1. Lipid Metabolism

3. http://www.expasy.org/cgi_bin/ search_biochem_index/ http://www.tcd.ie/Biochemistry /IUBMB_Nicholson/ http://www.genome.ad.jp/kegg /metabolism.html/

4. Metabolic Functions of Eukaryotic Organelles

5. Lipid Digestion, Absorption, and Transport triacylglycerols (fats) constitute 90% of dietary lipid triacylglycerols (fats) constitute 90% of dietary lipid major form of metabolic energy storage in animals major form of metabolic energy storage in animals ~6x energy yield vs CHO and protein since it is nonpolar and stored in anhydrous state ~6x energy yield vs CHO and protein since it is nonpolar and stored in anhydrous state

6. Lipid Digestion occurs at lipid-water interfaces occurs at lipid-water interfaces enhanced by the emulsifying action of bile salts (bile acids) enhanced by the emulsifying action of bile salts (bile acids) pancreatic lipase requires activation pancreatic lipase requires activation triacylglycerol lipase triacylglycerol lipase hydrolysis of TAGs at 1 and 3 positions hydrolysis of TAGs at 1 and 3 positions complex with colipase (1:1) complex with colipase (1:1) mixed micelles of phosphatidylcholine mixed micelles of phosphatidylcholine bile salts bile salts 1LPA.pdb

7. Major Bile Acids and their Conjugates amphipathic detergent-like molecules that solubilize fat globules amphipathic detergent-like molecules that solubilize fat globules cholesterol derivatives cholesterol derivatives synthesized in the liver and secreted as glycine or taurine conjugates synthesized in the liver and secreted as glycine or taurine conjugates exported into gallbladder for storage exported into gallbladder for storage secreted into small intestine, where lipid digestion and absorption occur secreted into small intestine, where lipid digestion and absorption occur

8. Substrate Binding to Phospholipase A 2

9. Bile Acids and Fatty Acid-binding Protein Facilitate Absorption of Lipids Rat Intestinal Fatty Acid-Binding Protein (2IFB.pdb) Rat Intestinal Fatty Acid-Binding Protein (2IFB.pdb) a cytoplasmic protein that increases the “solubility” of lipids and protects the cell from their detergent-like effects a cytoplasmic protein that increases the “solubility” of lipids and protects the cell from their detergent-like effects 131-residue protein with 10 antiparallel β sheets 131-residue protein with 10 antiparallel β sheets palmitate (yellow) occupies a gap between two β strands palmitate (yellow) occupies a gap between two β strands palmitate’s carboxyl group interacts with Arg 106, Gln 115, and two bound H 2 O molecules while its tail is encased by aromatic side chains palmitate’s carboxyl group interacts with Arg 106, Gln 115, and two bound H 2 O molecules while its tail is encased by aromatic side chains 2IFB.pdb

10. Lipoproteins lipoproteins are globular micelle-like particles lipoproteins are globular micelle-like particles nonpolar core of TAGs and cholesteryl esters nonpolar core of TAGs and cholesteryl esters surrounded by amphiphilic coating of protein, phospholipid, and cholesterol surrounded by amphiphilic coating of protein, phospholipid, and cholesterol intestinal mucosal cells convert FA to TAGs and package them with cholesterols, into lipoproteins called chylomicrons intestinal mucosal cells convert FA to TAGs and package them with cholesterols, into lipoproteins called chylomicrons LDL (Low Density Lipoprotein) has 1500 cholesteryl ester surrounded by ~800 phospholipid, ~500 cholesterol molecules and 1 apolipoprotein B-100

12. Characteristics of the Major Classes of Lipoproteins in Human Plasma VLDL, IDL, and LDL are synthesized by the liver to transport endogenous TAGs and cholesterol from liver to other tissues VLDL, IDL, and LDL are synthesized by the liver to transport endogenous TAGs and cholesterol from liver to other tissues HDL transport cholesterol and other lipids from tissues back to the liver HDL transport cholesterol and other lipids from tissues back to the liver

13. Apolipoproteins protein components of lipoprotein (apoproteins) protein components of lipoprotein (apoproteins) apoproteins coat lipoprotein surfaces apoproteins coat lipoprotein surfaces Apolipoprotein A-I (apoA-I), in chylomicrons and HDL Apolipoprotein A-I (apoA-I), in chylomicrons and HDL apoA-I is a 29-kD polypeptide with a twisted elliptical shape (1AV1.pdb) apoA-I is a 29-kD polypeptide with a twisted elliptical shape (1AV1.pdb) pseudocontinuous  -helix that is punctuated by a kink at Pro residues pseudocontinuous  -helix that is punctuated by a kink at Pro residues 1AV1.pdb

14. Transport of Plasma TAGs and Cholesterol

16. Receptor-Mediated Endocytosis of LDL

17. Fatty Acid Oxidation

18. Fatty Acid Activation FA must be “primed” before it can be oxidized FA must be “primed” before it can be oxidized ATP-dependent acylation reaction to form fatty acyl-CoA ATP-dependent acylation reaction to form fatty acyl-CoA activation is catalyzed by acyl-CoA synthetases (thiokinanases) activation is catalyzed by acyl-CoA synthetases (thiokinanases) Fatty acid + CoA + ATP  acyl-CoA + AMP + PP i Fatty acid + CoA + ATP  acyl-CoA + AMP + PP i

20. Transport across Mitochondrial Membrane FAs are activated for oxidation in the cytosol but they are oxidized in the mitochondrion FAs are activated for oxidation in the cytosol but they are oxidized in the mitochondrion Long-chain fatty acyl-CoA cannot directly cross the inner membrane of mitochondrion Long-chain fatty acyl-CoA cannot directly cross the inner membrane of mitochondrion Acyl group is first transferred to carnitine Acyl group is first transferred to carnitine

21. Translocation process is mediated by a specific carrier protein Translocation process is mediated by a specific carrier protein (1) the acyl group of a cytosolic acyl-CoA is transferred to carnitine, thereby releasing the CoA to its cytosolic pool (2) the resulting acyl-carnitine is transported into the mitochondrial matrix by the carrier protein (3) the acyl group is transferred to a CoA molecule from the mitochondrial pool (4) the product carnitine is returned to the cytosol Transport across Mitochondrial Membrane

23. β Oxidation

24. Acyl-CoA Dehydrogenase mitochondria contain 4 acyl-CoA dehydrogenases mitochondria contain 4 acyl-CoA dehydrogenases FADH 2 resulting from the oxidation of the fatty acyl-CoA substrate is reoxidized by the mitochondrial ETC FADH 2 resulting from the oxidation of the fatty acyl-CoA substrate is reoxidized by the mitochondrial ETC ribbon diagram of the active site of the enzyme (MCAD) with flavin ring (green), octanoyl-CoA substate (blue-white) ribbon diagram of the active site of the enzyme (MCAD) with flavin ring (green), octanoyl-CoA substate (blue-white) the octanoyl-CoA binds such that its C  -C β bond is sandwhiched between the carboxylate group of Glu 376 (red) the octanoyl-CoA binds such that its C  -C β bond is sandwhiched between the carboxylate group of Glu 376 (red) 3MDE.pdb

25. Enoyl-CoA Hydratase Adds water across the double bond at least three forms of the enzyme are known at least three forms of the enzyme are known aka crotonases aka crotonases Normal reaction converts trans-enoyl-CoA to L-  -hydroxyacyl-CoA Normal reaction converts trans-enoyl-CoA to L-  -hydroxyacyl-CoA

26. Hydroxyacyl-CoA Dehydrogenase Oxidizes the  -Hydroxyl Group This enzyme is completely specific for L-hydroxyacyl-CoA This enzyme is completely specific for L-hydroxyacyl-CoA D-hydroxylacyl-isomers are handled differently D-hydroxylacyl-isomers are handled differently

27. Mechanism of β-Ketoacyl-CoA Thiolase Thiolase reaction occurs via Claisen ester cleavage Thiolase reaction occurs via Claisen ester cleavage (1) an active site thiol group adds to the β- keto group of the substrate acyl-CoA (2) C-C bond cleavage forms an acetyl- CoA carbanion intermediate that is stabilized by e - withdrawal into this thioester’s carbonyl group (Claisen ester cleavage) (3) an enzyme acidic group protonoates the acetyl-CoA carbanion, yielding acetyl CoA (4) & (5) CoA displaces the enzyme thiol group from the enzyme-thioester intermediate, yielding an acyl-CoA that is shortened by two C atoms

29. FA Oxidation is Highly Exergonic each round of β oxidation produces: each round of β oxidation produces: 1 NADH (3 ATP) 1 NADH (3 ATP) 1 FADH 2 (2 ATP) 1 FADH 2 (2 ATP) 1 acetyl-CoA (12 ATP) 1 acetyl-CoA (12 ATP) oxidation of acetyl-CoA via CAC generates additional : oxidation of acetyl-CoA via CAC generates additional : 1GTP (1ATP) + 3 NADH (9 ATP) + 1 FADH 2 (2 ATP) 1GTP (1ATP) + 3 NADH (9 ATP) + 1 FADH 2 (2 ATP) e.g. complete oxidation of palmitoyl-CoA (C16 fatty acyl group) involves 7 rounds e.g. complete oxidation of palmitoyl-CoA (C16 fatty acyl group) involves 7 rounds 7 NADH + 7 FADH 2 + 8 acetyl-CoA (8 GTP + 24 NADH + 8 FADH 2 ) 7 NADH + 7 FADH 2 + 8 acetyl-CoA (8 GTP + 24 NADH + 8 FADH 2 ) 31 NADH (93 ATP) + 15 FADH 2 (30 ATP) + 8 ATP 31 NADH (93 ATP) + 15 FADH 2 (30 ATP) + 8 ATP Net yield: 131 ATP – 2 ATP (fatty acyl-CoA formation) = 129 ATP Net yield: 131 ATP – 2 ATP (fatty acyl-CoA formation) = 129 ATP

30. Oxidation of Unsaturated FAs almost all unsat FAs of biological origin contain only cis double bond between C9-C10 (  9 ) almost all unsat FAs of biological origin contain only cis double bond between C9-C10 (  9 ) additional double bonds occur at 3-C intervals additional double bonds occur at 3-C intervals

31. Oxidation of Unsaturated FAs Problem 1: A  Double bond Problem 1: A  Double bond Solution: enoyl-CoA isomerase Solution: enoyl-CoA isomerase Problem 2: A  4 Double Bond Inhibits Hydratase Action Problem 2: A  4 Double Bond Inhibits Hydratase Action Solution: 2,4-dienoyl-CoA reductase Solution: 2,4-dienoyl-CoA reductase Problem 3: Unanticipated Isomerization of 2,5-enoyl- CoA by 3,2-enoyl-CoA Isomerase Problem 3: Unanticipated Isomerization of 2,5-enoyl- CoA by 3,2-enoyl-CoA Isomerase 3,5-2,4-dienoyl-CoA isomerase 3,5-2,4-dienoyl-CoA isomerase

34. Oxidation of Odd-Chain FAs some plants and marine organisms synthesize fatty acids with an odd number of carbon atoms some plants and marine organisms synthesize fatty acids with an odd number of carbon atoms final round of  oxidation forms propionyl-CoA final round of  oxidation forms propionyl-CoA propionyl-CoA is converted to succinyl-CoA for entry into the CAC propionyl-CoA is converted to succinyl-CoA for entry into the CAC

35. Succinyl-CoA cannot be directly consumed by the CAC Succinyl-CoA is converted to malate via CAC Succinyl-CoA is converted to malate via CAC at high [malate], it transported to the cytosol (Recall: Malate- Aspartate Shuttle) at high [malate], it transported to the cytosol (Recall: Malate- Aspartate Shuttle) where it is oxidatively decarboxylated to pyruvate and CO 2 by malate dehydrogenase where it is oxidatively decarboxylated to pyruvate and CO 2 by malate dehydrogenase Pyruvate is then completely oxidized via pyruvate dehydrogenase and the CAC Pyruvate is then completely oxidized via pyruvate dehydrogenase and the CAC

36. Peroxisomal  Oxidation In animals,  oxidation of FAs occurs both in peroxisome and mitochondrion In animals,  oxidation of FAs occurs both in peroxisome and mitochondrion Peroxisomal  oxidation shortens very long chain FAs (> 22 C atoms) in order to facilitate mitochondrial  oxidation Peroxisomal  oxidation shortens very long chain FAs (> 22 C atoms) in order to facilitate mitochondrial  oxidation In yeast and plants, FA oxidation occurs exclusively in the peroxisomes and glyoxysomes In yeast and plants, FA oxidation occurs exclusively in the peroxisomes and glyoxysomes

37. Ketone Bodies

38. Acetyl-CoA produced by oxidation of FAs in mitochondria can be converted to acetoacetone or D-  -Hydroxybutyrate Acetyl-CoA produced by oxidation of FAs in mitochondria can be converted to acetoacetone or D-  -Hydroxybutyrate important metabolic fuels for heart and skeletal muscle important metabolic fuels for heart and skeletal muscle brain uses only glucose as its energy source but during starvation, ketone bodies become the major metabolic fuel brain uses only glucose as its energy source but during starvation, ketone bodies become the major metabolic fuel ketone bodies are water- soluble equivalents of fatty acids ketone bodies are water- soluble equivalents of fatty acids

39. Ketogenesis (1) 2 Acetyl-CoAs condense to form acetoacetyl-CoA in a thiolase-catalyzed reaction (2) a Claisen ester condensation of the acetoacetyl-CoA with a third acetyl-CoA to form HMG- CoA as catalyzed by HMG- CoA synthase (3) degradation of HMG-CoA to acetoacetate and acetyl- CoA in a mixed aldol- Claisen ester cleavage catalyzed by HMG-CoA lyase

40. Metabolic Conversion of Ketone Bodies to Acetyl-CoA liver releases acetoacetate and  - hydroxybutyrate, which are carried by the bloodstream to peripheral tissues for use as alternative fuel liver releases acetoacetate and  - hydroxybutyrate, which are carried by the bloodstream to peripheral tissues for use as alternative fuel reduction of acetoacetate to D-  - hydroxybutyrate by  -hydroxybutyrate dehydrogenase reduction of acetoacetate to D-  - hydroxybutyrate by  -hydroxybutyrate dehydrogenase a stereoisomer of L-  -hydroxyacyl- CoA that occurs in the  -oxidation pathway a stereoisomer of L-  -hydroxyacyl- CoA that occurs in the  -oxidation pathway acetoacetate undergoes nonenzymatic convertion to acetone + CO 2 acetoacetate undergoes nonenzymatic convertion to acetone + CO 2 ketosis or ketoacidosis, individuals with sweet smell (acetone) produces acetoacetate faster than it can metabolize ketosis or ketoacidosis, individuals with sweet smell (acetone) produces acetoacetate faster than it can metabolize

41. Ketone Bodies and Diabetes "Starvation of cells in the midst of plenty" Glucose is abundant in blood, but uptake by cells in muscle, liver, and adipose cells is low Glucose is abundant in blood, but uptake by cells in muscle, liver, and adipose cells is low Cells, metabolically starved, turn to gluconeogenesis and fat/protein catabolism Cells, metabolically starved, turn to gluconeogenesis and fat/protein catabolism In type I diabetics, OAA is low, due to excess gluconeogenesis, so Ac-CoA from fat/protein catabolism does not go to TCA, but rather to ketone body production In type I diabetics, OAA is low, due to excess gluconeogenesis, so Ac-CoA from fat/protein catabolism does not go to TCA, but rather to ketone body production Acetone can be detected on breath of type I diabetics Acetone can be detected on breath of type I diabetics

42. Fatty Acid Biosynthesis

43. A Comparison of Fatty Acid  Oxidation and Fatty Acid Biosynthesis

44. The Differences Between fatty acid biosynthesis and breakdown Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as compared to -SH groups of CoA Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as compared to -SH groups of CoA Synthesis in cytosol; breakdown in mitochondria Synthesis in cytosol; breakdown in mitochondria Enzymes of synthesis are one polypeptide Enzymes of synthesis are one polypeptide Biosynthesis uses NADPH/NADP + ; breakdown uses NADH/NAD + Biosynthesis uses NADPH/NADP + ; breakdown uses NADH/NAD +

45. Transfer of Acetyl-CoA from Mitochondrion to Cytosol via Tricarboxylate Transport System Acetyl-CoA is generated in the mitochondrion Acetyl-CoA is generated in the mitochondrion when demand for ATP is low, minimal oxidation of Acetyl-CoA via CAC and oxidative phosphorylation, Acetyl-CoA is stored as fat when demand for ATP is low, minimal oxidation of Acetyl-CoA via CAC and oxidative phosphorylation, Acetyl-CoA is stored as fat but fatty acid synthesis occurs in cytosol and inner membrane is impermeable to acetyl-CoA but fatty acid synthesis occurs in cytosol and inner membrane is impermeable to acetyl-CoA acetyl-CoA enters the cytosol in the form of citrate via the tricarboxylate system acetyl-CoA enters the cytosol in the form of citrate via the tricarboxylate system

46. Activation by Malonyl-CoA Acetate Units are Activated for Transfer in Fatty Acid Synthesis by Malonyl-CoA Fatty acids are built from 2-C units - acetyl-CoA Fatty acids are built from 2-C units - acetyl-CoA Acetate units are activated for transfer by conversion to malonyl-CoA Acetate units are activated for transfer by conversion to malonyl-CoA Decarboxylation of malonyl-CoA and reducing power of NADPH drive chain growth Decarboxylation of malonyl-CoA and reducing power of NADPH drive chain growth Chain grows to 16-carbons Chain grows to 16-carbons Other enzymes add double bonds and more Cs Other enzymes add double bonds and more Cs

47. Acetyl-CoA Carboxylase Catalyzes the first committed step of FA biosynthesis (also a rate-controlling step) Catalyzes the first committed step of FA biosynthesis (also a rate-controlling step) Reaction mechanism is similar propionyl-CoA carboxylase and pyruvate carboxylase Reaction mechanism is similar propionyl-CoA carboxylase and pyruvate carboxylase 2 Steps: 2 Steps: a CO 2 activation a CO 2 activation a carboxylation a carboxylation

48. Phosphopantetheine Group in Acyl-Carrier Protein and in CoA ACP, like CoA, forms thioesters with acyl groups ACP, like CoA, forms thioesters with acyl groups Ser (OH) of ACP is esterified to the phosphopantetheine group, whereas in CoA it is esterified to AMP Ser (OH) of ACP is esterified to the phosphopantetheine group, whereas in CoA it is esterified to AMP

49. Acetyl-CoA Carboxylase The "ACC enzyme" commits acetate to fatty acid synthesis Carboxylation of acetyl-CoA to form malonyl- CoA is the irreversible, committed step in fatty acid biosynthesis Carboxylation of acetyl-CoA to form malonyl- CoA is the irreversible, committed step in fatty acid biosynthesis ACC uses bicarbonate and ATP (AND biotin!) ACC uses bicarbonate and ATP (AND biotin!) E.coli enzyme has three subunits E.coli enzyme has three subunits Animal enzyme is one polypeptide with all three functions - biotin carboxyl carrier, biotin carboxylase and transcarboxylase Animal enzyme is one polypeptide with all three functions - biotin carboxyl carrier, biotin carboxylase and transcarboxylase

50. Reaction Cycle for the Biosynthesis of Fatty Acids Fatty acid synthesis (mainly palmitic acid) from acetyl-CoA and malonyl-CoA involves 7 enzymatic reactions Fatty acid synthesis (mainly palmitic acid) from acetyl-CoA and malonyl-CoA involves 7 enzymatic reactions In E. coli, 7 different enzymes In E. coli, 7 different enzymes In yeast and animal, fatty acid synthase (FAS), a multifunctional enzyme catalyzes FAs synthesis In yeast and animal, fatty acid synthase (FAS), a multifunctional enzyme catalyzes FAs synthesis

51. Fatty Acid Biosynthesis I

52. Fatty Acid Biosynthesis II

53. Fatty Acid Biosynthesis III

54. Fatty Acid Synthesis in Bacteria and Plants Separate enzymes in a complex See Figure 25.7 See Figure 25.7 Pathway initiated by formation of acetyl-ACP and malonyl-ACP by transacylases Pathway initiated by formation of acetyl-ACP and malonyl-ACP by transacylases Decarboxylation drives the condensation of acetyl-CoA and malonyl-CoA Decarboxylation drives the condensation of acetyl-CoA and malonyl-CoA Other three steps are VERY familiar! Other three steps are VERY familiar! Only differences: D configuration and NADPH Only differences: D configuration and NADPH Check equations on page 811! Check equations on page 811!

55. Fatty Acid Synthesis in Animals Fatty Acid Synthase - a multienzyme complex Dimer of 250 kD multifunctional polypeptides Dimer of 250 kD multifunctional polypeptides Note the roles of active site serines on AT & MT Note the roles of active site serines on AT & MT Study the mechanism in Figure 25.11 - note the roles of ACP and KSase Study the mechanism in Figure 25.11 - note the roles of ACP and KSase Steps 3-6 repeat to elongate the chain Steps 3-6 repeat to elongate the chain

56. Fatty Acid Synthase MAT (malonyl/acetyl-CoA-ACP transacetylase) MAT (malonyl/acetyl-CoA-ACP transacetylase) KS (  -ketoacyl-ACP synthase) KS (  -ketoacyl-ACP synthase) DH (  -hydroxyacyl-ACP dehydrase) DH (  -hydroxyacyl-ACP dehydrase) ER (enoyl-ACP reductase) ER (enoyl-ACP reductase) KR (  -ketoacyl-ACP reductase) KR (  -ketoacyl-ACP reductase) TE (Palmitoyl thioesterase) TE (Palmitoyl thioesterase)

58. Further Processing of FAs Additional elongation - in mitochondria and ER Additional elongation - in mitochondria and ER Introduction of cis double bonds - do you need O 2 or not? Introduction of cis double bonds - do you need O 2 or not? E.coli add double bonds while the site of attack is still near something functional (the thioester) E.coli add double bonds while the site of attack is still near something functional (the thioester) Eukaryotes add double bond to middle of the chain - and need power of O 2 to do it Eukaryotes add double bond to middle of the chain - and need power of O 2 to do it Polyunsaturated FAs - plants vs animals... Polyunsaturated FAs - plants vs animals...

59. Desaturation Unsaturated fatty acids are produced by terminal desaturases Unsaturated fatty acids are produced by terminal desaturases  9 -,  6 -,  5 -,  4 -fatty acyl-CoA desaturases  9 -,  6 -,  5 -,  4 -fatty acyl-CoA desaturases

61. Regulation of Fatty Acid Metabolism

62. Sites of Regulation of Fatty Acid Metabolisms Hormones regulate fatty acid metabolism Hormones regulate fatty acid metabolism [glucose]  :  cells  glucagon (fatty acid oxidation) [glucose]  :  cells  glucagon (fatty acid oxidation) [glucose]  :  cells  insulin (fatty acid biosynthesis) [glucose]  :  cells  insulin (fatty acid biosynthesis) Short-term regulation Short-term regulation substrate availability, allosteric interactions and covalent modifications substrate availability, allosteric interactions and covalent modifications response time < 1 min response time < 1 min Long-term regulation Long-term regulation [enzyme] depends on rates of protein synthesis and/or breakdown [enzyme] depends on rates of protein synthesis and/or breakdown process requires hours or days process requires hours or days Epinephrine & norepinephrine activate “hormone-sensitive” lipase, releases FAs, which are exported to the liver for degradation Epinephrine & norepinephrine activate “hormone-sensitive” lipase, releases FAs, which are exported to the liver for degradation

63. Regulation of FA Synthesis Allosteric modifiers, phosphorylation and hormones Malonyl-CoA blocks the carnitine acyltransferase and thus inhibits beta-oxidation Malonyl-CoA blocks the carnitine acyltransferase and thus inhibits beta-oxidation Citrate activates acetyl-CoA carboxylase Citrate activates acetyl-CoA carboxylase Fatty acyl-CoAs inhibit acetyl-CoA carboxylase Fatty acyl-CoAs inhibit acetyl-CoA carboxylase Hormones regulate ACC Hormones regulate ACC Glucagon activates lipases/inhibits ACC Glucagon activates lipases/inhibits ACC Insulin inhibits lipases/activates ACC Insulin inhibits lipases/activates ACC

66. Biosynthesis of Complex Lipids Synthetic pathways depend on organism Sphingolipids and triacylglycerols only made in eukaryotes Sphingolipids and triacylglycerols only made in eukaryotes PE accounts for 75% of PLs in E.coli PE accounts for 75% of PLs in E.coli No PC, PI, sphingolipids, cholesterol in E.coli No PC, PI, sphingolipids, cholesterol in E.coli But some bacteria do produce PC But some bacteria do produce PC

67. Glycerolipid Biosynthesis CTP drives formation of CDP complexes Phosphatidic acid (PA) is the precursor for all other glycerolipids in eukaryotes Phosphatidic acid (PA) is the precursor for all other glycerolipids in eukaryotes See Figure 25.18 See Figure 25.18 PA is made either into DAG or CDP-DAG PA is made either into DAG or CDP-DAG Note the roles of CDP-choline and CDP- ethanolamine in synthesis of PC and PE in Figure 25.19 Note the roles of CDP-choline and CDP- ethanolamine in synthesis of PC and PE in Figure 25.19 Note exchange of ethanolamine for serine (25.21) Note exchange of ethanolamine for serine (25.21)

72. Other PLs from CDP-DAG Figure 25.22 CDP-diacylglycerol is used in eukaryotes to produce: CDP-diacylglycerol is used in eukaryotes to produce: PI in one step PI in one step PG in two steps PG in two steps Cardiolipin in three steps Cardiolipin in three steps

74. Plasmalogen Biosynthesis Dihydroxyacetone phosphate is the precursor Acylation activates and an exchange reaction produces the ether linkage Acylation activates and an exchange reaction produces the ether linkage Ketone reduction is followed by acylation Ketone reduction is followed by acylation CDP-ethanolamine delivers the headgroup CDP-ethanolamine delivers the headgroup A desaturase produces the double bond in the alkyl chain A desaturase produces the double bond in the alkyl chain

76. Sphingolipid Biosynthesis High levels made in neural tissue Initial reaction is a condensation of serine and palmitoyl-CoA Initial reaction is a condensation of serine and palmitoyl-CoA 3-ketosphinganine synthase is PLP-dependent 3-ketosphinganine synthase is PLP-dependent Ketone is reduced with help of NADPH Ketone is reduced with help of NADPH Acylation is followed by double bond formation Acylation is followed by double bond formation See Figure 25.25 See Figure 25.25 Resulting ceramide is precursor for other sphingolipids Resulting ceramide is precursor for other sphingolipids

79. Eicosanoid Biosynthesis PLA 2 releases arachidonic acid - a precursor of eicosanoids Eicosanoids are local hormones Eicosanoids are local hormones The endoperoxide synthase oxidizes and cyclizes The endoperoxide synthase oxidizes and cyclizes Tissue injury and inflammation triggers arachidonate release and eicosanoid synthesis Tissue injury and inflammation triggers arachidonate release and eicosanoid synthesis

82. Eicosanoid Biosynthesis Aspirin and other nonsteroid anti- inflammatory agents inhibit the cyclooxygenase Aspirin and other nonsteroid anti- inflammatory agents inhibit the cyclooxygenase Aspirin covalently Aspirin covalently Others noncovalently Others noncovalently

86. Cholesterol Biosynthesis Occurs primarily in the liver Biosynthesis begins in the cytosol with the synthesis of mevalonate from acetyl-CoA Biosynthesis begins in the cytosol with the synthesis of mevalonate from acetyl-CoA First step is a thiolase reaction First step is a thiolase reaction Second step makes HMG-CoA Second step makes HMG-CoA Third step - HMG-CoA reductase - is the rate-limiting step in cholesterol biosynthesis Third step - HMG-CoA reductase - is the rate-limiting step in cholesterol biosynthesis HMG-CoA reductase is site of action of cholesterol-lowering drugs HMG-CoA reductase is site of action of cholesterol-lowering drugs

90. Regulation of HMG-CoA Reductase As rate-limiting step, it is the principal site of regulation in cholesterol synthesis 1) Phosphorylation by cAMP-dependent kinases inactivates the reductase 1) Phosphorylation by cAMP-dependent kinases inactivates the reductase 2) Degradation of HMG-CoA reductase - half-life is 3 hrs and depends on cholesterol level 2) Degradation of HMG-CoA reductase - half-life is 3 hrs and depends on cholesterol level 3) Gene expression (mRNA production) is controlled by cholesterol levels 3) Gene expression (mRNA production) is controlled by cholesterol levels

92. The thiolase brainteaser... An important puzzle If acetate units can be condensed by thiolase to give acetoacetate in the 1st step of cholesterol biosynthesis, why not also use thiolase for FA synthesis, avoiding complexity of FA synthase? If acetate units can be condensed by thiolase to give acetoacetate in the 1st step of cholesterol biosynthesis, why not also use thiolase for FA synthesis, avoiding complexity of FA synthase? Solution: Subsequent reactions drive cholesterol synthesis, but eight successive thiolase reactions would be very unfavorable energetically for FA synthesis Solution: Subsequent reactions drive cholesterol synthesis, but eight successive thiolase reactions would be very unfavorable energetically for FA synthesis

93. Squalene from Mevalonate Driven by ATP hydrolysis, decarboxylation and PP i hydrolysis Six-carbon mevalonate makes five carbon isopentenyl PP i and dimethylallyl PP i Six-carbon mevalonate makes five carbon isopentenyl PP i and dimethylallyl PP i Condensation of 3 of these yields farnesyl PP i Condensation of 3 of these yields farnesyl PP i Two farnesyl PP i s link to form squalene Two farnesyl PP i s link to form squalene Bloch and Langdon were first to show that squalene is derived from acetate units and that cholesterol is derived from squalene Bloch and Langdon were first to show that squalene is derived from acetate units and that cholesterol is derived from squalene

95. Cholesterol from Squalene At the endoplasmic reticulum membrane Squalene monooxygenase converts squalene to squalene-2,3-epoxide Squalene monooxygenase converts squalene to squalene-2,3-epoxide A cyclase converts the epoxide to lanosterol A cyclase converts the epoxide to lanosterol Though lanosterol looks like cholesterol, 20 more steps are required to form cholesterol! Though lanosterol looks like cholesterol, 20 more steps are required to form cholesterol! All at/in the endoplasmic reticulum membrane All at/in the endoplasmic reticulum membrane

97. Inhibiting Cholesterol Synthesis Merck and the Lovastatin story... HMG-CoA reductase is the key - the rate- limiting step in cholesterol biosynthesis HMG-CoA reductase is the key - the rate- limiting step in cholesterol biosynthesis Lovastatin (mevinolin) blocks HMG-CoA reductase and prevents synthesis of cholesterol Lovastatin (mevinolin) blocks HMG-CoA reductase and prevents synthesis of cholesterol Lovastatin is an (inactive) lactone Lovastatin is an (inactive) lactone In the body, the lactone is hydrolyzed to mevinolinic acid, a competitive (TSA!) inhibitor of the reductase, K i = 0.6 nM! In the body, the lactone is hydrolyzed to mevinolinic acid, a competitive (TSA!) inhibitor of the reductase, K i = 0.6 nM!

99. Biosynthesis of Bile Acids Carboxylic acid derivatives of cholesterol Essential for the digestion of food, especially for solubilization of ingested fats Essential for the digestion of food, especially for solubilization of ingested fats Synthesized from cholesterol Synthesized from cholesterol Cholic acid conjugates with taurine and glycine to form taurocholic and glycocholic acids Cholic acid conjugates with taurine and glycine to form taurocholic and glycocholic acids First step is oxidation of cholesterol by a mixed-function oxidase First step is oxidation of cholesterol by a mixed-function oxidase

102. Steroid Hormone Synthesis Desmolase (in mitochondria) forms pregnenolone, precursor to all others Pregnenolone migrates from mitochondria to ER where progesterone is formed Pregnenolone migrates from mitochondria to ER where progesterone is formed Progesterone is a branch point - it produces sex steroids (testosterone and estradiol), and corticosteroids (cortisol and aldosterone) Progesterone is a branch point - it produces sex steroids (testosterone and estradiol), and corticosteroids (cortisol and aldosterone) Anabolic steroids are illegal and dangerous Anabolic steroids are illegal and dangerous Recall the Ben Johnson story.... Recall the Ben Johnson story....