Chapter 24

1. Chapter 24 Lipid Biosynthesis Biochemistry by Reginald Garrett and Charles Grisham

2. Outline • How Are Fatty Acids Synthesized? • How Are Complex Lipids Synthesized? • How Are Eicosanoids Synthesized, and What Are Their Functions? • How Is Cholesterol Synthesized? • How Are Lipids Transported Throughout the Body? • How Are Bile Acids Biosynthesized? • How Are Steroid Hormones Synthesized and Utilized?

3. 24.1 – How Are Fatty Acids Synthesized? The Biosynthesis and Degradation Pathways are Different • As in cases of glycolysis/gluconeogenesis and glycogen synthesis/breakdown, fatty acid synthesis and degradation go by different routes • There are fivemajor differences between fatty acid breakdown and biosynthesis

4. 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) • Synthesis in cytosol; breakdown in mitochondria • Enzymes of synthesis are one polypeptide (fatty acid synthase) in animals • Biosynthesis uses NADPH/NADP+;breakdown uses NADH/NAD+ & FAD • Stereochemistry (D-b-hydroxyacyl)

5. 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 • Acetate units are activated for transfer by conversion to malonyl-CoA • Decarboxylation of malonyl-CoA and reducing power of NADPH drive chain growth • Chain grows to 16-carbons (palmitate) • Other enzymes add double bonds and more Cs

6. The sources of Acetyl-CoA • Amino acid degradation produces cytosolic acetyl-CoA • Fatty acid oxidation produces mitochondrial acetyl-CoA • Glycolysis yields cytosolic pyruvate which is converted to acetyl-CoA in mitochondria • Citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents for fatty acid synthesis

8. The reducing power: NAPDH • Produced by malic enzyme • Produced in pentose phosphate pathway (chapter 22)

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

10. Figure 24.2(a) The acetyl-CoA carboxylase reaction produces malonyl-CoA for fatty acid synthesis. (b) A mechanism for the acetyl-CoA carboxylase reaction. Bicarbonate is activated for carboxylation reactions by formation of N-carboxybiotin. ATP drives the reaction forward, with transient formation of a carbonylphosphate intermediate (Step 1). In a typical biotin-dependent reaction, nucleophilic attack by the acetyl-CoA carbanion on the carboxyl carbon of N-carboxybiotin - a transcarboxylation - yields the carboxylated product (Step 2). Malonyl-CoA

11. Figure 24.3In the acetyl-CoA carboxylase reaction, the biotin ring, on its flexible tether, acquires carboxyl groups from carbonylphosphate on the carboxylase subunit and transfers them to acyl-CoA molecules on the transcarboxylase subunits.

12. Acetyl-CoA Carboxylase ACC forms long, active filamentous polymers from inactive protomers • As a committed step, ACC is carefully regulated • Palmitoyl-CoA (product) favors monomers • Citrate favors the active polymeric form Citrate Inactive protomers active polymer Acyl-CoA

13. Figure 24.4Models of the acetyl-CoA carboxylase polypeptide, with phosphorylation sites indicated, along with the protein kinases responsible. Phosphorylation at Ser1200 is primarily responsible for decreasing the affinity for citrate.

14. Phosphorylation of ACC modulates activation by citrate and inhibition by palmitoyl-CoA • Unphosphorylated E has high affinity for citrate and is active at low [citrate] • Unphosphorylated E has low affinity for palm-CoA and needs high [palm-CoA] to inhibit • Phosphorylated E has low affinity for citrate and needs high [citrate] to activate • Phosphorylated E has high affinity for palm-CoA and is inhibited at low [palm-CoA]

15. Figure 24.5The activity of acetyl-CoA carboxylase is modulated by phosphorylation and dephosphorylation. The dephospho form of the enzyme is activated by low [citrate] and inhibited only by high levels of fatty acyl-CoA. In contrast, the phosphorylated form of the enzyme is activated only by high levels of citrate, but is very sensitive to inhibition by fatty acyl-CoA.

16. The acyl carrier protein carry the intermediates in fatty acid synthesis • ACP is a 77 residue protein in E. coli - with a phosphopantetheine • The same functional group of CoA

17. Fatty Acid Synthesis • Acetyl-CoA-ACP transacylases (ATase) • Malonyl-CoA-ACP transacylases (MTase) • b-ketoacyl-ACP synthase (KSase) • b-ketoacyl-ACP reductase (KRase) • b-hydroxyacyl-ACP dehydratase (DH) • Enoyl-ACP reductase (ERase) • Thioesterase (TEase) in animals

18. Figure 24.7The pathway of palmitate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl building blocks are introduced as acyl carrier protein conjugates. Decarboxylation drives the b-ketoacyl-ACP synthase and results in the addition of two-carbon units to the growing chain. Concentrations of free fatty acids are extremely low in most cells, and newly synthesized fatty acids exist primarily as acyl-CoA esters.

19. Fatty Acid Synthesis in Bacteria and Plants • The separate enzymes in bacteria and plants • Pathway initiated by formation of acetyl-ACP and malonyl-ACP by transacylases • Decarboxylation drives the condensation of acetyl-CoA and malonyl-CoA by KSase • Next three steps look very similar to the fatty acid oxidation in reverse • Only differences: D configuration and NADPH

20. Fatty Acid Synthesis Acetyl-CoA + 7 malonyl-CoA + 14 NAPDH + 14 H+ Palmitoyl-CoA + 7 HCO3- + 14 NAPD+ + 7 CoA-SH The formation of malonyl-CoA: 7 Acetyl-CoA + 7 HCO3- + 7 ATP4- 7 malonyl-CoA + 7 ADP3- + 7Pi2- +7H+ The overall reaction: 8 Acetyl-CoA + 7 ATP4- + 14 NAPDH + 14 H+ Palmitoyl-CoA + 14 NAPD+ + 7 CoA-SH + 7 ADP3- + 7Pi2-

21. (213 KD) In yeast: a6b6 (203 KD) Figure 24.8In yeast, the functional groups and enzyme activities required for fatty acid synthesis are distributed between a- and b-subunits.

22. Fatty Acid Synthase in Animals Fatty Acid Synthase - a multienzyme complex • Dimer of 250 kD multifunctional polypeptides • Domain 1: AT, MT & KSase • Domain 2: ACP, KRase, DH, ERase • Domain 3: Thioesterase

23. Figure 24.9Fatty acid synthase in animals contains all the functional groups and enzyme activities on a single multifunctional subunit. The active enzyme is a head-to-tail dimer of identical subunits. (Adapted from Wakil, S. J., Stoops, J. K., and Joshi, V. C., 1983.Fatty acid synthesis and its regulation. Annual Review of Biochemistry52:537-579.)

24. Figure 24.10Acetyl units are covalently linked to a serine residue at the active site of the acetyl transferase in eukaryotes. A similar reaction links malonyl units to the malonyl transferase.

26. Further Processing of FAs Additional elongation & desaturation • Additional elongation occurs in mitochondria and the surface of ER • The reducing coenzyme for the second step is NADH, whereas the reductant for the fourth step is NADPH. • In ER, involving malonyl-CoA

27. Figure 24.12Elongation of fatty acids in mitochondria is initiated by the thiolase reaction. The b-ketoacyl intermediate thus formed undergoes the same three reactions (in reverse order) that are the basis of b-oxidation of fatty acids. Reduction of the b-keto group is followed by dehydration to form a double bond. Reduction of the double bond yields a fatty acyl-CoA that is elongated by two carbons. Note that the reducing coenzyme for the second step is NADH, whereas the reductant for the fourth step is NADPH. ( in mitochondria)

28. Further Processing of FAs Introduction of cis double bonds • E.coli add double bonds during the fatty acid synthesis • After four normal cycles, b-hydroxydecanoyl thioester dehydrase forms a double bond b, g to the thioester • O2-independent

29. Figure 24.13Double bonds are introduced into the growing fatty acid chain in E. coli by specific dehydrases. Palmitoleoyl-ACP is synthesized by a sequence of reactions involving four rounds of chain elongation, followed by double bond insertion by b-hydroxydecanoyl thioester dehydrase and three additional elongation steps. Another elongation cycle produces cis-vaccenic acid.

30. Further Processing of FAs Introduction of cis double bonds • Eukaryotes add double bond until the fatty acyl chain has reached its full length (usually 16 to 18 carbons) • Desaturase • Cytochrome b5 reductase & Cytochrome b5 • All three proteins are ssociated with the ER membrane • NADH & O2 are required; O2-dependent

31. Figure 24.14The conversion of stearoyl-CoA to oleoyl-CoA in eukaryotes is catalyzed by stearoyl-CoA desaturase in a reaction sequence that also involves cytochrome b5 and cytochrome b5 reductase. Two electrons are passed from NADH through the chain of reactions as shown, and two electrons are also derived from the fatty acyl substrate. linoleic acid in eukaryotes. This is the only means by which animals can synthesize fatty acids with double bonds at positions beyond C-9.

32. Mammals cannot synthesize most polyunsaturated fatty acids • Essential fatty acids • Introduce double bonds between the double bond at the 8- or 9- position and the carboxyl group • form a double bond at 5-position (6) if one already exists at the 8-position (9)

33. Arachidonic acid • mammals can also add double bonds to unsaturated fatty acids • synthesized from linoleic acid in eukaryotes • The precursor for prostaglandins and other biologically active derivatives

34. Figure 24.15Arachidonic acid is synthesized from linoleic acid in eukaryotes. This is the only means by which animals can synthesize fatty acids with double bonds at positions beyond C-9.

35. Regulation of Fatty Acid Synthesis Allosteric modifiers, phosphorylation and hormones • Allosteric regulation • Malonyl-CoA blocks the carnitine acyltransferase and thus inhibits b-oxidation • Citrate activates acetyl-CoA carboxylase • Fatty acyl-CoAs inhibit acetyl-CoA carboxylase

36. Figure 24.16Regulation of fatty acid synthesis and fatty acid oxidation are coupled as shown. Malonyl-CoA, produced during fatty acid synthesis, inhibits the uptake of fatty acylcarnitine (and thus fatty acid oxidation) by mitochondria. When fatty acyl-CoA levels rise, fatty acid synthesis is inhibited and fatty acid oxidation activity increases. Rising citrate levels (which reflect an abundance of acetyl-CoA) similarly signal the initiation of fatty acid synthesis.

37. Regulation of FA Synthesis • Phosphorylation and hormones • Citrate activation and acyl-CoA inhibition of ACC are dependent on the phosphorylation state • Phosphorylation causes inhibition of fatty acid biosynthesis • Hormone signals regulate ACC and fatty acid biosynthesis • Glucagon activates lipases/inhibits ACC • Insulininhibits lipases/activates ACC

38. Figure 24.17Hormonal signals regulate fatty acid synthesis, primarily through actions on acetyl-CoA carboxylase. Availability of fatty acids also depends upon hormonal activation of triacylglycerol lipase.

39. 24.2 – How Are Complex Lipids Synthesized? Complexed lipids: • Glycerolipid • Triacylglycerols • Glycerophospholipids • Sphingolipids Phospholipids (membrane components) • Glycerophospholipids • Sphingolipids

40. Synthetic pathways depend on different organism • Sphingolipids and triacylglycerols only made in eukaryotes • Phosphatidylethanolamine (PE) accounts for 75% of phospholipids in E.coli • No PC, PI, sphingolipids, cholesterol in E.coli • But some bacteria do produce PC

41. Glycerolipid Biosynthesis Glycerolipids are synthesized by phosphorylation & acylation of glycerol • Phosphatidic acid (PA) is the precursor for all other glycerolipids in eukaryotes • Eukaryotic systems can also utilize DHAP as a starting point

42. Unsatuated Fatty Acid Figure 24.18Synthesis of glycerolipids in eukaryotes begins with the formation of phosphatidic acid, which may be formed from dihydroxyacetone phosphate or glycerol as shown. Satuated Fatty Acid

43. Glycerolipid Biosynthesis • PA is converted either to DAG or CDP-DAG • DAG is a precursor for synthesis of TAG, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) • TAG is synthesized mainly in adipose tissue, liver, and intestines

44. Figure 24.19Diacylglycerol and CDP-diacylglycerol are the principal precursors of glycerolipids in eukaryotes. Phosphatidylethanolamine and phosphatidylcholine are formed by reaction of diacylglycerol with CDP-ethanolamine or CDP-choline, respectively.

45. Glycerolipid Biosynthesis • PE synthesis • begins with phosphorylation of ethanolamine to form phosphoethanolamine • Transfer of a cytidylyl group from CTP to from CDP-ethanolamine • Phosphoethanolaminetransferase link phosphoethanolamine to the DAG • Synthesis of PC is entirely analogous • PC can also be converted from PE by methylation reactions

46. Exchange of ethanolamine for serine converts PE to PS (phosphatidylserine) Figure 24.21The interconversion of phosphatidylethanolamine and phosphatidylserine in mammals.

47. Other PLs from CDP-DAG Figure 24.22 • CDP-diacylglycerol is used in eukaryotes to produce: • Phosphatiylinositol (PI) in one step • 2-8% in animal membrane • Breakdown to form inositol-1,4,5-triphosphate & DAG (second messengers) • Phosphatiylglycerol (PG) in two steps • Cardiolipin in three steps

48. Figure 24.22CDP-diacylglycerol is a precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin in eukaryotes.

50. Plasmalogen Biosynthesis Dihydroxyacetone phosphate (DHAP) is the precursor to the plasmalogens • Acylation of DHAP • Exchange reaction produces the ether linkage by long-chain alcohol (acyl-CoA reductase) • Ketone reduction • Acylation again • CDP-ethanolamine delivers the head group • A desaturase produces the double bond in the alkyl chain