyl CoA • Reaction catalyzed by acyl-CoA oxidase rat liveracyl CoA oxidaseEC 1.3.3.6PDB 1IS2, 2.2Å151kDa dimer Lipid Anabolism & Catabolism

  28. Perixosomes • Peroxisomes don’t have ETS so the reducing equivalents used in other ways • Compartmentation keeps H2O2 away from ETS Lipid Anabolism & Catabolism

  29. Similarities and differences • … between synthesis and oxidation of fatty acids • Acetyl CoA  Malonyl ACP  fatty acid • Fatty acid  FA CoA  acetyl CoA • Text gives clear exposition: read it! • This could easily result in a final-exam question! Lipid Anabolism & Catabolism

  30. Moving acyl CoA into the mitochondrial matrix • Carnitine shuttle moves them: • Fatty acyl CoA gets into the intermembrane space • Carnitine acyltransferase II acylates carnitine • Acylcarnitine crosses inner membrane • Similar enzyme inside gets acyl CoA back Carnitine:3-hydroxy-4-trimethyl-ammonio-butanoate Lipid Anabolism & Catabolism

  31. Bookkeeping • Remember:mitochondrial or bacterial FA oxidation yields one NADH and one QH2 per pair of carbons, e.g. • Stearoyl CoA + 8HS-CoA + 8Q + 8NAD+  9 acetyl CoA + 8QH2 + 8NADH +8H+ • That adds up to 12+20+90-2 = 120 ATP! Lipid Anabolism & Catabolism

  32. Comparisons with glucose • Glucose is 6 carbons, not 18, so the 120 ATPs vs. 32 comes out more like 120 vs.96 on a per-carbon basis… • But per gram:1 g * 32 mol ATP (mol glucose) / 180 g/mol = 32/180 = 0.178 moles ATP per gram glucose • 1 g * 120 mol ATP /(mol stearate) / 288 g/mol) = 120/288 = 0.417 moles ATP per gram stearate • Hydration matters too! Lipid Anabolism & Catabolism

  33. Efficiency of fatty acyl synthesis • 8 acetyl CoA -> 8 malonyl ACP = 8 ATP • 8 syntheses * 5 ATP/synthesis = 40 • 9 Acetyl CoA * 17 ATP/AcCoA = 153 • Total: 201 ATP • So efficiency = 120/201 = 0.597 • That’s a typical efficiency defined as(energy derived from oxidation) /(energy required for synthesis) Lipid Anabolism & Catabolism

  34. Methylmalonyl CoA Odd-chain fatty acids • Rarer than even-chain but they do exist • Broken down as with even-chains but with propionyl CoA as end-product • Condenses with bicarbonate to form D-methylmalonyl CoA • Racemized to L-methylmalonyl CoA • Mutated to succinoyl CoA via an adenosylcobalamin-dependent reaction • This can actually be a source of sugars! Lipid Anabolism & Catabolism

  35. Catabolism of cis-unsaturated fatty acids • Normal beta-oxidation until we encounter a double bond • Double bond moves from cis-3,4 to trans-2,3 via 3,2-enoyl-CoA isomerase reaction • Further beta oxidation proceeds until we encounter the next double bond; • Cis double bonds at even positions get modified by 2,4-dienoyl-CoA reductase from trans,cis-2,4 to trans-3 • 3,2-enoyl-CoA isomerase moves trans-3 to trans-2 and then we can -oxidize again Lipid Anabolism & Catabolism

  36. Regulation I • Key hormones:insulin, glucagon, ephinephrine • Under low-glucose conditions: • glucagon and epinephrine circulate at high concentrations • -oxidation encouraged • Glucose not needed for fuel so it’s conserved • High glucose conditions: • insulin, glucagon & epinephrine , • FA synthesis dominates • Glucose used as fuel for making fatty acids Lipid Anabolism & Catabolism

  37. Regulation II • Main regulatory enzyme:acetyl-CoA carboxylase • High insulin levels after meal stimulates formation of malonyl CoA • Product allosterically inhibits carnitine acyltransferase so FAs stay in cytosol Carnitine palmitoyl -transferasePDB 2RCUEC 2.3.1.21 1.8Å148 kDa dimer Monomer shown Lipid Anabolism & Catabolism

  38. Mobilization of fatty acids • Triacylglycerols transported through circulatory system in lipoprotein masses (cholesterol + various MW proteins forming shell around lipid) • Lipoproteins hydrolyzed via lipoprotein lipase extracellularly • Fatty acids & glycerol released extracellularly, FAs re-esterified Lipid Anabolism & Catabolism

  39. Fates of triacylglycerols • What happens next depends on needs: • Triacylglycerols hydrolyzed to FAs and monoacylglycerols, and sometimes further • High [insulin] inhibits hydrolysis Lipid Anabolism & Catabolism

  40. GlucagonPDB 1GCN, 3Å3.3kDa monomer Glycerol and free fatty acids • Some of them diffuse through the adipocyte plasma membrane & enter blood • Glycerol metabolized in liver to (…) glucose • FAs travel bound to serum albumin to heart, skeletal muscle, & liver—energy source, esp. in fasting • Glucagon  means inhibition of acetyl CoA carboxylase, so less malonyl CoA made • Meanwhile: high [acetyl CoA],[NADH] means inhibition of pyruvate dehydrogenase Lipid Anabolism & Catabolism

  41. Absorption of lipids from food • Majority of dietary lipids are triacylglyerols;Smaller amounts of phospholipids & cholesterol • Suspended fat particles are coated with bile salts, amphipathic cholesterol derivatives From Y.Shi & P.Burn (2004) Nature Rev. Drug Discov.3: 695. Lipid Anabolism & Catabolism

  42. Lipase and colipase • Pancreatic lipase secreted into small intestine degrades triacylglycerols (in fat particles) at C-1&3 • Colipase: 10.4 kDa protein that helps bind the lipase to its substrates Lipase-colipase complexPDB 1LPB10.4 kDa (colipase)+ 50kDa (lipase)heterodimerEC 3.1.1.3, 2.46Å Pig / human Lipid Anabolism & Catabolism

  43. What bile salts do Taurocholate, a bile salt • Bile-salt micelles travel to intestinal wall • Monoacylglycerols & free FAs are absorbed and bile salts are released • Bile salts recirculate rapidly • When fully formed triglycerides are made, those travel via chylomicrons for transport to other tissues Lipid Anabolism & Catabolism

  44. Dietary phospholipids • Phospholipase A2 in intestine hydrolyzes ester bond at C2; the resulting lysophosphoglycerides get re-esterified in the intestine • High [lysophosphoglyceride] disrupts membranes: that’s how snake venoms work on erythrocytes PDB 1G4I13.5 kDa monomerBovine pancreasEC 3.1.1.40.97Å Lipid Anabolism & Catabolism

  45. Dietary cholesterol • Cholesterol esters are hydrolyzed in the lumen of the intestine • Free cholesterol is solubilized by bile-salts for absorption • Free cholesterol often esterified in the intestine to form cholesteryl esters Cholesterol esterasePDB 2BCE64 kDa monomerBovineEC 3.1.1.16 1.6Å Lipid Anabolism & Catabolism

  46. Core Lipoproteins • Spherical vehicles fortransport of fats • Several sizes • Biggest, least dense:chylomicrons • Others are smaller,more dense Cartoon courtesyU. WisconsinStevens Point Lipid Anabolism & Catabolism

  47. Chylomicrons • Largest, least dense oflipoproteins • found in bloodonly after a meal • Deliver triacylglycerol &cholesterol to muscle and adipose tissue • Remaining cholesterol-rich particles deliver cholesterol to liver • Contains Apolipoprotein E -binds to specific receptor in liver cells Lipid Anabolism & Catabolism

  48. Types of lipoproteins(cf. table 16.1 & fig. 16.30) Type Chylo- VLDLs IDLs LDLs HDLs microns MW*10-6 >400 10-80 5-10 2.3 .18-.36 , g cm-3 <0.95 <1.006 <1.019 <1.063 <1.21 Composition (%) Protein 2 10 18 25 33 Triacylglycerol 85 50 31 10 8 Cholesterol 4 22 29 45 30 Phospholipid 9 18 22 20 29 Lipid Anabolism & Catabolism

  49. % protein and density Lipid Anabolism & Catabolism

  50. Protein components • Structural amphipathic crust proteins: • ApoB-100 (513 kDa) bound to outer layer of VLDLs, IDLs, LDLs. • ApoB-48 (241 kDa): N-terminal end of ApoB-100, found in chylomicrons • Smaller, less strongly bound proteins • Some are responsible for specific binding to receptors in cells Kringle domain of ApoA1 PDB 3KIV 8.7 kDa monomer Human 1.8Å Lipid Anabolism & Catabolism

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