Bài giảng Biochemistry 2/e - Chapter 25: Lipid Biosynthesis

Chapter 25Lipid Biosynthesisto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline25.1 Fatty Acid Biosynthesis & Degradation25.2 Biosynthesis of Complex Lipids25.3 Eicosanoid Biosynthesis and Function25.4 Cholesterol Biosynthesis25.5 Transport via Lipoprotein Complexes25.6 Biosynthesis of Bile Acids25.7 Synthesis and Metabolism of Steroids Fatty Acid PathwaysThe Biosynthesis and Degradation Pathways are DifferentAs in cases of glycolysis/gluconeogenesis and glycogen synthesis/breakdown, fatty acid synthesis and degradation go by different routes There are four major differences between fatty acid breakdown and biosynthesis The DifferencesBetween fatty acid biosynthesis and breakdown Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as compared to -SH groups of CoASynthesis in cytosol; breakdown in mitochondriaEnzymes of synthesis are one polypeptideBiosynthesis uses NADPH/NADP+; breakdown uses NADH/NAD+Activation by Malonyl-CoAAcetate Units are Activated for Transfer in Fatty Acid Synthesis by Malonyl-CoAFatty acids are built from 2-C units - acetyl-CoAAcetate units are activated for transfer by conversion to malonyl-CoADecarboxylation of malonyl-CoA and reducing power of NADPH drive chain growthChain grows to 16-carbonsOther enzymes add double bonds and more CsChallenge: Ac-CoA in CytosolWhat are the sources?Amino acid degradation produces cytosolic acetyl-CoAFA oxidation produces mitochondrial acetyl-CoAGlycolysis yields cytosolic pyruvate which is converted to acetyl-CoA in mitochondriaCitrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents for fatty acid synthesisAcetyl-CoA CarboxylaseThe "ACC enzyme" commits acetate to fatty acid synthesisCarboxylation of acetyl-CoA to form malonyl-CoA is the irreversible, committed step in fatty acid biosynthesisACC uses bicarbonate and ATP (AND biotin!)E.coli enzyme has three subunitsAnimal enzyme is one polypeptide with all three functions - biotin carboxyl carrier, biotin carboxylase and transcarboxylaseAcetyl-CoA Carboxylase IIACC forms long, active filamentous polymers from inactive protomers As a committed step, ACC is carefully regulatedPalmitoyl-CoA (product) favors monomersCitrate favors the active polymeric formPhosphorylation modulates citrate activation and palmitoyl-CoA inhibitionThe Effect of PhosphorylationUnphosphorylated E has low Km for citrate and is active at low citrateUnphosphorylated E has high Ki for palm-CoA and needs high palm-CoA to inhibitPhosphorylated E has high Km for citrate and needs high citrate to activatePhosphorylated E has low Ki for palm-CoA and is inhibited at low palm-CoAThe Acyl Carrier ProteinCarrier of intermediates in fatty acid synthesisDiscovered by P. Roy Vagelos - a 77 residue protein in E.coli - with a phosphopantetheineIn terms of function, it’s a large CoASee Figure 25.6 to compare ACP and CoAFatty Acid Synthesis in Bacteria and PlantsSeparate enzymes in a complexSee Figure 25.7Pathway initiated by formation of acetyl-ACP and malonyl-ACP by transacylasesDecarboxylation drives the condensation of acetyl-CoA and malonyl-CoAOther three steps are VERY familiar!Only differences: D configuration and NADPHCheck equations on page 811!Fatty Acid Synthesis in AnimalsFatty Acid Synthase - a multienzyme complexDimer of 250 kD multifunctional polypeptidesNote the roles of active site serines on AT & MTStudy the mechanism in Figure 25.11 - note the roles of ACP and KSaseSteps 3-6 repeat to elongate the chainFurther Processing of FAsAdditional elongation - in mitochondria and ERIntroduction of cis double bonds - do you need O2 or not?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 O2 to do itPolyunsaturated FAs - plants vs animals...Regulation of FA SynthesisAllosteric modifiers, phosphorylation and hormonesMalonyl-CoA blocks the carnitine acyltransferase and thus inhibits beta-oxidationCitrate activates acetyl-CoA carboxylaseFatty acyl-CoAs inhibit acetyl-CoA carboxylaseHormones regulate ACCGlucagon activates lipases/inhibits ACCInsulin inhibits lipases/activates ACCBiosynthesis of Complex LipidsSynthetic pathways depend on organismSphingolipids and triacylglycerols only made in eukaryotesPE accounts for 75% of PLs in E.coliNo PC, PI, sphingolipids, cholesterol in E.coliBut some bacteria do produce PCGlycerolipid BiosynthesisCTP drives formation of CDP complexes Phosphatidic acid is the precursor for all other glycerolipids in eukaryotesSee Figure 25.18PA is made either into DAG or CDP-DAGNote the roles of CDP-choline and CDP-ethanolamine in synthesis of PC and PE in Figure 25.19Note exchange of ethanolamine for serine (25.21)Other PLs from CDP-DAGFigure 25.22CDP-diacylglycerol is used in eukaryotes to produce:PI in one stepPG in two stepsCardiolipin in three stepsPlasmalogen BiosynthesisDihydroxyacetone phosphate is the precursorAcylation activates and an exchange reaction produces the ether linkageKetone reduction is followed by acylationCDP-ethanolamine delivers the headgroupA desaturase produces the double bond in the alkyl chainSphingolipid BiosynthesisHigh levels made in neural tissueInitial reaction is a condensation of serine and palmitoyl-CoA3-ketosphinganine synthase is PLP-dependentKetone is reduced with help of NADPHAcylation is followed by double bond formationSee Figure 25.25Resulting ceramide is precursor for other sphingolipidsEicosanoid BiosynthesisPLA2 releases arachidonic acid - a precursor of eicosanoidsEicosanoids are local hormonesThe endoperoxide synthase oxidizes and cyclizesTissue injury and inflammation triggers arachidonate release and eicosanoid synthesisEicosanoid BiosynthesisAspirin and other nonsteroid anti-inflammatory agents inhibit the cyclooxygenaseAspirin covalentlyOthers noncovalentlyCholesterol BiosynthesisOccurs primarily in the liverBiosynthesis begins in the cytosol with the synthesis of mevalonate from acetyl-CoAFirst step is a thiolase reactionSecond step makes HMG-CoAThird step - HMG-CoA reductase - is the rate-limiting step in cholesterol biosynthesisHMG-CoA reductase is site of action of cholesterol-lowering drugsRegulation of HMG-CoA ReductaseAs rate-limiting step, it is the principal site of regulation in cholesterol synthesis1) Phosphorylation by cAMP-dependent kinases inactivates the reductase2) Degradation of HMG-CoA reductase - half-life is 3 hrs and depends on cholesterol level3) Gene expression (mRNA production) is controlled by cholesterol levelsThe thiolase brainteaser...An important puzzleIf 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 synthesisSqualene from MevalonateDriven by ATP hydrolysis, decarboxylation and PPi hydrolysisSix-carbon mevalonate makes five carbon isopentenyl PPi and dimethylallyl PPiCondensation of 3 of these yields farnesyl PPiTwo farnesyl PPi s link to form squaleneBloch and Langdon were first to show that squalene is derived from acetate units and that cholesterol is derived from squaleneCholesterol from SqualeneAt the endoplasmic reticulum membraneSqualene monooxygenase converts squalene to squalene-2,3-epoxideA cyclase converts the epoxide to lanosterolThough lanosterol looks like cholesterol, 20 more steps are required to form cholesterol!All at/in the endoplasmic reticulum membraneInhibiting Cholesterol SynthesisMerck and the Lovastatin story...HMG-CoA reductase is the key - the rate-limiting step in cholesterol biosynthesisLovastatin (mevinolin) blocks HMG-CoA reductase and prevents synthesis of cholesterolLovastatin is an (inactive) lactoneIn the body, the lactone is hydrolyzed to mevinolinic acid, a competitive (TSA!) inhibitor of the reductase, Ki = 0.6 nM!Lipid Transport & LipoproteinsLipoproteins are the carriers of most lipids in the body Lipoprotein - a cluster of lipids, often with a monolayer membrane, together with an apolipoproteinSee Table 25.1 on lipoproteinsHDL, VLDL assemble in the ER of liver cellsChylomicrons form in the intestinesLDL not made directly, but evolves from VLDLLipoproteinsThe division of laborChylomicrons' main task is to carry triglyceridesLDLs are main carriers of cholesterol and cholesterol estersRelative amounts of HDL and LDL affect disposition of cholesterol and formation of arterial plaquesThe cholesterol/HDL ratio is key: greater than 4.5 is a risk factor for heart diseaseTypical values for HDL, LDLfor males, females 15-29Cholesterol: females - 157-167, males - 150-174HDL: females - 52-55, males 45LDL: females - 100-106, males 97-116However, with age, total cholesterol rises,and HDLs may fall, so exercise and diet become keysRegular, vigorous exercise raises HDLs and a low fat diet that avoids red meat reduces serum cholesterol levelsLipoproteins in CirculationProgressive degradation by lipasesMostly in the capillaries of muscle and adipose cells, lipoprotein lipases hydrolyze triglycerides from lipoproteins, making the lipoproteins smaller and raising their densityThus chylomicrons and VLDLs are progressively converted to IDL and then LDL, which either return to the liver for reprocessing or are redirected to adipose tissues and adrenal glandsThe LDL ReceptorA complex plasma membrane proteinLDL binding domain on N-terminusN-linked and O-linked oligosaccharide domainsA single TMSA cytosolic domain essential to aggregation of receptors in the membrane during endocytosisDysfunctions in or absence of LDL receptors lead to familial hypercholesterolemiaBiosynthesis of Bile AcidsCarboxylic acid derivatives of cholesterolEssential for the digestion of food, especially for solubilization of ingested fats Synthesized from cholesterolCholic acid conjugates with taurine and glycine to form taurocholic and glycocholic acidsFirst step is oxidation of cholesterol by a mixed-function oxidaseSteroid Hormone SynthesisDesmolase (in mitochondria) forms pregnenolone, precursor to all othersPregnenolone migrates from mitochondria to ER where progesterone is formedProgesterone is a branch point - it produces sex steroids (testosterone and estradiol), and corticosteroids (cortisol and aldosterone) Anabolic steroids are illegal and dangerousRecall the Ben Johnson story....