2013 W. H. Freeman and Company. 21 Lipid Biosynthesis

Transcription

1 2013 W. H. Freeman and Company 21 Lipid Biosynthesis

2 CHAPTER 21 Lipid Biosynthesis Key topics: Biosynthesis of fatty acids and eicosanoids Assembly of fatty acids and glycerol into triacylglycerols Biosynthesis of cholesterol

3 Lipids fulfill a variety of biological functions Energy storage Constituents of membranes Anchors for membrane proteins Cofactors for enzymes Signaling molecules Pigments Detergents Transporters Antioxidants

4 Catabolism and anabolism of fatty acids proceed via different pathways Catabolism of fatty acids produces acetyl CoA produces reducing power (NADH) takes place in the mitochondria Anabolism of fatty acids requires acetyl CoA and malonyl CoA requires reducing power from NADPH takes place in cytosol in animals, chloroplast in plants

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7 Lipid Biosynthesis Lipid의역할 1저장E 2 세포막의구성성분 3 색소, 보조인자 (Vit.E), 계면활성제 (bile acid), carrier(dolichol), Vt.D 유도체, 성 Hormone 세포내 / 외 messenger (Eicosanoid/phosphatidyl inositol 유도체 ), 막단백 anchor( 닻 ) 지방질의생합성반응 E 투입 (ATP), NADPH( 전자운반체 )

8 Fatty acid 의생합성 Malonyl-CoA 의 active form : 간, 뇌, 신장, 유선, 지방조직의 cytosol 1) Malonyl-CoA 의합성 O O C-CH 2 -C - O S-CoA (1) Acetyl-CoA carboxylase (ACC) 1 Biotin carrier protein 2 Biotin carboxylase: ATP-dependent 반응 CO 2 를 biotin 에부착시켜 CO 2 를활성화 3 Transcarboxylase: 활성화된 CO 2 를 acetyl-coa 에전하여 malonyl-coa 생성 (2) Malonyl-CoA 합성 ATP ADP+P i Biotin carrier O acetyl-coa protein + HCO - 3 biotin N C O - malonyl-coa transcarboxylase carboxylase 일시적 CO 2 운반체

9 2) Lipid 생성효소 (1) Fatty acid synthase(fas) complex β-ketoacyl-acp synthase (KS), malonyl/acetyl-coa ACP transferase (MAT), β-hydroxyacyl-acp dehydratase (DH), enoyl- ACP reductase (ER), and β-ketoacyl-acp reductase (KR), ACP is the acyl carrier protein, thioesterase(te) COO - 1ACP thioester 결합된 acyl 기운반 최초의 acyl 기 -ketoacyl -ACP synthase (KS) CH 3 C=O S CO 2 CH 2 C=O Malonyl gr S Acyl Carrier Protein (ACP) 2TE 3KS 4MAT 5KR 6DH 합성된 acyl 기유리 Acyl 기와 malonyl 기축합 Malonyl, acetyl 기전달 -keto 기 -hydroxyl 기 -hydroxyacyl-acp 의 H 2 O 제거 생성 7ER = 환원, 포화 acyl-acp 생성 (2) 역할 Acetyl 기와 malonyl 기를받아들여지방산사슬을늘리기위한반응촉매

10 FIGURE 21-4 The overall process of palmitate synthesis.

11 Overview of Fatty Acid Synthesis Fatty acids are built in several passes, processing one acetate unit at a time. The acetate is coming from activated malonate in the form of malonyl CoA. Each pass involves reduction of a carbonyl carbon to a methylene carbon.

12 The Acetyl CoA Carboxylase Reaction ATP

13 Malonyl CoA is formed from acetyl CoA and bicarbonate Rx carboxylates acetyl CoA Catalyzed by acetyl CoA carboxylase Enz has three subunits: One unit has Biotin covalently linked to Lys Biotin carries CO 2 In animals, all three subunits are on one polypeptide chain HCO 3 (bicarbonate) is the source of CO 2

14 Biotin carries CO 2 Two step rx similar to carboxylations catalyzed by pyruvate carboxylase and propionyl CoA carboxylase CO 2 binds to biotin - CO 2 is activated by attachment to N in ring of biotin - Rx with ATP produces carbamoyl phosphate

15 Synthesis of fatty acids is catalyzed by fatty acid synthase (FAS) Catalyzes a repeating four step sequence that elongates the fatty acyl chain by two carbons at each step See Fig Uses NADPH as as the electron donor Uses two enzyme bound SH groups as activating groups FAS I in vertebrates and fungi FAS II in plants and bacteria

16 FAS I vs. FAS II FAS I Single polypeptide chain in vertebrates Leads to single product: palmitate 16:0 C 15 and C 16 are from the acetyl CoA used to prime the Rx FAS II Made of separate, diffusible enzymes Makes many products (saturated, unsaturated, branched, many lengths, etc.) Mostly in plants and bacteria

17 Fatty Acid Synthase Type I Systems

18 Fatty Acid Synthesis Overall goal: attach two C acetate unit from malonyl CoA to a growing chain and then reduce it Reaction involves cycles of four enzyme catalyzed steps Condensation of the growing chain with activated acetate Reduction of carbonyl to hydroxyl Dehydration of alcohol to trans alkene Reduction of alkene to alkane The growing chain is initially attached to the enzyme via a thioester linkage During condensation, the growing chain is transferred to the acyl carrier protein After the second reduction step, the elongated chain is transferred back to fatty acid synthase

19 The General Four Step Fatty Acid Synthase I Reaction in Mammals (1) Prep: Malonyl CoA and acetyl CoA (or longer fatty acyl chain) are bound to FAS I bind via thioester terminus or a Cys of the FAS activates the acyl group Step 1: Condensation rx attaches two C from malonyl CoA to the attached acetyl CoA (or longer fatty acyl chain) also releases CO 2 from malonyl CoA the decarboxylation facilitates the rx creates keto intermediate

20 Step 1 of FAS I: Elongation(condensation)

21 The General Four Step Fatty Acid Synthase I Reaction in Mammals (2) Step 2: 1 st Reduction: NADPH reduces the keto intermediate to an alcohol Step 3: Dehydration: OH group from C 2 and H from neighboring CH 2 are eliminated, creating double bond (trans alkene) Step 4: 2 nd Reduction: NADPH reduces double bond to yield saturated alkane

22 Steps 2 4 of the FAS I rx

23 Overall Palmitate Synthesis

24 Acyl Carrier Protein (ACP) serves as a shuttle in fatty acid synthesis Contains a covalently attached prosthetic group 4 phosphopantetheine Flexible arm to tether acyl chain while carrying intermediates from one enzyme subunit to the next Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase Shuttles the growing chain from one active site to another during the fourstep reaction

25 Charging ACP and FAS I with acyl groups activates them Two thiols must be charged with the correct acyl groups before condensation rx can begin Thiol from 4 phosphopantethine in ACP Thiol from Cys in fatty acid synthase 1) Acetyl group of acetyl CoA is transferred to ACP Catalyzed by malonyl/aceyl CoA transferase (MAT) ACP passes this acetate to the Cys of the ketoacyl ACP synthase (KS) domain of FAS 1 2) ACP SH group is re charged with malonyl from malonyl CoA

26 MAT β-ketoacyl-acp synthase (KS), malonyl/acetyl- CoA ACP transferase (MAT), β-hydroxyacyl-acp dehydratase (DH), enoyl-acp reductase (ER), and β-ketoacyl-acp reductase (KR). ACP is the acyl carrier protein, thioesterase(te) MAT Charging, Activation with ACP, and the Four Step Sequence of Mammalian Fatty Acid Synthesis

27 Fatty Acid Synthesis in Detail: Step 1:Condensation Activated acetyl and malonyl groups form acetoacetyl ACP and CO 2 Claisen condensation rx Catalyzed by ketoacyl ACP synthase Coupling condensation to decarboxylation of malonyl CoA makes the rx energetically favorable

28 Fatty Acid Synthesis in Detail: Step 2: Reduction Carbonyl at C 3 is reduced to form D hydroxybutyryl ACP NADPH is e donor Catalyzed by ketoacyl ACP reductase (KR)

29 Fatty Acid Synthesis in Detail: Step 3: Dehydration OH and H removed from C 2 and C 3 of hydroxybutyryl ACP to form trans 2 butenoyl ACP Catalyzed by hydroxyacyl ACP dehydratase (DH)

30 Fatty Acid Synthesis in Detail: Step 4: Reduction (2 nd ) NADPH is the electron donor to reduce double bond of trans 2 butenoyl ACP to form butyryl ACP Catalyzed by enoyl ACP reductase (ER)

31 Enzymes in Fatty Acid Synthase Condensation with acetate ketoacyl ACP synthase (KS) Reduction of carbonyl to hydroxyl ketoacyl ACP reductase (KR) Dehydration of alcohol to alkene hydroxyacyl ACP dehydratase (DH) Reduction of alkene to alkane enoyl ACP reductase (ER) Chain transfer/charging Malonyl/acetyl CoA ACP transferase(mat) Release of acyl group Thiosterase

32 The Transferase and FAS rx s are repeated in new rounds Product of first round is butyryl ACP (bound to phosphopantetheine SH group of ACP) Butyrul gp is transferred to the Cys of ketoacyl ACP synthase In the first round, acetyl CoA was bound here New malonyl CoA binds to ACP After new round of four steps, six C product is made (bound to ACP)

33 Beginning of the Second Round of Fatty Acid Synthesis

34 Stoichiometry of Synthesis of Palmitate (16:0) 1) 7 acetyl CoAs are carboxylated to make 7 malonyl CoAs using ATP 7 AcCoA + 7 CO ATP 7 malcoa + 7 ADP + 7 P i 2) Seven cycles of condensation, reduction, dehydration and reduction using NADPH to reduce the keto group and trans double bond AcCoA + 7 malcoa + 14 NADPH + 14 H + Palmitate + 7 CO CoA + 14 NADP H 2 O Note: Eukaryotes have one additional energy cost. (Next slide)

35 Acetyl CoA is transported into the cytosol for fatty acid synthesis In nonphotosynthetic eukaryotes Acetyl CoA is made in the mitochondria But fatty acids are made in the cytosol So Acetyl CoA is transported into the cytosol with a cost of 2 ATPs Therefore, cost of FA synthesis is 3 ATPs per 2 C unit

36 Fatty acid synthesis occurs in cell compartments where NADPH levels are high Cytosol for animals, yeast Chloroplast for plants Sources of NADPH: In adipocytes: pentose phosphate pathway and malic enzyme (Fig. 21 9) NADPH made as malate converts to pyruvate + CO 2 In hepatocytes and mammary gland: pentose phosphate pathway NADPH made as glucose 6 phosphate converts to ribulose 6 phosphate In plants: photosynthesis

37 Pathways for NADPH Production

38 Acetyl CoA, generated in the mitochondria, is shuttled to the cytosol as citrate In most eukaryotes, the acetyl CoA for lipid synthesis is made in the mitochondria But lipid synthesis occurs in the cytosol And there is no way for acetyl CoA to cross mitochondrial inner membrane to the cytosol So acetyl CoA is converted to citrate Acetyl CoA + oxaloacetate citrate Same rx as occurs in CAC Catalyzed by citrate synthase Citrate passes through citrate transporter

39 Shuttle for Transfer of Acetyl Groups from Mitochondria to Cytosol

40 Citrate is cleaved to regenerate acetyl CoA Citrate (now in cytosol) is cleaved by citrate lyase Regenerates acetyl CoA and oxaloacetate Rx requires ATP Acetyl CoA can now be used for lipid synthesis What happens to the oxaloacetate because there is no oxaloacetate transporter either? (Next slide)

41 Oxaloacetate cyt is converted to malate Malate dehydrogenase in cytosol reduces oxaloacetate to malate Two potential fates for malate: Can be converted to NADPH cyt and pyruvate cyt via the malic enzyme NADPH used for lipid synthesis Pyruvate cyt sent back to mt via pyruvate transporter Converted back to oxaloacetate mt by pyruvate carboxylase, reqires ATP Can be transported back to mt via malate ketoglutarate transporter Malate mt is reoxidized to oxaloacetate mt

42 Fatty acid synthesis is tightly regulated via ACC Acetyl CoA carboxylase (ACC) catalyzes the ratelimiting step ACC is feedback inhibited by palmitoyl CoA ACC is activated by citrate Remember citrate is made from acetyl CoA mt Citrate signals excess energy to be converted to fat When [acetyl CoA] mt, converted to citrate citrate exported to cytosol

43 Regulation of Fatty Acid Synthesis in Vertebrates

44 Importance of Citrate to Regulation of Fatty Acid Synthesis In animals, citrate stimulates fatty acid synthesis! Precursor for acetyl CoA Sent to cytosol and cleaved to become AcCoA when AcCoA and ATP (energy excess) Allosteric activator of ACC Inhibitor of PFK 1 Reduces glycolysis

45 ACC is also regulated by covalent modification Inhibited when energy is needed Glucagon and epinephrine: reduce sensitivity of citrate activation lead to phosphorylation and inactivation of ACC ACC is active as dephosphorylated monomers When dephosphorylated, ACC polymerizes into long inactive filaments Phosphorylation reverses the polymerization

46 Regulation of Fatty Acid Synthesis and Breakdown

47 MAT β-ketoacyl-acp synthase (KS), malonyl/acetyl- CoA ACP transferase (MAT), β-hydroxyacyl-acp dehydratase (DH), enoyl-acp reductase (ER), and β-ketoacyl-acp reductase (KR). ACP is the acyl carrier protein, thioesterase(te) MAT Charging, Activation with ACP, and the Four Step Sequence of Mammalian Fatty Acid Synthesis

48 FIGURE Routes of synthesis of other fatty acids.

49 3) 다양한지방산생합성의조절 (1) Palmitate 를전구체로하는생합성 ( unsaturation, elongation) arachidonate Palmitate 16 : 0 (2) 조절 palmitoleic acid 16:1( 9 ) stearate 18:0 길어진포화지방산 oleic acid 18:1( 9 ) C CH3: = 불가 linoleic acid 18:2( 9,12 ) eicosatriene -linolenic acid * * -linolenic acid 18:3( 9,12,15 ) EPA citrate 분해효소 +insulin Acetyl-CoA carboxylase Citrate acetyl-coa malonyl-coa palmitoyl-coa -glucagon, epinephrine +citrate, insulin -palmitoyl-coa

50 Additional Modes of Regulation in Fatty Acid Synthesis Changes in gene expression SREBP : 합성 ChREBP Example: Fatty acids (and eicosanoids) bind to transcription factors called Peroxisome Proliferator Activated Receptors (PPARS): 분해 Reciprocal regulation Malonyl CoA inhibits fatty acid import into mt One of many ways to ensure that fat synthesis and oxidation don t occur simultaneously

51 Palmitate can be lengthened to longer chain fatty acids Elongation systems in the endoplasmic reticulum and mitochondria create longer fatty acids As in palmitate synthesis, each step adds units of 2 C Stearate (18:0) is the most common product

52 Palmitate and stearate can be desaturated Palmitate(16:0) palmitoleate(16:1; 9 ) Stearate (18:0) oleate (18;1; 9 ) Catalyzed by fatty acyl CoA desaturase in animals Also known as the fatty acid desaturases Requires NADPH; enzyme uses cytochrome b 5 and cytochrome b 5 reductase See Fig Note that this is a 9 desaturase! It reduces the bond between C 9 and C 10.

53 Desaturation of a Fatty Acid by Fatty Acyl CoA Desaturase SCD1: stearoyl-coa desaturase, 비만과밀접

54 Vertebrate fatty acyl desaturase is a nonheme, iron containing, mixed function oxidase O 2 accepts four electrons from two substrates Two electrons come from saturated fatty acid Two electrons come from ferrous state of cytochrome b 5

55 Plants can desaturate positions beyond C 9 Humans have 4, 5, 6, and 9 desaturases but cannot desaturate beyond 9 Plants can produce: linoleate 18:2( 9,12 ) linolenate 18:3 ( 9,12,15 ) These fatty acids are essential to humans Polyunsaturated fatty acids (PUFAs) help control membrane fluidity PUFAs are precursors to eicosanoids

56 Plant desaturases act on fatty acids in a phospholipid Unlike mammal desaturases, plant desaturases do not oxidize free fatty acids They oxidize fatty acids that are bound to glycerol in phosphatidylcholine See Fig Free desaturated fatty acids can then be hydrolyzed from the glycerol backbone

57 Desaturases Operating on Fatty Acids in Phosphatidyl Choline

58 Oxidases, Monooxygenases, and Dioxygenases Many enzymes use oxygen as an e acceptor, but not all of them incorporate oxygen into the product. Oxidases do not incorporate oxygen into the product Oxygen atoms usually end up in H 2 O 2 Oxygenases do incorporate oxygen into the product Monooxygenases incorporate one of the oxygen atoms into the product Dioxygenases incorporate both oxygen atoms into the product

59 Monooxygenases incorporate one oxygen into the product AH + BH 2 + O O A OH + B + H 2 O Product is often hydroxylated, so also called hydroxylases or mixed function oxygenases Example: Phenylanine hydroxylase hydroxylates phenylalanine to form tyrosine Deficiency causes phenylketonuria

60 Cytochrome P450s are monooxygenases Important in drug metabolism Hydroxylate nonpolar molecules usually inactivating them and making them more H 2 O solubile for excretion If two drugs (or alcohol and a drug) use the same P450, they will compete, and levels of the drug or alcohol will not be cleared as quickly Can be deadly