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Molecular Biochemistry II

Fatty Acid Synthesis

Copyright © 1999-2009 by Joyce J. Diwan.
All rights reserved.

O

H3C C SCoA
acetyl-CoA
The input to fatty acid O
synthesis is acetyl-CoA,

which is carboxylated to OOC CH2 C SCoA

malonyl-CoA. malonyl-CoA

ATP-dependent carboxylation provides energy input.
The CO2 is lost later during condensation with the
growing fatty acid.
The spontaneous decarboxylation drives the
condensation reaction.

Enzyme-biotin
-
Acetyl-CoA HCO3 + ATP
1
Carboxylase ADP + Pi
-
catalyzes the Enzyme-biotin-CO2
2-step reaction O
ll 2
by which CH3-C-SCoA
Enzyme-biotin
acetyl-CoA is acetyl-CoA
carboxylated O
-
ll
to form O2C-CH2-C-SCoA
malonyl-CoA. malonyl-CoA

As with other carboxylation reactions, the enzyme
prosthetic group is biotin.
ATP-dependent carboxylation of the biotin, carried out at
one active site 1 , is followed by transfer of the carboxyl
group to acetyl-CoA at a second active site 2 .

which is spontaneous. may be summarized as: HCO3 + ATP + acetyl-CoA  ADP + Pi + malonyl-CoA . Enzyme-biotin - HCO3 + ATP 1 ADP + Pi - Enzyme-biotin-CO2 O ll 2 CH3-C-SCoA Enzyme-biotin acetyl-CoA O - ll O2C-CH2-C-SCoA malonyl-CoA The overall reaction.

The combined biotin and lysine side chains act as a long flexible arm that allows the biotin ring to translocate between the 2 active sites. O O C C N NH O CH CH O O C H2C CH S (CH2)4 C NH (CH2)4 CH Carboxybiotin lysine NH residue Biotin is linked to the enzyme by an amide bond between the terminal carboxyl of the biotin side chain and the e-amino group of a lysine residue. .

.Acetyl-CoA Carboxylase. by  phosphorylation  allosteric control by local metabolites. which converts acetyl-CoA to malonyl-CoA. is the committed step of the fatty acid synthesis pathway. The mammalian enzyme is regulated.

 Transition to the inactive conformation is associated with dissociation to yield the monomeric form of the enzyme (protomer).Conformational changes associated with regulation:  In the active conformation. Acetyl-CoA Carboxylase associates to form multimeric filamentous complexes. .

a higher portion of a cell's adenine nucleotide pool is in the form of AMP.g. ..AMP functions as an energy sensor and regulator of metabolism. E. When ATP production does not keep up with needs. AMP regulates fatty acid synthesis and catabolism by controlling availability of malonyl-CoA.  AMP promotes catabolic pathways that lead to synthesis of ATP.  AMP inhibits energy-utilizing synthetic pathways.

ADP + Pi AMP-Activated Kinase) O This causes inhibition of  ATP-utilizing of malonyl. . O H3C C SCoA AMP-Activated Kinase acetyl-CoA Acetyl-CoA catalyzes phosphorylation ATP + HCO3 Carboxylase of Acetyl-CoA (inhibited by Carboxylase.  As discussed earlier. OOC CH2 C SCoA CoA production. malonyl-CoA  Fatty acid synthesis is diminished by lack of the substrate malonyl-CoA. fatty acid oxidation is stimulated due to decreased inhibition by malonyl-CoA of transfer of fatty acids into mitochondria.

the alternative metabolic fuel used when blood glucose is low. . With Acetyl-CoA Carboxylase inhibited. O activated by glucagon &  OOC CH2 C SCoA epinephrine when blood malonyl-CoA glucose is low. may also result in phosphorylation of Acetyl-CoA Carboxylase via cAMP-Dependent Protein Kinase. acetyl-CoA remains available for synthesis of ketone bodies. O H3C C SCoA acetyl-CoA Acetyl-CoA ATP + HCO3 Carboxylase (inhibited by ADP + Pi AMP-Activated Kinase) A cAMP cascade.

g. Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate Palmitoyl-CoA Dephosphorylated. e. via e.g.. by insulin. . produced when blood glucose is high.. Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) Regulation of Acetyl-CoA Carboxylase The antagonistic effect of insulin. is attributed to activation of Protein Phosphatase. Phosphorylated. AMP-activated Kinase activated Protein when cellular stress or Phosphatase exercise depletes ATP.

by insulin. via e. Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate Palmitoyl-CoA Dephosphorylated. AMP-activated Kinase activated Protein when cellular stress or Phosphatase exercise depletes ATP.. This is an example of feedback inhibition.g. diminishing production of malonyl-CoA. Regulation of Acetyl-CoA Carboxylase by Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) local metabolites: Regulation of Acetyl-CoA Carboxylase Palmitoyl-CoA (product of Fatty Acid Synthase) promotes the inactive conformation. the precursor of fatty acid synthesis.. Phosphorylated. e.g. .

. Excess acetyl-CoA is then converted via malonyl-CoA to fatty acids for storage. Glucose-6-phosphatase glucose-6-P glucose Gluconeogenesis Glycolysis pyruvate fatty acids acetyl CoA ketone bodies Citrate cholesterol oxaloacetate citrate allosterically activates Acetyl- Krebs Cycle CoA Carboxylase. [Citrate] is high when there is adequate acetyl-CoA entering Krebs Cycle.

The NADPH is produced mainly by the Pentose Phosphate Pathway.Fatty acid synthesis from acetyl-CoA & malonyl-CoA occurs by a series of reactions that are:  in bacteria catalyzed by 6 different enzymes plus a separate acyl carrier protein (ACP)  in mammals catalyzed by individual domains of a very large polypeptide that includes an ACP domain. Evolution of the mammalian Fatty Acid Synthase apparently has involved gene fusion. NADPH serves as electron donor in the two reactions involving substrate reduction. .

H3C C CH3 O O N N  the thiol of H2C O P O P O CH2 O H H phosphopantetheine.  O P O O . O O H H equivalent in structure phosphopantetheine O OH to part of coenzyme A. H SH Coenzyme A H3N+ C COO CH2 CH2 CH2 -mercaptoethylamine SH NH Fatty Acid cysteine C O Synthase CH2 prosthetic groups: CH2 pantothenate  the thiol of the side. NH NH2 chain of a cysteine C O N ADP-3'. N residue of Condensing HO C H phosphate Enzyme domain.

O C O phosphate . SH phosphopantetheine CH2 of acyl carrier protein Phosphopantetheine CH2 (Pant) is covalently -mercaptoethylamine linked via a phosphate NH ester to a serine OH of C O the acyl carrier protein CH2 domain of Fatty Acid CH2 Synthase. pantothenate NH The long flexible arm C O of phosphopantetheine HO C H helps its thiol to move H3C C CH3 O NH from one active site to serine another within the H2C O P O CH2 CH residue complex.

. the initial attacking group is the oxygen of a serine hydroxyl group of the Malonyl/acetyl-CoA Transacylase enzyme domain.Condensing Malonyl/acetyl-CoA Dehydratase Enoyl Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant) -C Order of domains in primary structure of mammalian Fatty Acid Synthase As each of the substrates acetyl-CoA & malonyl-CoA bind to the complex. -Ketoacyl ACP Thioesterase N. Each acetyl or malonyl moiety is transiently in ester linkage to this serine hydroxyl. Acetate is subsequently transferred to a cysteine thiol of the Condensing Enzyme domain. before being transferred into thioester linkage with the phosphopantetheine thiol of the acyl carrier protein (ACP) domain.

followed by attack of the resultant carbanion on the carbonyl carbon of the acetyl (or acyl) moiety. acetyl-S-CoA HS-CoA malonyl-S-CoA HS-CoA CO2 Pant Cys Pant Cys Pant Cys Pant Cys 1 2 3 SH SH SH S S S S SH C O C O C O C O CH3 CH2 CH3 CH2 1 Malonyl/acetyl-CoA-ACP Transacylase COO C O 2 Malonyl/acetyl-CoA-ACP Transacylase CH3 3 Condensing Enzyme (-Ketoacyl Synthase) The condensation reaction (step 3) involves decarboxylation of the malonyl moiety. .

Dehydration yields a trans double bond. The -ketone is reduced to an alcohol by e transfer from NADPH. 5. NADPH NADP+ H2O NADPH NADP+ Pant Cys Pant Cys Pant Cys Pant Cys S SH 4 S SH 5 S SH 6 S SH C O C O C O C O CH2 CH2 CH CH2 C O HC OH HC CH2 CH3 CH3 CH3 CH3 4 -Ketoacyl-ACP Reductase 5 -Hydroxyacyl-ACP Dehydratase 6 Enoyl-ACP Reductase 4. . 6. Reduction by NADPH yields a saturated chain.

with another malonyl-CoA. Following transfer of the growing fatty acid from phosphopantetheine to the Condensing Enzyme's cysteine sulfhydryl. Malonyl-S-CoA HS-CoA Pant Cys Pant Cys Pant Cys S SH 7 SH S 2 S S C O C O C O C O CH2 CH2 CH2 CH2 CH2 CH2 COO CH2 CH3 CH3 CH3 7 Condensing Enzyme 2 Malonyl/acetyl-CoA-ACP Transacylase (repeat). . the cycle begins again.

Product release: When the fatty acid is 16 carbon atoms long. . The 16-C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex. a Thioesterase domain catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine.

Fatty Acid Synthase in mammals is a homo-dimer. X-Ray crystallographic analysis at 3.Condensing Malonyl/acetyl-CoA Dehydratase Enoyl Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant) -C Order of domains in primary structure of mammalian Fatty Acid Synthase The primary structure of the mammalian Fatty Acid Synthase protein is summarized above.2 Å resolution shows the dimeric Fatty Acid Synthase to have an X-shape. -Ketoacyl ACP Thioesterase N. Fatty Acid Synthase PDB 2VZ8 . The 2 copies of the protein are displayed at right in different colors.

Each copy of the dimeric protein has an S shape.Condensing Malonyl/acetyl-CoA Dehydratase Enoyl Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant) -C Order of domains in primary structure of mammalian Fatty Acid Synthase The domain arrangement is shown below. with the N-terminal KS (Condensing Enzyme / -Ketoacyl Synthase) domain folded back to form part of the central interaction domain. MAT MAT MAT = Malonyl/Acetyl-CoA “leg” Transacylase. KS = -Ketoacyl Synthase KS KS (Condensing Enzyme). Arrangement of domains in Fatty Acid Synthase . -Ketoacyl ACP Thioesterase N. “arm” ER = Enoyl Reductase. DH DH DH = Dehydratase. KR ER ER KR KR = -Ketoacyl Reductase.

KS = -Ketoacyl Synthase KS KS (Condensing Enzyme). These domains may be too flexible to be resolved by crystallography. KR ER ER KR KR = -Ketoacyl Reductase. predicted from the primary structure to be near the KR domains.Condensing Malonyl/acetyl-CoA Dehydratase Enoyl Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant) -C Order of domains in primary structure of mammalian Fatty Acid Synthase The X-ray analysis does not resolve the C-terminal ACP (acyl carrier protein) & Thioesterase domains. Arrangement of domains in Fatty Acid Synthase . “arm” ER = Enoyl Reductase. DH DH DH = Dehydratase. MAT MAT MAT = Malonyl/Acetyl-CoA “leg” Transacylase. -Ketoacyl ACP Thioesterase N.

requires subscription to Science). There is evidence for conformational changes relating to catalysis.Fatty Acid Synthase complex is somewhat asymmetric. For images see: website (ETH Zurich) website (Asturias lab. Protein flexibility may facilitate transfer of ACP-attached reaction intermediates among the several active sites in each half of the complex. Scripps) article (Maier. Fatty Acid Synthase PDB 2VZ8 . Leibundgut & Ban.

Explore with Chime the structure of the Mammalian Fatty Acid Synthase III. .

Total NADPH used per synthesis of one 16-C palmitate. 1. How many acetyl-CoA used for synthesis of each malonate? ____ 1 c.Palmitate. How many malonate used (how many reaction cycles) per 7 synthesis of one 16-C palminate? ________ d. 7 2a(1c): ________ 3. How many ~P bonds of ATP used for synthesis of each 1 malonate? ________ b. a + b(c): ________ 8 2. a. a. is the final product of the Fatty Acid Synthase reactions. Total acetyl-CoA used for priming & for syntheisis of malonate. a. 3a(1c): 14 _________ . How many acetyl-CoA used for initial priming of enzyme? _____ 1 b. How many NADPH used per reaction cycle? __________ 2 b. Total ~P bonds of ATP used for synthesis of one 16-C palmitate. a 16-C saturated fatty acid.

Acetyl-CoA generated in mitochondria is transported to the cytosol via a shuttle mechanism involving citrate. . listing net inputs and outputs: 8 acetyl-CoA + 14 NADPH + 7 ATP  palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi Summary based on malonate as an input: acetyl-CoA + 7 malonyl-CoA + 14 NADPH  palmitate + 7 CO2 + 14 NADP+ + 8 CoA Fatty acid synthesis occurs in the cytosol.Summary (ignoring H+ & water): Write a balanced equation for synthesis of palmitate from acetyl-CoA.

-Oxidation & Fatty Acid Synthesis Compared  Oxidation Pathway Fatty Acid Synthesis pathway location mitochondrial matrix cytosol acyl carriers phosphopantetheine Coenzyme-A (thiols) (ACP) & cysteine e acceptors/donor FAD & NAD+ NADPH -OH intermediate L D malonyl-CoA 2-C product/donor acetyl-CoA (& acetyl-CoA) .

Thus excess glucose is stored as fat. In liver:  Insulin. SREBPs (sterol response element binding proteins) were first identified for their regulation of cholesterol synthesis. a hormone produced when blood glucose is high. stimulates Fatty Acid Synthase expression.  Polyunsaturated fatty acids diminish transcription of the Fatty Acid Synthase gene in liver cells. Transcription factors that that mediate the stimulatory effect of insulin include USFs (upstream stimulatory factors) and SREBP-1. .Fatty Acid Synthase is transcriptionally regulated. by suppressing production of SREBPs.

Leptin is produced by fat cells in response to excess fat storage. Leptin regulates body weight by decreasing food intake. a hormone that has a role in regulating food intake and fat metabolism. .In fat cells: Expression of SREBP-1 and of Fatty Acid Synthase is inhibited by leptin. and inhibiting fatty acid synthesis. increasing energy expenditure.

Elongation beyond the 16-C length of the palmitate product of Fatty Acid Synthase is mainly catalyzed by enzymes associated with the endoplasmic reticulum (ER). ER enzymes lengthen fatty acids produced by Fatty Acyl Synthase as well as dietary polyunsaturated fatty acids. A family of enzymes designated Fatty Acid Elongases or ELOVL (elongation of very long chain fatty acid) catalyze the initial condensation step. Fatty acids esterified to coenzyme A serve as substrates. . Malonyl-CoA is the donor of 2-carbon units in a reaction sequence similar to that of Fatty Acid Synthase except that individual steps are catalyzed by separate proteins.

D12.. e.g. . 18:2 cis D9. Mammalian cells are unable to produce double bonds at certain locations.12 (18 C atoms long. Thus some polyunsaturated fatty acids are dietary essentials. e. O 10 9 C OH oleate 18:1 cis D9 Desaturases introduce double bonds at specific positions in a fatty acid chain. with cis double bonds at carbons 9-10 & 12-13).. linoleic acid.g.

. with an active site that contains two iron atoms complexed by histidine residues.  Desaturase.  Cytochrome b5. which may be a separate protein or a domain at one end of the desaturase. O 10 9 C OH oleate 18:1 cis D9 Formation of a double bond in a fatty acid involves the following endoplasmic reticulum membrane proteins in mammalian cells:  NADH-cyt b5 Reductase. a flavoprotein with FAD as prosthetic group.

. There is a 4-electron reduction of O2  2 H2O as a fatty acid is oxidized to form a double bond.g. the order of electron transfer being: NADH  FAD  cyt b5  desaturase  2e are extracted from the fatty acid as the double bond is formed. E. the overall reaction for desaturation of stearate (18:0) to form oleate (18:1 cis D9) is: stearate + NADH + H+ + O2  oleate + NAD+ + 2H2O .  2e pass from NADH to the desaturase via the FAD-containing reductase & cytochrome b5.The desaturase catalyzes a mixed function oxidation reaction.