Fatty Acid Synthesis

Comparison of fatty acid biosynthesis and degradation

Sites of regulation of fatty acid metabolism

The formation of malonyl~CoA is the committed step in fatty acid synthesis

Irreversible reaction catalyzed by acetyl CoA carboxylase (ACC): malonyl~CoA + ADP + Pi + H+

acetyl~CoA + ATP + HCO3-

ACC contains a biotin prosthetic group, and the reaction mechanism is similar to pyruvate carboxylase and propionyl~CoA carboxylase reactions.

The two steps in the reaction: <===> CO2-Biotin----Enzyme + ADP + Pi malonyl~CoA + Biotin----Enzyme

Biotin----Enzyme + ATP + HCO3-

CO2-Biotin----Enzyme + acetyl~CoA

Notes: In E. coli, 3 subunits: biotin carboxylase, transcarboxylase, biotin-carboxyl carrier protein. In birds and mammals a single polypeptide chain has all three functions.

ACC is subject to hormonal regulation. Dephosphorylated form is active, phosphorylated form is less active. Glucagon and epinephrine stimulate phosphorylation, Insulin stimulates dephosphorylation.

Citrate activates and palmitoyl~CoA inhibits the enzyme

Phosphorylation leads to depolymerization.

O H3C C SCoA

The input to fatty acid O synthesis is acetyl OOC CH2 C SCoA CoA, which is malonyl-CoA carboxylated to malonyl-CoA. ATP-dependent carboxylation provides energy input.

acetyl-CoA

The CO2 is lost later during condensation with the growing fatty acid.

The spontaneous decarboxylation drives the condensation reaction.

Enzyme-biotin HCO3 + ATP ADP + Pi Enzyme-biotin-CO2 O

ll

2
Enzyme-biotin O
ll

CH3-C-SCoA acetyl-CoA
-

Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetylCoA is carboxylated to form malonylCoA.
O2C-CH2-C-SCoA 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 .

Enzyme-biotin HCO3 + ATP ADP + Pi Enzyme-biotin-CO2 O

ll

2
Enzyme-biotin O
ll

CH3-C-SCoA acetyl-CoA
-

O2C-CH2-C-SCoA malonyl-CoA

The overall reaction, which is spontaneous, may be summarized as: ADP + Pi + malonyl-

HCO3 + ATP + acetyl-CoA CoA

O C N CH CH H2C S (CH2)4 C NH (CH2)4 CH CH O O C NH

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 amino group of a lysine residue. 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.

Acetyl-CoA Carboxylase, which converts acetyl-CoA to malonyl-CoA, is the committed step of the fatty acid synthesis pathway.

The mammalian enzyme is regulated, by phosphorylation allosteric control by local metabolites.

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

enzyme (protomer).

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate Palmitoyl-CoA

Phosphorylated, e.g., via AMP-activated Kinase when cellular stress or exercise depletes ATP. Dephosphorylated, e.g., by insulinactivated Protein Phosphatase

AMP-Activated Kinase catalyzes phosphorylation of Acetyl-CoA Carboxylase, causing inhibition.

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) Regulation of Acetyl-CoA Carboxylase

The decreased production of malonyl-CoA prevents energyutilizing fatty acid synthesis when cellular energy stores are depleted. (AMP is abundant only when ATP has been extensively dephosphorylated.)

O H3C C

SCoA

AMP-Activated Kinase has a significant role even in tissues (e.g., cardiac muscle) that do not significantly synthesize fatty acids.
acetyl-CoA ATP + HCO3 ADP + Pi

O OOC CH2

Acetyl-CoA Carboxylase (inhibited by AMP-Activated Kinase)

C SCoA

In such tissues malonylCoA, produced via one isoform of Acetyl-CoA Carboxylase, functions mainly as an inhibitor of fatty acid oxidation.
malonyl-CoA

When AMP is high (ATP low), malonyl-CoA production is diminished, releasing fatty acid oxidation from inhibition. This will lead to increased ATP production.

O H3C C SCoA

acetyl-CoA
O

OOC

CH2

SCoA

malonyl-CoA

A cAMP cascade, activated by glucagon & epinephrine when blood glucose is low, may also result in phosphorylation of Acetyl-CoA Carboxylase via cAMP-Dependent Protein Kinase.

With Acetyl-CoA Carboxylase inhibited, acetyl-CoA remains available for synthesis of ketone bodies, the alternative metabolic fuel used when blood glucose is low.

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate Palmitoyl-CoA

Phosphorylated, e.g., via AMP-activated Kinase when cellular stress or exercise depletes ATP.

Dephosphorylated, e.g., by insulinactivated Protein Phosphatase

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active)

Regulation of Acetyl-CoA Carboxylase

The antagonistic effect of insulin, produced when blood glucose is high, is attributed to activation of Protein Phosphatase.

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive) Citrate Palmitoyl-CoA

Phosphorylated, e.g., via AMP-activated Kinase when cellular stress or exercise depletes ATP. Dephosphorylated, e.g., by insulinactivated Protein Phosphatase

Regulation of Acetyl-CoA Carboxylase by local metabolites:

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active) Regulation of Acetyl-CoA Carboxylase

Palmitoyl-CoA (product of Fatty Acid Synthase) promotes the inactive conformation, diminishing production of malonyl-CoA, the precursor of fatty acid synthesis. This is an example of feedback inhibition.

Glucose-6-phosphatase glucose-6-P glucose Gluconeogenesis Glycolysis pyruvate fatty acids acetyl CoA oxaloacetate Krebs Cycle ketone bodies cholesterol citrate

Citrate allosterically activates AcetylCoA Carboxylase.

[Citrate] is high when there is adequate acetyl-CoA entering Krebs Cycle.

Excess acetyl-CoA is then converted via malonylCoA to fatty acids for storage.

An hypothesis for the regulation of ACC

Active, polymerized ACC AMPK palmitoyl~CoA

protein phosphatase 2A other insulin-dependent phosphatase? inactive, depolymerized ACC

palmitoyl~CoA

phosphoryl group

citrate

partially polymerized, partially active

Energy charge of cell controls AMPK. Epinephrine and glucagon act by causing inhibition of PP2A Insulin acts through unknown phosphatase palmitoyl~CoA disperses filaments phosphorylation inhibits formation of filaments citrate promotes polymerization

Fatty acid synthesis from acetyl-CoA & malonylCoA occurs by a series of reactions that are:

in bacteria catalyzed by seven separate enzymes.

in mammals catalyzed by individual domains of a single large polypeptide. Evolution of the mammalian Fatty Acid Synthase apparently has involved gene fusion.

NADPH serves as electron donor in two reactions involving substrate reduction.

The NADPH is produced mainly by the Pentose Phosphate Pathway.

Mammalian Fatty Acid Synthase (FAS)

ketoacyl-ACP synthase enoyl-ACP reductase

malonyl/acetyl-CoA ACP transacylase

palmitoyl thioesterase

ketoacyl-ACP reductase hydroxyacyl-ACP dehydratase

Mammalian enzyme is a homodimer of 272 kDa subunits Member of the megasynthase family. In E. coli 7 different enzymes + ACP

H
SH

Coenzyme A
-mercaptoethylamine
O

H3N+ C CH2 SH
C CH2 CH2 NH CH2 CH2

COO

Fatty Acid cysteine Synthase prosthetic groups:

pantothenate
NH C HO H3C H2C C C H CH3 O O P O O O

NH2 N

the thiol of the sidechain of a cysteine residue of Condensing Enzyme domain.

ADP-3'phosphate
O P O O

N CH2 H H O O H

the thiol of phosphopantetheine, equivalent in structure to part of coenzyme A.

phosphopantetheine

H OH

P O

The phosphopantetheine prosthetic group of ACP is derived from CoA

In E. coli, ACP is a separate 125-amino acid protein, in mammals it is part of FAS

SH CH2 CH2

phosphopantetheine of acyl carrier protein -mercaptoethylamine

O

NH C CH2 CH2 NH C HO H3C C C H2C O H CH3 O O P O O CH2 NH CH C

Phosphopantetheine (Pant) is covalently linked via a phosphate ester to a serine OH of the acyl carrier protein domain of Fatty Acid Synthase.
pantothenate

serine residue
O

The long flexible arm of phosphopantetheine allows its thiol to move from one active site to another within the complex.

phosphate

Individual steps of the Fatty Acid Synthase reaction pathway are catalyzed by the catalytic domains listed.

Fatty Acid Synthase complex is an obligate dimer.

Within each monomer, the order of enzyme domains along the primary sequence of the protein is summarized below.

There is still debate over the arrangement of domains in 3D within the complex. An atomic resolution structure of the entire complex has not yet been achieved.

Malonyl/acetyl-CoA Dehydratase Enoyl -Ketoacyl ACP Thioesterase N- Condensing -C Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant)

Order of domains in primary structure of mammalian Fatty Acid Synthase

Malonyl/acetyl-CoA Dehydratase Enoyl -Ketoacyl ACP Thioesterase N- Condensing -C Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant)

Order of domains in primary structure of mammalian Fatty Acid Synthase

As each of the substrates acetyl-CoA & malonylCoA bind to the complex, the initial attacking group is the oxygen of a serine hydroxyl group of the Malonyl/acetyl-CoA Transacylase enzyme domain.

Each acetyl or malonyl moiety is transiently in ester linkage to this serine hydroxyl, before being transferred into thioester linkage with the phosphopantetheine thiol of the acyl carrier protein (ACP) domain.

Acetate is subsequently transferred to a cysteine thiol of the Condensing Enzyme domain.

acetyl-S-CoA HS-CoA malonyl-S-CoA HS-CoA Cys S C CH3 CH2 CH3 O C O C O Pant Cys S Pant CO2 Pant SH

Pant

Cys

Cys SH

SH

SH

1
S

2
S C

O CH2 C CH3 O

1 Malonyl/acetyl-CoA-ACP Transacylase COO 2 Malonyl/acetyl-CoA-ACP Transacylase 3 Condensing Enzyme ( -Ketoacyl Synthase)

The condensation reaction (step 3) involves decarboxylation of the malonyl moiety, followed by attack of the resultant carbanion on the carbonyl carbon of the acetyl (or acyl) moiety.

Mechanism of carbon-carbon bond formation in fatty acid biosynthesis

Notes: The CO2 that was added in the ACC reaction is lost as CO2 and does not appear in the fatty acid.

Loss of CO2 results in resonance-stabilized carbanion

NADPH NADP+ H2O Cys Cys SH O SH O CH OH CH3 HC CH2 CH2 CH3 C O C Pant Cys SH Pant Pant S C CH2 HC CH3

NADPH NADP+

Pant

Cys

SH

4
S S

CH2

CH3

4 -Ketoacyl-ACP Reductase 5 -Hydroxyacyl-ACP Dehydratase 6 Enoyl-ACP Reductase

4. The -ketone is reduced to an alcohol by e transfer from NADPH. 5. Dehydration yields a trans double bond. 6. Reduction by NADPH yields a saturated chain.

Malonyl-S-CoA HS-CoA Pant Cys S C CH2 CH2 CH3 COO CH2 O C O C CH2 CH2 CH3 O Pant Cys S

Pant

Cys

SH

7
SH S

CH2

CH2

CH3

7 Condensing Enzyme 2 Malonyl/acetyl-CoA-ACP Transacylase (repeat).

Following transfer of the growing fatty acid from phosphopantetheine to the Condensing Enzyme's cysteine sulfhydryl, the cycle begins again, with another malonyl-CoA.

Malonyl/acetyl-CoA Dehydratase Enoyl -Ketoacyl ACP Thioesterase N- Condensing -C Enzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant)

Order of domains in primary structure of mammalian Fatty Acid Synthase

Product release:

When the fatty acid is 16 carbon atoms long, a Thioesterase domain catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine.

The 16-C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex.

There is some evidence that the 2 copies of the multi-domain enzyme are aligned antiparallel, as below. In the transfer step the growing fatty acid is preferentially passed from the ACP phosphopantetheine thiol of one subunit to the Condensing Enzyme cysteine thiol of the other subunit of the dimer.

However intra-subunit substrate transfers also occur.

Pant-SH HS-Cys

Cys-SH HS-Pant

Fatty Acid Synthase dimer

Palmitate, a 16-C saturated fatty acid, is the final product of the Fatty Acid Synthase reactions.

Summary (ignoring H+ & water): acetyl-CoA + 7 malonyl-CoA + 14 NADPH palmitate + 7 CO2 + 14 NADP+ + 8 CoA Accounting for ATP-dependent synthesis of malonate: 8 acetyl-CoA + 14 NADPH + 7 ATP palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi

Fatty acid synthesis occurs in the cytosol. AcetylCoA generated in mitochondria is transported to the cytosol via a shuttle mechanism involving citrate.

A molecule of palmitate costs ~136 ATP

palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 ADP 80 ATP 49 ATP 7 ATP 136 ATP

8 acetyl~CoA + 14 NADPH + 6 H+ + 7 ATP @ 10 ATP/acetyl~CoA @ 3.5 ATP/NADPH

8 acetyl~CoA 14 NADPH 7 ATP

Total

-Oxidation & Fatty Acid Synthesis Compared

Oxidation Pathway Fatty Acid Synthesis mitochondrial matrix Coenzyme-A FAD & NAD+ L acetyl-CoA cytosol phosphopantetheine (ACP) & cysteine NADPH D malonyl-CoA (& acetyl-CoA)

pathway location

acyl carriers (thiols)

e acceptors/donor

-OH intermediate

2-C product/donor

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

Elongation beyond the 16-C length of the palmitate product of Fatty Acid Synthase occurs in mitochondria and endoplasmic reticulum (ER).

Fatty acid elongation within mitochondria involves the -oxidation pathway running in reverse, except that NADPH serves as electron donor for the final reduction step.

Polyunsaturated fatty acids esterified to coenzyme A are substrates for the ER elongation machinery, which uses malonylCoA as donor of 2-carbon units.

10 9 C OH

oleate 18:1 cis 9

Desaturases introduce double bonds at specific positions in a fatty acid chain.

Mammalian cells are unable to produce double bonds at certain locations, e.g., 12.

Thus some polyunsaturated fatty acids are dietary essentials, e.g., linoleic acid, 18:2 cis 9,12 (18 C atoms long, with cis double bonds at carbons 9-10 & 12-13).

10 9 C OH

oleate 18:1 cis 9

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.

Cytochrome b5, which may be a separate protein or a domain at one end of the desaturase.

Desaturase, with an active site that contains two iron atoms complexed by histidine residues.

The desaturase catalyzes a mixed function oxidation reaction.

There is a 4-electron reduction of O2 2 H2O as a fatty acid is oxidized to form a double bond.

2e pass from NADH to the desaturase via the FAD-containing reductase & cytochrome b5, 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.g., the overall reaction for desaturation of stearate (18:0) to form oleate (18:1 cis 9) is: stearate + NADH + H+ + O2 oleate + NAD+ + 2H2O

Citrate carries acetyl groups from the mitochondrion to the cytosol for fatty acid synthesis
Acetyl~CoA is generated in the mitochondrion by pyruvate dehydrogenase. Excess acetyl~CoA is shipped to the cytosol for storage as fat. Citrate is passed to the ctytosol. ATP-citrate lyase regenerates the acetyl~CoA and OAA in cytosol. Malic enzyme produces the NADPH that may be used in fatty acid synthesis.

tricarboxylate transport system for transfer of acetyl CoA from mitochondrion to cytosol

Regulation of Fatty Acid Metabolism

Fatty acid metabolism is coordinated with other aspects of metabolism. Hormonally regulated by pancreatic a and b cells, which sense metabolic status and secrete glucagon and insulin, respectively. The simultaneous synthesis and oxidation of fatty acids in liver cells is prevented. Malonyl~CoA inhibits carnitine palmitoyl transferase I. Long term regulation-synthesis of ACC adipose lippoprotein lipase, and FAS stimulated by insulin, inhibited by glucagon, epinephrine. A diet high in polyunsaturated FA also causes decreased FAS, ACC.