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1.FattyAcids - BIOCHEMISTRY BIOCHEMISTRY 441 Part A Winter...

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Unformatted text preview: BIOCHEMISTRY BIOCHEMISTRY 441, Part A Winter, 2012 1. 2. 3. 4. 5. 6. Biosynthesis of fatty acids Triacylglycerols, phospholipids & complex lipids Cholesterol & lipoproteins Photosynthesis: antennas & reaction centers Photosynthesis: electron transfer & photophosphorylation Photosynthesis: carbon fixation by C3 and C4 pathways 7. Amino acid metabolism: transamination and NH3 transport 8. 9. 10. 11. 12. 13. Urea cycle, amino acid catabolism & biosynthesis Aromatic amino acids & neurotransmitters One-carbon metabolism Biosynthesis of pyrimidines & purines Deoxyribonucleotide biosynthesis & nucleotide catabolism DNA & RNA structure 1. 1. Biosynthesis of Fatty acids Fatty acids have extended hydrocarbon chains 1 CO2H 16 palmitic acid, C16:0 1 18 CO2H 10 9 1 CO2H stearic acid, C18:0 palmitoleic acid, C16:1(∆ 9) 16 10 9 1 CO2H oleic acid, C18:1(∆ 9) 18 Most natural fatty acids have an even number of carbons. Most natural unsaturated fatty acids have cis double bonds. Fatty acids are components of phospholipids and triacylglycerols glycerophospholipids O O CH2OCR RC-O-CH O+ CH2OPOCH2CH2N(CH3)3 O sphingolipids sphingolipids O HOCH-CH=CH-CH2R R-C-NH-CH O+ CH2OPOCH2CH2N(CH3)3 O O CH2OCR RC-O-CH O CH2OCR O Sphingolipids on cell surfaces are sites of cell recognition. Inositol phospholipids participate in intracellular signaling. Phospholipid bilayers are the central structural elements of biological membranes. triacylglycerols Triacylglycerols are stored as energy reserves. Fatty acids also are found as cholesterol Fatty esters in lipoproteins, and are attached covalently to some proteins. Triacylglycerols are stored as energy reserves in adipose tissue and other tissues capillary cytosol lipid droplet Cross section of four adipocytes from Cross a guinea pig. Lipid droplets fill most of the volume of the cells. Lipid droplets are dynamic organelles consisting of triacylglycerols surrounded by a phospholipid monolayer with numerous bound proteins. Little is known about how cells control their number and size. Fatty acids are synthesized from acetyl-CoA in adipose tissue & the liver O CH3-C-S-CoA ATP, NADPH, CO2 Why is CO2 needed? O C-S-CoA H3C palmitic acid (C16:0) palmitoylCoA O C-S-CoA H3C stearic acid (C18:0) stearoylCoA 9 O C-S-CoA oleic acid (C18:1 ∆ 9) 1 oleoylCoA H3C 18 The labeling patterns suggest that the fatty acid chain forms by successive addition of two-carbon units Malonyl-CoA serves as the donor of two-carbon units bound biotin HN O C ATP NH HCO3- Pi HN N-CO2 O HN - S S O=C C O O2C-CH2-C-S-CoA O CH3-C-S-CoA O ADP malonyl-CoA O=C NH biotincarboxylase site NH S O=C NH biotin attachment site C NH carboxyltransferase site Malonyl-CoA is formed from acetyl-CoA and CO2 by a multifunctional enzyme, Malonyl-CoA acetylCoA carboxylase (biotin carboxylase-transcarboxylase). Biotin is attached covalently to a Lys residue of the enzyme. In bacteria, the three domains are in separate subunits; in animals, they are on a single, multifunctional polypeptide. Pyruvate carboxylase, a similar multifunctional enzyme, has four domains with different functions The active form of pyruvate carboxylase is a tetramer. biotin carboxylase (BC, blue), Zn2+ CT BC ATP carboxyl-transferase (CT, yellow) biotin carboxylcarrier protein (red) 65 Å allosteric regulatory domain (green) CT BC ATP M. St. Maurice et al. Science 317, 1076 (2007). 2qf7.pdb Zn2+ The biotin carboxyl-carrier protein moves carboxybiotin between sites on different monomers. How could you test this? Hint: make a mutant with an inactive BC site, and another mutant with an inactive CT site. Then make hybrid tetramers. one monomer is outlined (To view or download pdb structures go to www.rcsb.org/pdb) The fatty acid chain is synthesized attached to a protein -- acyl-carrier protein (ACP) Malonyl and acetyl groups are transferred from CoA to a thiol sulfur atom of ACP. malonyl-CoA O O2C-CH2-C-S-CoA HS-ACP O O2C-CH2-C-S-ACP HS-CoA malonyl/acetylCoA-ACP transferase malonyl/acetylCoA-ACP acetyl-CoA O CH3-C-S-CoA HS-ACP O CH3-C-S-ACP HS-CoA malonyl-ACP Bacteria Bacteria have separate malonyl & acetyl transferases. In animals, one enzyme catalyzes both reactions. acetyl-ACP The functional thiol group of ACP is in a 4’-phosphopantetheine group linked to a Ser residue of the protein O O H3C CH3 O O P O Ser HS N H N H O OH 4’-phosphopantetheine NH2 C N CoA-SH ACP of Bacillus subtilis. 1f80.pdb O H3C CH3 O In animals, ACP is a domain of a large protein with multiple catalytic centers; in bacteria, it’s a separate, small protein. A groove in the protein shelters the growing fatty acid chain. O OPOPO HS N H O N H O HC C N CH2 O C N CH N O OH 4’-phosphopantetheine also provides the thiol group of coenzyme A. Pantetheine is vitamin B5. O PO32 OH The condensation reaction 3-ketoacyl-ACP malonyl-ACP O S O C HO - HCO3 O H+ O- ACP S O C ACP CH3 O S-C-CH3 β-ketoacyl-ACP synthase The acetyl group first moves from ACP to a Cys residue of the ketoacyl-ACP synthase, and then combines with malonyl-ACP to give a 3-ketoacyl-ACP. SH β-ketoacyl-ACP synthase Release of bicarbonate is exothermic and pulls the reaction in the direction of condensation. O CH3-C-S- O KS O -O-C-CH2-C-S-ACP HCO3 β-ketoacyl-ACP synthase (KS) Reduction by NADPH, followed by dehydration and a second reduction, generates butyryl-ACP KS -SH NADPH β-ketoacyl-ACP reductase H enoyl-ACP reductase H O CH3-C-CH2-C-S-ACP O CH3-CH2CH2-C-S-ACP NADP+ O O CH3-C=C-C-S-ACP H hydroxyacylACP dehydrase NADPH NADP+ O CH3-C-CH2-C-S-ACP OH HOH To continue the cycle, the butyrl group must move from ACP to β-ketoacyl-ACP synthase O CH3-C-S- β-ketoacyl-ACP synthase catalyzes this transfer O CH3-CH2CH2-C-S- KS O -O-C-CH2-C-S-ACP β-ketoacyl-ACP synthase KS KS -SH ACP-SH O CH3-CH2CH2-C-S-ACP NADPH CO2 O O CH3-C-CH2-C-S-ACP β-ketoacyl-ACP synthase NADP+ O reductase NADP+ enoyl reductase H NADPH O CH3-C=C-C-S-ACP H H O dehydrase CH3-C-CH2-C-S-ACP OH HOH A second turn of the cycle generates hexanoyl-ACP O O -O-C-CH2-C-S-ACP O CH3-CH2CH2-C-S- β-ketoacyl-ACP synthase KS KS -SH CO2 O O CH3CH2 CH2-C-CH2-C-S-ACP O CH3CH2CH2-CH2CH2-C-S-ACP reductase NADP+ enoyl reductase NADP+ NADPH H NADPH O CH3CH2CH2-C=C-C-S-ACP H dehydrase H O CH3CH2 CH2-C-CH2-C-S-ACP OH HOH The cycle continues until the fatty acid chain on ACP reaches 16 carbons Thioesterase hydrolyzes palmitoyl-ACP, releasing free palmitate (C16:0) C14:0 O CH3-(CH2)11CH2-C-S- O -O-C-CH2-C-S-ACP β-ketoacyl-ACP synthase KS KS -SH ACP-SH thioesterase palmitate O CO2 O O CH3-(CH2)11CH2-C-CH2-C-S-ACP H2O NADPH O CH3-(CH2)11CH2-CH2CH2-C-S-ACP NADP+ C16:0 H NADP+ H O NADPH CH3-(CH2)11CH2-C=C-C-S-ACP H O CH3-(CH2)11CH2-C-CH2-C-S-ACP OH HOH Ketoacyl synthase internal acyltransferase C16:0 C14:0 C12:0 C10:0 C8:0 C6:0 C4:0 C2:0 malonyl Thioesterase Malonyl/acetyltransferase Vmax (µmol/min/mg) Chain-length specificity of the substrate-loading, chain-elongation and chain-termination activities of mammalian fatty-acid synthase S. Smith et al. Prog. Lipid Res. 42: 289 (2003) The enzymes of fatty acid synthesis have fused into a single protein during evolution KS H3N + H3N CO2- MT CO2- OH H3N + SH KR H3N + ER CO2H3N + H3N + AT CO 2 H3N + DH CO2 - CO2- TE H3N + ACP + CO2- OH CO2- O-phosphopantetheine OH E. coli: eight separate proteins 400 H3N + 320 140 600 220 230 75 300 KS MAT DH core ER KR ACP Approx. number of amino acid residues in each domain TE SH OH Animals: one multifunctional protein OH O-phosphopantetheine How does the growing fatty acid chain bound to ACP reach all the active sites? CO2- Architecture of the mammalian fatty acid synthase The active form of the enzyme is a dimer. The ACP & TE domains are not resolved in this crystal structure, probably because they are very flexible. The white and blue spheres indicate the active sites. Hollow spheres in the domain colors represent the length of phosphopantheteine, showing how closely ACP must approach each site during the catalytic cycle. Maier et al. Science 311: 1258 (2006) 2cf2.pdb One of the two monomeric subunits of mammalian fatty acid synthase β-ketoacyl-ACP reductase enoyl-ACP reductase hydroxyacyl-ACP dehydrase Linker and structural domains are shown in gray. The ACP & thioesterase domains are not resolved in the crystal structure. ACP β-ketoacylACP synthase Maier et al. Science 321: 1315 (2008). 2vz8.pdb malonyl/acetylCo A-ACP transferase Yeast fatty acid synthase contains two proteins, each of which has 4 different types of catalytic sites Six copies of each subunit pack together to form reaction chambers surrounding anchor points for the ACP domains. Lomakin et al. Cell 129: 319 (2007) Jenni et al. Science 316: 254 (2007) >70% of the proteins in the eukaryotic proteome have multiple domains. What are the potential advantages and disadvantages of fusing proteins to make a multifunctional enzyme? Are special mechanisms needed to prevent such proteins from misfolding? Differences between fatty acid synthesis and oxidation oxidation synthesis CO-S-ACP CO-S-CoA FAD ACP-SH NADP+ FADH2 NADPH CO-S-CoA H2O HO H CO-S-ACP mitochondrion H2O cytosol CO-S-CoA H OH NAD+ O NADP+ NADH NADPH CO-S-CoA O CO-S-ACP CO2 CoA-SH CH3-CO-S-CoA CO-S-CoA CO-S-ACP CO-S-ACP CO2CO-S-ACP Fatty acids with longer chains (C18:0 & C20:0) are synthesized from palmitoyl-CoA by addition of 2-carbon units from malonyl-CoA (“elongation”) CO-S-CoA palmitoyl-CoA (C16:0) - O C-CH CO-S-CoA 2 2 These reactions occur in mitochondria & the smooth ER. CoA-SH, CO2 NADPH NADP H2O NADPH NADP CO-S-CoA stearoyl-CoA (C18:0) Different enzymes are involved, and CoA is used in place of ACP, but the reactions are otherwise formally the same as in synthesis of palmitate. Citrate carries 2-carbon units from mitochondria to the cytosol citrate synthase acetyl-CoA CO2- O=C-CO2 CH2 CO2 Cytosol CoA-SH CH3CO-S-CoA oxaloacetate Mitrochondrion - - CO2- CH2 CH2 HO-C-CO2- HO-C-CO2CH2 CO2citrate CH2 citrate citrate transporter CoA-SH + ATP CO2- citrate lyase ADP + Pi O=C-CO2 + - CH3CO-S-CoA CH2 CO2- Citrate Citrate lyase uses ATP to drive the breakdown of citrate to acetyl-CoA & oxaloacetate in the cytosol Integration of fatty acid synthesis with carbohydrate metabolism Mitochondrion Cytosol citrate CoA-SH pyruvate oxaloacetate ATP ADP + Pi CoA-SH acetyl-CoA ADP + Pi oxaloacetate malate dehydrogenase NADH NAD + malate pyruvate carboxylase pyruvate NADH NAD + malate malic enzyme ATP + CO2 NADPH ATP citrate lyase TCA cycle ox. ox. phos. citrate ATP acetyl-CoA amino acids fatty acids pyruvate NADP+ CO2CHOH CH2 CO2- NADPH + CO2 glucose Acetyl-CoA carboxylase (biotin carboxylase/transcarboxylase) is the main control point for fatty acid synthesis in animals the enzyme is regulated by allosteric effects, phosphorylation, and controls on expression of the gene citrate citrate lyase acetyl-CoA acetyl-CoA carboxylase malonyl-CoA X insulin stimulates dephosphorylation (activation) the phosphorylated enzyme is inactive acetyl-CoA -O--P carboxylase AMP- and cAMPdependent protein kinases AMP, glucagon, epinephrine, adiponectin and leptin stimulate phosphorylation (inactivation) palmitoyl-CoA X carnitine-acyltransferase I malonylCoA inhibits carnitine-acyltransferase I, blocking transport of palmitoylCoA into mitochondria for oxidation The active (unphosphorylated) form of acetylCoA carboxylase forms long filaments o 400 Å An imbalance between energy input and output can lead to obesity Food adipose tissue Work or Growth fatty acids & triacylglcerols ADP Obesity ATP Heat CO2 + H2O ~65% ~65% of the adult U.S. population are considered to be overweight (BMI* > 25); ~30% are obese (BMI > 30). Obesity raises the risk of heart disease, stroke, type2 diabetes and cancer, and has become a leading cause of global ill health.** Individual susceptibility to obesity is strongly influenced by heredity. *Body mass index = (weight in kg)/(height in m)2 = 703x(weight in pounds)/(height in inches)2. **P.G. Kopelman Nature 404: 63 (2000) The risk of type-2 diabetes increases with obesity BMI relative risk < 21 1 25 5 30 28 ≥ 35 93 G.A. Colditz et al. Arch. Int. Med. 122: 481 (1995). Fat in the abdominal region appears to have more bearing on health than subcutaneous fat. Leptin and adiponectin convey long-term signals of nutritional excess other parts of the brain sympathetic nervous system neuronal signals hypothalamus Increase catabolism & thermogenesis Increase blood pressure & heart rate (express gene for uncoupling protein) heart, muscle, liver leptin, fatty acids adipose tissue blood leptin, adiponectin Decrease fatty acid synthesis and increase catabolism (phosphorylate acetylCoA carboxylase and decrease expression of fatty-acid synthase gene) In obesity, leptin also decreases synthesis of insulin, which can lead to diabetes. Defects in leptin or its receptor can cause obesity weight 67 g weight 35 g These mice are the same age. Both are homozygous for a defective variant of leptin. The mouse on the right received daily injections of purified leptin; the mouse on the left was not treated. The name “leptin” comes from Greek leptos, meaning thin. But most obese humans do not have a deficiency in leptin. More than 20 other genes have been associated with obesity. Ghrelin and PYY convey short-term signals of hunger or satiety other parts of the brain Ghrelin concentrations in the blood change dramatically during the day, peaking just before each meal and then falling rapidly. PYY increases after a meal. You’re full! Stop eating! You’re hungry! Eat! hypothalamus ghrelin stomach PYY3-36 intestine It is very difficult to avoid regaining weight that is lost by dieting and exercise. For more about this, see J. Thaler et al. J. Clinical Invest. (2012). ...
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