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1、Chapter 21 Lipid Biosynthesis,1. Fatty acids; 2. Eicosanoids; 3. Triacylglycerols; 4. Membrane phospholipids; 5. Cholesterol, steroids, and isoprenoids;,1. Fatty acid synthesis takes a different pathway from its degradation,Occurs in the cytosol (chloroplasts in plants). Acetyl-CoA provides the firs
2、t two carbons, which is elongated by sequential addition of two-carbon units donated from malonyl-CoA. Intermediates are attached to the -SH groups of an acyl carrier protein (ACP). NADPH is the reductant. The enzymes are associated as a multi-enzyme complex or even being in one polypeptide chain in
3、 higher organisms (fatty acid synthase).,Elongation by the fatty acid synthase complex stops upon formation of palmitate (C16), further elongation and desaturation are carried out by other enzyme systems.,2. Malonyl-CoA is formed from acetyl-CoA and bicarbonate,Salih Wakil discovered that HCO3- is r
4、equired for fatty acid synthesis. Acetyl-CoA carboxylase (being trimeric in bacteria, monomeric in animals and both in plants) catalyzes this carboxylation reaction. The enzyme has three functional parts: a biotin carrier protein; an ATP-dependent biotin carboxylase; and a transcarboxylase. The enzy
5、me exemplifies a ping-pong reaction mechanism.,This irreversible reaction commits acetyl-CoA to fatty acid synthesis.,biotin carboxylase,Trans- carboxylase,Acetyl-CoA carboxylase catalyzes the two-step carboxylation reaction of acetyl-CoA in two active sites.,3. The acetyl and malony groups are firs
6、t transferred to two SH groups of the fatty acid synthase complex,The acetyl group of acetyl-CoA is first transferred to the SH group of a Cys residue on the b-ketoacyl-ACP synthase (KS) in a reaction catalyzed by acetyl-CoA-ACP transacetylase (AT). The malonyl group is transferred from malonyl-CoA
7、to the SH group of the 4-phosphopantetheine covalently attached to a Ser residue of the acyl carrier protein (ACP).,The acyl carrier protein (ACP) is very similar to CoA (thus can be regarded as “macro CoA”),4. Fatty acids are synthesized by a repeating four-step reaction sequence,In the condensatio
8、n reaction (step 1), catalyzed by b-ketoacyl-ACP synthase, the methylene group of malonyl-CoA (linked to ACP) undergoes a nucleophilic attack on the carbonyl carbon of the acetyl group linked to KS, forming the b-ketobutyryl-ACP with simultaneous elimination of CO2. the b-ketobutyryl-ACP is then red
9、uced to D-b-hydroxybutyryl-ACP (step 2), using NADPH and the b-ketobutyryl-ACP reductase (KR).,A water molecule is then removed from the b-hydroxybutyryl-ACP to produce trans-2-butenoyl-ACP in a reaction catalyzed by b-hydroxybutyryl-ACP dehydratase (step 3). A further reduction (step 4), also using
10、 NADPH, of the carbon-carbon double in trans-2-butenoyl-ACP, catalyzed by enoyl-ACP reductase produces a saturated acyl on ACP (butyryl-ACP). The butyryl group is then transferred to the Cys SH group of b-ketoacyl-ACP synthase for another round of four reactions, which will extend the chain by two m
11、ore carbons.,Seven rounds of the four-step lengthening reactions produces palmitoyl-ACP, which will be hydrolyzed to release a free palmitate. The flexible 4-phosphopantetheine group covalently attached to ACP is believed to act as a switch arm to move the intermediates from one active site to the n
12、ext on the enzyme complex (i.e., the substrates are channeled). A total of 7 ATP and 14 NADPH will be consumed for making one palmitate molecule.,5. The seven activities of fatty acid synthesis from different organisms have different level of integration,Each activity resides in a separate polypepti
13、de chain in bacteria and higher plants. The seven activities reside in two separate polypeptide chains, with the synthase present as dodecamers (a6b 6). The seven activities reside in one large polypeptide chain in vertebrates, with the synthase present as dimers.,The seven activities of fatty acid
14、synthase are integrated to different levels in different organisms.,6. Fatty acid synthesis occurs in cellular compartments having a high NADPH/NADP+ ratio,NAD and NADP have selected for functioning as electron carriers in oxidative catablism and reductive anabolism respectively. In the hepatocytes
15、and adipocytes, NADPH is mainly produced in the cytosol via the pentose phosphate pathway and by the malic enzyme. In photosynthetic plants, fatty acid synthesis occur in the chloroplast stroma, using NADPH made from photophosphorylation.,Malic enzyme,Pentose phosphate pathway,NADPH in the cytosol o
16、f animal cells is largely produced by the oxidative decarboxylation of malate and the pentose phosphate pathway,7. The acetyl groups of the mitochondrion are transported into the cytosol in the form of citrate,The acetyl-CoA molecules are made from glucose and amino acids in mitochondria. The are sh
17、uttled into the cytosol in the form of citrate via the citrate transporter of the inner membrane. Acetyl-CoA is regenerated by the action of ATP-dependent citrate lyase in the cytosol. Oxaloacetate is shuttled back into the mitochondria as malate or pyruvate.,8. The rate of fatty acid biosynthesis i
18、s controlled by acetyl-CoA carboxylase,Excess fuel is generally converted to fatty acids/triacylglycerol for longer term storage. Acetyl-CoA carboxylase, catalyzing the committing and rate-limiting step of fatty acid synthesis, is allosterically inhibited by palmitoyl-CoA and activated by citrate. G
19、lucagon and epinephrine triggers the phosphorylation and disassociation of the polymeric enzyme subunits, which inactivates the enzyme.,Citrate partially activate the phosphorylated acetyl-CoA carboxylase (similar to how AMP partially active the dephosphorylated glycogen phosphorylase). In plants, a
20、cetyl-CoA carboxylase is activated by a increase of Mg 2+ concentration and decrease of H+ concentration that accompany illumination. (Malonyl-CoA inhibits carnitine acyltransferase I),Dephosphorylated acetyl-CoA Carboxylase (active),Acetyl-CoA carboxylase is regulated by allosteric effectors and re
21、versible phosphorylation,Citrate partially activate the phosphorylated acetyl-CoA carboxylase,9. Palmitate can be further elongated and desaturated in smooth ER,Palmitoyl-CoA can be further elongated by the fatty acid elongation system present mainly in the smooth endoplasmic reticulum, with two-car
22、bon units also donated by malonyl-CoA. Palmitoyl-CoA and Stearoyl-CoA can be desaturated between C-9 and C-10 to produce palmitoleate, 16:1(9), and oleate, 18:1(9) respectively.,The double bonds are introduced by the catalysis of fatty acyl-CoA desaturase (a mixed-function oxidase), where both the f
23、atty acyl group and NADPH are oxidized by O2. The electrons of NADPH are transferred to O2 via Cyt b5 reductase and cytochrome b5. Further desaturation of oleate occur on phosphatidylcholine and is catalyzed by another desaturase, which is present only in plant cells. Linoleate and linolenate, neede
24、d to make other polyunsaturated fatty acids like arachidonate are essential fatty acids for mammals.,Palmitate is the Precursor for the biosynthesis of other fatty acids,Fatty acyl-CoA is desaturated (oxidized) by O2 and NADPH.,Oleate can be desaturated on Phosphatidylcholine (often attaching to C-2
25、) to form linoleate and linolenate,10. Eicosanoids are derived from arachidonate, 20:4 (5,8,11,14),The arachidonate (花生四烯酸) is first cleaved off from membrane phospholipids by phospolipase A2, in response to hormonal or other stimuli. Arachidonate is then converted to PGH2 by the catalysis of the bi
26、functional cyclooxygenase (COX): the cyclooxygenase activity converts arachidonate to PGG2; the peroxidase activity then converts PGG2 to PGH2. PGH2 is the immediate precursor of other prostaglandins and thromboxanes.,Aspirin (acetylsalicylate) irreversibly inhibits the cyclooxygenase by acetylating
27、 an active site Ser, thus blocking the synthesis of prostglandins and thromboxanes; Ibuprofen also inhibit the same enzyme. Arachidonate can also be modified by adding hydroperoxy groups at various positions to form various hydroperoxyeicosatetraenoates (HPETEs) in reactions catalyzed by various lip
28、ooxygenases with the incorporation of O2. The HPETEs will be further converted to leukotrienes (白细胞三烯).,Prostaglandins and thromboxanes are synthesized from arachidonate,Cyclooxygenase activity of COX,Peroxidase activity of COX,The dimeric bifunctional cyclooxygenase (COX-1),Heme for the peroxidase
29、active site,Tyr385, a key residue for the cyclooxygenase activity,flurbiprofen,Ser530,11. Newly synthesized fatty acids have mainly two alternative fates in cells,Fate I: be incorporated into triacylglycerols as a form to store metabolic energy in long terms. Fate II: be incorporated into membrane p
30、hospholipids (during rapid growth).,12. Phosphatidic acid is the common precursor for the syntheses of both triacylglycerols and glycerophospholipids,Phosphatidic acid (or diacylglycerol 3-phosphate) is made by transferring two acyl groups from two acyl-CoAs to L-glycerol 3-phosphate, which is deriv
31、ed from either glycerol or dihydroxyacetone phosphate. A phosphatidic acid is converted to a triacylglycerol via a dephosphorylation reaction (catalyzed by phosphatidic acid phosphatase) and a acyl transferring reaction.,Phosphatidic acid is derived from L-glycerol 3- phosphate and two acyl-CoAs.,Of
32、ten unsaturated,Often saturated,Phosphatidic acid phosphatase,Phosphatidic acid is the common precursor for both triacylglycerols and glycerophospholipids,13. Insulin stimulates conversion of dietary carbohydrates/proteins into fat,Diabetes patients due to lack of insulin would neither be able to us
33、e glucose properly, nor to synthesize fatty acids from carbohydrates and amino acids. They show increased rates of fatty acid oxidation and ketone body formation, thus losing weight.,14. Two strategies are taken for converting phosphatidic acid to glycerophospholipid,Eugene Kennedy revealed in 1960s
34、 that either the OH group of the diacylglycerol (strategy 1) or that of the polar head (strategy II) is first activated by attaching to cytidine nucleotide. The CMP moiety is displaced by the other OH group in a nucleophilic attack reaction to synthesize a glycerophospholipid. Both strategies are us
35、ed in eukaryotic cells, but only strategy I is use in bacterial cells.,Bacteria mainly use this strategy,Eukaryotic cells use both strategies (occurring on sER and inner membrane of mitochondria),phosphatase,decarboxylase,Phospholipid synthesis in E. coli employs CDP-diacylglcerol,15. Acidic (anioni
36、c) phospholipids in eukaryotic cells are synthesized using CDP-diacylglycerol,These include phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine. eukaryotic cardiolipin is synthesized from one phosphatidylglycerol and one CDP-diacylglycerol (from two phosphatidylglycerols in b
37、acteria).,4,5,16. Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) are often made from the salvage (reuse) pathway in mammals,Diet choline and ethanolamine are first converted to CDP-choline and CDP-ethanolamine after an initial phosphorylation step. The CMP moiety is then replaced by a
38、diacylglycerol, forming PC and PE. Phosphatidylserine (PS) is often made from PE by a head exchange reaction (reversible).,PC can be made from PE by three methylation reactions using S-adenosylmethionine (adoMet) in the liver cells. PS can also be converted to PE by a decarboxylation reaction.,(etha
39、nolamine),(Phosphoethanolamine),(CDP-ethanolamine),(Phosphatidylthanolamine),PC and PE are made from the salvage pathway in mammals,The synthesis of PE, PC, PS in eukaryotic cells.,17. The synthesis of ether lipids involves a displacement of fatty acyl by fatty alcohol step and a desaturation step,B
40、oth plasmalogen (缩醛磷脂) and platelet-activating factor are made using this pathway. The acyl group on 1-acyldihydroxyacetone 3-phosphate is replaced by a long chain alcohol group to form the ether linkage. The double bond in plasmalogen is introduced at the end by the catalysis of a mixed-funciton ox
41、idase.,Synthesis of the ether lipids (醚脂类）,1-alkylglycerol 3-phosphate,18. The sphingosine backbone of spingolipids is derived from palmitoyl-CoA and Ser,Palmitoyl-CoA condenses with serine (PLP is needed for decarboxylate serine) to form b-ketosphinganine, which is then reduced to sphinganine (二氢鞘氨
42、醇). Sphinganine is then acylated and desaturated to form ceramide (containing sphingosine). Addition of sugar(s) or phosphocholine heads leads to the synthesis of cerebroside, gangliosides, or sphingomyelin.,The ways for the membrane lipids (glycerolphospholipids and spingolipids) synthesized at smo
43、oth endoplasmic reticulum or inner membrane of Mitochondria to be transported to specific cellular locations are not well understood yet.,PLP,Spingolipid synthesis begins with the condensation between palmitoyl-CoA and Ser.,(not CDP-choline!),A glycolipid, not a phospholipid,19. Radioisotope tracer
44、experiments revealed that all the 27 carbons of cholesterol is derived from acetyl-CoA,The origin of the carbon atoms of cholesterol was deduced from tracer experiments where either with the methyl carbon or the carboxyl carbon in acetate is labeled with 14C (1940s). The pattern of labeling provided
45、 the blueprint for revealing the detail enzymatic steps for cholesterol biosynthesis occurring in mammals. The 30-carbon squalene (of six isoprene units) and later on mevalonate were found to be intermediates of cholesterol biosynthesis.,The biosynthetic pathway of cholesterol, being the most comple
46、x known, was elucidated mainly by Konrad Bloch and Feodor Lynen in the 1950s.,The carbon origins of cholesterol as revealed by radioisotope labeling studies.,20. The cholesterol biosynthesis pathway can be divided into four stages,Stage I: three acetyl-CoA molecules condense to form the 6-carbon mev
47、alonate (甲羟戊酸). Stage II: mevalonate is converted to activated 5-carbon isoprene (异戊二烯) units. Stage III: Six isoprene units condense to form the linear 30-carbon squalene(鲨烯). Stage IV: The linear squalene is cyclized to form a four-ring structure, which is eventually converted to the 27-carbon cho
48、lesterol through a series of complicated reactions.,(6C),(5C),(30C),(27C),(2C),Reactions assembling cholesterol from 18 molecules of acetyl-CoA can be divided into four stages.,21. Mevalonate commits the acetyl groups for cholesterol synthesis,One molecule of b-hydroxy-b-methylglutaryl-CoA (HMG-CoA)
49、 is formed from three acetyl-CoA molecules in the cytosol via the same reactions as occurring in mitochondria for ketone body formation. HMG-CoA reductase (an integrated membrane protein in the smooth ER) catalyzes the irreversible reduction of HMG-CoA (using two molecules of NADPH) to form mevalonate: committing the acetyl groups for cholesterol synthesis (thus being a major regulation step).,The irreversible committing step for cholesterol biosynthesis,HMG-CoA Reductase In cytosol,One mevalonate is synthesized from three acetyl-CoA molecules