BI/CH 422/622 Liver cytosol ANABOLISM OUTLINE: Photosynthesis Carbohydrate Biosynthesis in Animals Biosynthesis of Fatty Acids and Lipids Fatty Acids Triacylglycerides contrasts Membrane lipids location & transport Glycerophospholipids Synthesis Sphingolipids acetyl-CoA carboxylase Isoprene lipids: fatty acid synthase Ketone Bodies ACP priming 4 steps Cholesterol Control of fatty acid metabolism isoprene synth. ACC Joining Reciprocal control of b-ox Cholesterol Synth. Diversification of fatty acids Fates Eicosanoids Cholesterol esters Bile acids Prostaglandins,Thromboxanes, Steroid Hormones and Leukotrienes Metabolism & transport Control ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Lipid Fat Biosynthesis Catabolism Fatty Acid Fatty Acid Synthesis Degradation Ketone body Utilization Isoprene Biosynthesis 1 Cholesterol and Steroid Biosynthesis mevalonate kinase Mevalonate to Activated Isoprenes • Two phosphates are transferred stepwise from ATP to mevalonate. • A third phosphate from ATP is added at the hydroxyl, followed by decarboxylation and elimination catalyzed by pyrophospho- mevalonate decarboxylase creates a pyrophosphorylated 5-C product: D3-isopentyl pyrophosphate (IPP) (isoprene). • Isomerization to a second isoprene dimethylallylpyrophosphate (DMAPP) gives two activated isoprene IPP compounds that act as precursors for D3-isopentyl pyrophosphate Isopentyl-D-pyrophosphate all of the other lipids in this class isomerase DMAPP Cholesterol and Steroid Biosynthesis mevalonate kinase Mevalonate to Activated Isoprenes • Two phosphates are transferred stepwise from ATP to mevalonate. • A third phosphate from ATP is added at the hydroxyl, followed by decarboxylation and elimination catalyzed by pyrophospho- mevalonate decarboxylase creates a pyrophosphorylated 5-C product: D3-isopentyl pyrophosphate (IPP) (isoprene). • Isomerization to a second isoprene dimethylallylpyrophosphate (DMAPP) gives two activated isoprene IPP compounds that act as precursors for D3-isopentyl pyrophosphate Isopentyl-D-pyrophosphate all of the other lipids in this class isomerase DMAPP 2 Cholesterol and Steroid Biosynthesis DMAPP IPP Joining Isoprenes HEAD HEAD TAIL TAIL • The two isoprenes join head to tail, displacing one set of diphosphates, forming 10-carbon geranyl-pyrophosphate geranyl pyrophosphate (GPP). synthase (I (DMAPP) Geranyl-PP synthase Geranyl-pyrophosphate Mechanism Carotenoids synthase Resonance Gibberellins stabilized carbonium ion Tocopherols Farnesyl-PP synthase Ionization Isopentyl Chlorophylls by pyrophosphate elimination (IPP) of PPi Squalene synthase Geranyl pyrophosphate (GPP) H+ deprotonation Cholesterol and Steroid Biosynthesis Formation of Squalene •Geranyl pyrophosphate (GPP) joins to another isopentenyl pyrophosphate. à forms 15-C farnesyl pyrophosphate (tail-to-tail) •Two farnesyl pyrophosphates (FPP) join tail to tail to form phosphate-free squalene. 3 Cholesterol and Steroid Biosynthesis Synthesis of Cholesterol Conversion of Squalene to Four-Ring Steroid Nucleus • Squalene monooxygenase (squalene epoxidase) adds one oxygen to the end of the squalene chain. (2,3-oxidosqualene) à forms squalene 2,3-epoxide • Here, pathways diverge in animal cells versus plant cells. • In plants, the epoxide cyclizes lanosterol to other sterols, such as synthase stigmasterol. ⑤ 1) C14-demethylation (5 rxns) 2) C14-reduction ② 3) C4-demethylation (10 rxns) • In fungi, it forms ergosterol. ④ ① 4) C8à7 isomerization ⑦ 5) C24-reduction ⑥ ③ 6) C5-desaturation • The cyclization product in 7) C7-reduction animals is lanosterol, which converts to cholesterol. Cholesterol and Steroid Biosynthesis Synthesis of Cholesterol Conversion of Squalene to Four-Ring Steroid Nucleus • Squalene monooxygenase (squalene epoxidase) adds one oxygen to the end of the squalene chain. (2,3-oxidosqualene) à forms squalene 2,3-epoxide • Here, pathways diverge in animal cells versus plant cells. • In plants, the epoxide cyclizes lanosterol to other sterols, such as synthase stigmasterol. ⑤ 1) C14-demethylation (5 rxns) 2) C14-reduction ② 3) C4-demethylation (10 rxns) • In fungi, it forms ergosterol. ④ ① 4) C8à7 isomerization ⑦ 5) C24-reduction ⑥ ③ 6) C5-desaturation • The cyclization product in 7) C7-reduction animals is lanosterol, which converts to cholesterol. 4 Cholesterol and Steroid Biosynthesis Fates of Cholesterol After Synthesis Vitamin D pre-VitD3 •In vertebrates, most 7-dehydro-Chol cholesterol is synthesized in the liver, then exported. -They are exported as bile (Oxysterols) acids, biliary cholesterol, or cholesteryl esters. From degrad- ation of Cys -Bile is stored in the gall bladder and secreted into the Lecithin-Cholesterol small intestine after fatty Acyl Transferase- Catalyzed Reaction meal. Occurs in HDL -Bile acids such as taurocholic (LCAT) acid emulsify fats. •Adrenal gland-synthesized steroids: –They surround droplets of fat, – mineralcorticoids increasing surface area for • control electrolyte balance, reabsorption of Na+, Cl−, − attack by lipases. HCO3 from kidney •Other tissues convert – glucocorticoids • regulate gluconeogenesis, reduce inflammation cholesterol into steroid •Gonad-synthesized steroids: hormones and Vitamin D – progesterone, testosterone, estrogens Steroid Biosynthesis Steroid Hormones Derived from Cholesterol ox=C3O • Takes place in & iso=C4/5 mitochondria • The “side chain” on C-17 of the D C-11 C-21 C-17 C-17 C-11 –acetateC-20/21 ring is modified, C-21 then cleaved. C-20 –C-19 aromatase C-22 C-18 ox-C18O • Two adjacent carbons are hydroxylated. • Desmolase cleaves • All three use mixed- function oxidases, NADPH and cytochrome P450 5 Steroid Biosynthesis Steroid Hormones Derived from Cholesterol ox=C3O • Takes place in & iso=C4/5 mitochondria • The “side chain” on C-17 of the D C-11 C-21 C-17 C-17 C-11 –acetateC-20/21 ring is modified, C-21 then cleaved. C-20 –C-19 aromatase C-22 C-18 ox-C18O • Two adjacent carbons are hydroxylated. • Desmolase cleaves • All three use mixed- function oxidases, NADPH and cytochrome P450 Cholesterol and Steroid Biosynthesis TABLE 21-1 Major Classes of Human Plasma Lipoproteins: Some Properties Composition (wt %) Free Cholesteryl Lipoprotein Density (g/ml) Protein PL Triacylglycerols cholesterol esters Chylomicrons <1.006 2 (ApoB-48,-E,-CII) 9 1 3 85 VLDL 0.95–1.006 10 (ApoC-I,CII) 18 7 12 50 LDL 1.006–1.063 23 (ApoB-100) 20 8 37 10 HDL 1.063–1.210 55 (ApoA-I,-II) 24 2 15 4 Source: Data from D. Kritchevsky, Nutr. Int. 2:290, 1986. •Lipids are carried through the plasma on spherical particles. –surface is made of protein (called apolipoprotein*) and a phospholipid monolayer –interior contains cholesterol, TAGs, and cholesteryl esters, which are more nonpolar than cholesterol • Named based on position of sedimentation (density) in centrifuge • Composition varies between class of lipoprotein • Includes four major *“apolipoprotein” refers to the protein part of a classes shown in Table lipoprotein particle. 6 Cholesterol and Steroid Biosynthesis TABLE 21-2 Apolipoproteins of the Human Plasma Lipoproteins Biological Polypeptide Roles and molecular Apolipoprotein weight Lipoprotein association Function (if known) Characteristics ApoA-I 28,100 HDL Activates LCAT; interacts of Lipoproteins with ABC transporter ApoA-II 17,400 HDL Inhibits LCAT ApoA-IV 44,500 Chylomicrons, HDL Activates LCAT; cholesterol transport/clearance ApoB-48 242,000 Chylomicrons Cholesterol transport/clearance ApoB-100 512,000 VLDL, LDL Binds to LDL receptor ApoC-I 7,000 VLDL, HDL ApoC-II 9,000 Chylomicrons, VLDL, Activates lipoprotein lipase HDL ApoC-III 9,000 Chylomicrons, VLDL, Inhibits lipoprotein lipase HDL ApoD 32,500 HDL ApoE 34,200 Chylomicrons, VLDL, Triggers clearance of VLDL HDL and chylomicron remnants ApoH 50,000 Possibly VLDL, binds Roles in coagulation, lipid cardiolipin metabolism, apoptosis, inflammation Source: Information from D. E. Vance and J. E. Vance (eds), Biochemistry of Lipids and Membranes, 5th edn, Elsevier Science Publishing, 2008. Cholesterol and Steroid Biosynthesis Biological Roles of Lipoproteins in Receptor-Mediated Trafficking Cholesterol and TAGs Endocytosis IN: Exogenous (diet) Pathway ❶–❹ HDL Pathway 10 – 13 OUT: Endogenous ❺ ❾ Pathway – Enterohepatic 14 Pathway 7 Cholesterol and Steroid Biosynthesis Cholesterol and Steroid Biosynthesis Regulation of Cholesterol Metabolism •Major regulation is by gene expression: Three levels – Sterol regulatory element-binding proteins (SREBPs) • When sterol levels are high, oxysterols are produced, which retain SREBPs in the ER membrane with other proteins. • When sterol levels fall, the complex is cleaved and moves to the nucleus. • It activates transcription of HMG-CoA reductase and LDL receptor, as well as other genes. – Translational control on mRNA stability • Increased mevalonate and other isoprenes destabilize the HMG-CoA mRNA – Post-translational control • High cholesterol produces oxysterol, which allosterically binds HMG-CoA reductase • Destabilizes by a conformational change that allows ubiquitination • Proteolytic degradation of HMG-CoA reductase •Minor regulatory mechanisms: Phosphorylation/de-phosphorylation – Covalent modification provides short-term regulation Other effects of high cholesterol – Glucagon, epinephrine Activation of ACAT, which increases esterification for storage • cascades lead to phosphorylation, ¯ activity Transcriptional regulation of the LDL • AMP-dependent protein kinase receptor gene activation • also when AMP rises, kinase phosphorylates the enzyme à activity ¯, cholesterol synthesis ¯ – Insulin: cascades