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1、Lipid Biosynthesis I Biosynthesis of fatty acids and eicosanoids Biosynthesis of other lipidsa. Triacylglycerolsb. Membrane phopholipidsc. Cholesterol, steroids and isoprenoidscarbohydratepyruvatepyruvateKetonebodiesAcetyl-CoAAcetyl-CoAketogenesis -oxidationDifference FA synthesis FA degradationSubc
2、ellular location cytosol mitochondrial matrix Carrier acyl carrier protein (ACP) Coenzyme A (CoA)Enzyme organization all activities on a single separate enzymes or polypeptide chain (mammals) multifunctional redox NADPH as reductant NAD+ and FAD as oxidantsBuilding block malonyl-CoA acetyl-CoA Fatty
3、 acid synthesis is not simply a reversal of the degradation pathway. Fatty acid synthesis and degradation pathways exemplify the principle that synthetic and degradation pathways are almost always distinct.Biosynthesis of fatty acidsOccurs through the condensation of C2 units;Occurs largely in adipo
4、cytes and liver3. Loading step: transfer of acetyl-CoA and malonyl-CoA to form acetyl-ACP and malonly-ACP Successive rounds of condensation, reduction, dehydration and reduction1.Acetyl-CoA is required to be transported from mitochondrial matrix to cytosol;2. Malonyl-CoA is formed from carboxylation
5、 of acetyl-CoAFatty acid synthaseBiosynthesis of fatty acid Acyl Carrier Protein（ACP）3,5-ADP Similarities between ACP and CoAMitochondrialmatrixCytosolshuttle for transfer ofacetyl groups fromMit. to the cytosolCytosol of heptocytes: NADPH/NADP+ ratio: highDifferences between NADPH and NADH NADH is
6、found in mitochondria, NADPH is not. NADH is involved in catabolic reactions; NADPH is involved in ANABOLIC reactions.Biosynthesis of biomoleculesNADH/NAD+ ratio: lowCatabolism of glucose The accumulation of precursors for fatty acid synthesis is a wonderful example of the coordinated use of multipl
7、e processes to fulfill a biochemical need. The citric acid cycle, subcellular compartmentalization, and the pentose phosphatepathway provide the carbon atoms and reducing power, whereas glycolysis and oxidative phosphorylation provide the ATP to meet the needs for fatty acid synthesis.Shuttle for tr
8、ansfer of acetyl-CoA from mitochondrial matrix to the cytosol1. The mitochondrial membranes are not permeable to acetyl-CoA; 2. It is shuttled out of the mitochondria in the form of citrate;3. Acetyl-CoA is regenerated by the action of ATP-citrate lyaseCitrate + ATP + CoA + H2O acetyl-CoA + ADP + Pi
9、 + oxaloacetateAcetyl CoA and oxaloacetate are transferred from mitochondria to the cytosol at the expense of the hydrolysis of one molecule of ATP. NADPH in adipocyte cytosol is largely generated from the oxidative decarboxylation of malate (catalyzed by malic enzyme, a reaction of acetyl shuttling
10、); NADPH in hepatocytes and mammary glands is supplied primarily from the pentose phosphate pathway; NADPH in chloroplast stroma is mainly produced by the light reactions.Sources of NADPH for fatty acid synthesis: Production of NADPHNADPH is produced in chloroplasts by the light reaction of photosyn
11、thesisIn plants, fatty acid occurs in chloroplast stroma, but not in cytosol.Formation of malonyl-CoA by acetyl-CoA Carboxylase 1. In bacteria，Acetyl-CoA carboxylase has three separatepolypeptide subunits, biotin carboxylase, biotin carrier protein, and transcarboxylase;2. In animal cells， All three
12、 activities of acetyl-CoA carboxylase are part of a single multifunctional polypeptide;Acetyl-CoA CarboxylaseThe reactions occur at two active sites, via a Ping-Pong mechanismAcetyl-CoA CarboxylaseAcetyl-CoA CarboxylaseMalonyl-CoA-ACPtransacylaseAcetyl-CoA-ACPtransacylaseLoading step: transfer of ac
13、etyl-CoA and malonyl-CoA to form acetyl-ACP and malonly-ACPSequence of events during synthesis of a fatty acidLoading stepLoading step-Ketoacyl-ACP synthase-Ketoacyl-ACP synthase: Acetyl-ACP + malonyl-ACP acetoacetyl-ACP + ACP + CO2 Why is the four-carbon unit not formed from 2 two-carbon units ? Th
14、e equilibrium is favorable if malonyl ACP is a reactant because its decarboxylation contributes a substantial decrease in free energy. ATP drives the condensation reaction, though ATP does not directly participate in the condensation reaction. Rather, ATP is used to carboxylate acetyl-CoA to malonyl
15、-CoA. The free energy stored in malonyl-CoA is released in the decarboxylation accompanying the formation of acetoacetyl ACP.Similar mechanism： Pyruvate PEP（gluconeogenesis）-Ketoacyl-ACPreductase-Hydroxyacyl-ACPdehydrataseEnoyl-ACP reducatse:inhibited by triclosan(a broad-spectrum antibacterial agen
16、t)Beginning of the second round of the fatty acid synthesis cycleThe overall process of palmitate synthesisMaier, T., Jenni, S. & Ban, N. (2006) Architecture of mammalian fatty acid synthase at 4.5 A resolution. Science 311, 1258-1262Jenni, S., Leibundgut, M., Maier, T., Ban, N. (2006) Architecture
17、of a fungal fatty acid synthase at 5 A resolution. Science 311, 1263-1267. Fatty acid synthase type I : Mammalian systemAll of the active sites in the mammalian system are located in different domains within a single large polypeptide chain.Dimer (2) (Mr 240,000)Fatty acid synthase type I : Fungal S
18、ystemDodecamers (66)Dome: trimers of subunits Wheel: six of subunits Science 311, 1258-1262FAS I (mammalian and fungal cells): Fatty acid synthesis leads to a single product, andno intermediates are released. FAS II (bacteria, plant cells and vertebrate mitochondria): FAS II is a dissociated system,
19、 generates a variety ofproducts. The flexible phosphopantetheinyl unit (20- maximal length ) of ACP carries substrate from one active site to another :The enzyme subunits do not need to undergo large structural rearrangements to interact with the substrate. Instead, the substrate is on a long, flexi
20、ble arm that can reach each of the numerous active sites. The organization of the fatty acid synthases of higher organisms enhances the efficiency of the overall process because intermediates are directly transferred from one active site to the next (substrate channeling). Fatty acid synthase inhibi
21、tors may be useful drugs:- Antitumor- AntiobesityThe overall process:8 Acetyl-CoA + 7ATP + 14NADPH + 14H+palmitate + 14 NADP+ + 8CoA + 6H2O + 7ADP + 7Pi1.Seven cycles of condensation and reduction :1Acetyl-CoA + 7 malonyl-CoA + 14NADPH + 14H+ palmitate + 7CO2 + 14 NADP+ + 8CoA + 6H2O2. Formation of
22、seven malonyl-CoA molecules: 7 Acetyl-CoA + 7CO2 + 7ATP 7 malonyl-CoA + 7ADP + 7Pi 3. Palmitate-ACP + H2O Palmitate + ACP + H2OPalmitoyl thioesteraseSubcellular localization of lipid metabolismRegulation of fatty acid synthesisshort-term regulation(substrate availability; allosteric effectors and/or
23、 enzyme modification)e.g. Acetyl-CoA carboxylase (ACC), Carnitine acyltransferase I (inhibited by malonyl-CoA)long-term regulation(regulation of the rate of enzyme synthesis and turn-over)e.g. regulated by hormones (insulin, glucagon)Coordinated regulation of FA synthesis and breakdown(ACC-P, inacti
24、ve)(ACC, active)“Fats burn in the flame of carbohydrates”Peroxisome Proliferator-activated receptors (PPARs) responding to changes in dietary lipid by changing gene expression levels in fat and carbohydrate metabolism; PPAR: expressed in liver, kidney, heart, skeletal muscle;PPAR/: key regulators of
25、 fat oxidation; PPAR: primarily in liver and adipose tissue; Peroxisome Proliferator-activated receptors (PPARs) PPAR heterdimerized with retinoid X receptor (RXR); Endogenous ligands: free fatty acids and eicosanoids. PPAR: target of type2 diabetes drugsThiazolidinediones (TZD,噻唑烷二酮) are a class of
26、 drugs used to treat type 2 diatetes, including Rosilitazone, Piolitazone, and Troglitazone.PPAR: target of type2 diabetes drugs TZD activates PPAR which induces the activity of PEP carboxykinase, increasing the rate of glyceroneogenesis in adipose tissue and reducing the amount of free FAs in the b
27、lood. TZD side effects: weight gain and heart failure. Regulation of acetyl-CoA carboxylase(allosteric regulation + hormone-dependentcovalent modification)Acetyl-CoA carboxylase from plants and bacteria is not regulated by citrate or by a phosphorylation dephosphorylation cycle.Acetyl-CoA carboxylas
28、e is regulated by covalent modificationAMP-activatedprotein kinase (AMPK)AMP (+), ATP(-) ProteinPhosphatase 2ACitrate activates the inactive phosphorylated enzyme rather than removing the phosphateInsulin Glucagon or Epinephrine +The active (dephosphorylated) form of acetyl-CoA carboxylase forms fil
29、aments(under electron microscope)Citrate partially activate the phosphorylated acetyl-CoA carboxylase (similar to how AMP partially activate the dephosphorylated glycogen phosphorylase) .Nature review 2004, Vol 3: 340-351AMP kinase (AMPK)(Nature review 2004, Vol 3: 340-351)Acetyl-CoA carboxylase (ke
30、y regulatory step): It is inhibited by palmitoyl-CoA and fatty acyl-CoAs, and activated by citrate (allosteric regulation, local regulation); Glucagon and epinepherine inactivate the enzyme by triggering its phosphorylation (AMPK); Insulin has the opposite effect (global regulation); The active (dep
31、hosphorylated) form of the enzyme forms filaments, while its inactive form dissociate into monomers or oligomers; Response to dietFatty acid synthesis and degradation are reciprocally regulated so that both are not simultaneously active; In starvation, the level of free fatty acids rises because hor
32、mones such as epinephrine and glucagon stimulate lipase. Insulin, in contrast, inhibits lipolysis. Fatty acid oxidation is blocked during synthesis: Malonyl-CoA effectively inhibits carnitine acyltransferase I, thus blocking the -oxidation of fatty acids in mitochondria; NADH inhibits 3-hydroxyacyl-
33、CoA dehydrogenase; acetyl-CoA inhibits thiolase Adaptive control: Animals that have fasted and are then fed high-carbohydrate, low-fat diets show marked increases in their amounts of acetyl CoA carboxylase and fatty acid synthase within a few days. In plant: Fatty acid synthesis occurs in chloroplas
34、t (stroma) in plants; The plant acetyl-CoA carboxylase is not regulated by phosphorylation; It is activated by increase in stroma pH and magnesiumFatty acid elongation and desaturationLong-chain saturated FAs aresynthesized from palmitateFormation of polyunsaturated FAsTwo systems: In smooth ER memb
35、ranes ：similar to the last cycle of FA synthesis ; (CoA replaces ACP as acyl carrier );In mitochondria: reverse reaction of FA -oxidation; Enoyl-CoA reductase: use NADPH； Acyl-CoA dehydrogenase: use FAD. Elongation of fatty acids: The double bonds of palmitoleate and oleate are introduced in vertebr
36、ates by fatty acyl-CoA desaturase, together with cytochrome b5 and cytochrome b5 reductase; Two substrates, fatty acyl-CoA and NADPH, are oxidized simultaneously by O2 . The desaturase is thus a mixed-function oxidase.Desaturation of fatty acids:Fatty acyl-CoA desaturase is a mixed-function oxidase
37、which oxidize two different substrates simultaneously.Differences between Oxidase and OxygenaseCytochrome P450: a family of proteins belonging to monooxygenase(mixed- function oxidase or oxygenase : here, the oxidase means oxygenase ) Desaturation of fatty acids：In vertebrates， Palmitate（C16） Palmit
38、oleate (C16,9 ） Stearate（C18） Oleate （C18,9 ） Mammalian hepatocytes cannot introduce double bonds beyond 9. Linoleate, 18:2(9,12）and -linolenate 18:3 (9,12,15）,cannot be synthesized by mammals, but plants can synthesize both.fatty acyl-CoA desaturase Further desaturation of oleate (to form linoleate
39、 and linolenate) occur on phosphatidylcholine; This is catalyzed by another desaturase, which is present only in plant cells, not in vertebrates; Therefore, linoleate and linolenate are essential fatty acids for mammals.Production of polyunsaturated fatty acids in plants:In plants:Oleate can be furt
40、her desaturated (beyondposition 9) on phosphatidylcholine(often attaching to C-2)to form linoleate and linolenate inplants.Linoleate and linolenate, needed to make other polyunsaturated fatty acids like arachidonateare essential fatty acids for mammals.Eicosanoids are a class of lipids that include
41、prostaglandins，thromboxanes and leucotrienes ;Eicosanoids derive their name from their common origin, that is, from C20 polyunsaturated fatty acids, the eicosanoic acids, particularly arachidonate, 20:4 ( 5,8,11,14), 5,8,11,14-eicosatetraenoic acid.Eicosanoids exert specific physiological effects on
42、 target cells, like hormones. However, eicosanoids are distinct from most hormones in that they act locally, near their sites of synthesis, and they are catabolized extremely rapidly. Thus, eicosanoids are considered to be locally acting hormones.EicosanoidsArachidonic acid is formed as arachidonyl-
43、CoA from 8,11,14 eicosatrienoyl-CoA. The essential fatty acid linoleic acid is an important precursor of arachidonic acid.Cyclooxygenase (COX), also called prostaglandin H2 synthase (bifunctional enzyme): - cyclooxygenase activity; - peroxidase activityProstaglandins stimulate inflammation,regulate
44、blood flow, control ion transport and modulate synaptic transmission.Aspirin inhibits the cyclooxgenase activity of COX by acetylating an essential Ser residue on the enzyme.Ibuprofen and Naproxen inhibit the same step, probably by mimicking the structure of the substrate or an intermediate in the r
45、eaction.They belong to Non-steroidal anti-inflammatory drugs ,NSAIDs .Side effect of Aspirin: Aspirin inhibits both isozymes (COX1 and COX2)equally, so a dose sufficient to reduce inflammationalso risks stomach irritation. New developing NSAIDSshould inhibit COX-2 specifically. COX1: for the synthes
46、is of the prostaglandins that regulate the secretion of gastric mucin;COX2: for the synthesis of prostaglandins that mediate inflammation, pain and fever. COX-2 specific cyclooxygenase inhibitorThis pathway is not inhibited by aspirin or other NSAIDsLeukotrienes are eicosanoid lipid mediators, actin
47、g as autocrine signalling and paracrine signalling to regulate the bodys response. Their production usually accompanies the production of histamine . Fatty acid biosynthesis takes a different pathway from the reverse of its degradation and takes place in different cellular compartments. The aceytl-C
48、oA units are transported out of mitochondrial matrix as citrate. Acetyl-CoA carboxylase catalyzes the rate-limiting step of fatty acid synthesis and is highly regulated by allosteric and covalent modifications.SummarylPalmitate, the usual final product of fatty acid synthesis, can be further elongated and desaturated.lEicosanoids are derived from arachidonate by the action of cyclooxygenases and peroxidases
生物化學(xué)：Chapter 21 Lipid Biosynthesis I
