Fatty Acid Synthesis

Fatty Acid Synthesis

Copyright © 1999-2005 by Joyce J. Diwan. All rights reserved.

Molecular Biochemistry II

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.

H 3C C SC o A

O

C H 2 C SC o A

O

O O C

acetyl-C oA

m alonyl-C oA

The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to 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 .

Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA.

ll

Enzyme-biotin HCO3

- + ATP

ADP + Pi

Enzyme-biotin-CO2-

O

CH3-C-SCoA acetyl-CoA O

-O2C-CH2-C-SCoA malonyl-CoA

ll

Enzyme-biotin

1

2

The overall reaction, which is spontaneous, may be summarized as:

HCO3 + ATP + acetyl-CoA ADP + Pi + malonyl-CoA

ll

Enzyme-biotin HCO3

- + ATP

ADP + Pi

Enzyme-biotin-CO2-

O

CH3-C-SCoA acetyl-CoA O

-O2C-CH2-C-SCoA malonyl-CoA

ll

Enzyme-biotin

1

2

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.

CHCH

H2CS

CH

NHC

N

O

(CH2)4 C NH (CH2)4 CH

CO

NH

O

CO

O

Carboxybiotin lysine residue

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).

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

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

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive)

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active)

Citrate

Dephosphorylated, e.g., by insulin-

activated Protein Phosphatase

Palmitoyl-CoA

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

Regulation of Acetyl-CoA Carboxylase

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

AMP-Activated Kinase has a significant role even in tissues (e.g., cardiac muscle) that do not significantly synthesize fatty acids.

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

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

ATP + HCO3

ADP + Pi

Acetyl-CoA Carboxylase (inhibited by

AMP-Activated Kinase)

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.

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

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)

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active)

Citrate

Dephosphorylated, e.g., by insulin-

activated Protein Phosphatase

Palmitoyl-CoA

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

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.

Regulation of Acetyl-CoA Carboxylase by local metabolites:

Phosphorylated protomer of Acetyl-CoA Carboxylase (inactive)

Dephosphorylated Polymer of Acetyl-CoA Carboxylase (active)

Citrate

Dephosphorylated, e.g., by insulin-

activated Protein Phosphatase

Palmitoyl-CoA

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

Regulation of Acetyl-CoA Carboxylase

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

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 cholesterol oxaloacetate citrate

Krebs Cycle

Citrate allosterically activates Acetyl-CoA Carboxylase.

Fatty acid synthesis from acetyl-CoA & malonyl-CoA 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.

Fatty AcidSynthase prosthetic groups:

the thiol of the side-chain of a cysteine residue of Condensing Enzyme domain.

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

N

N N

N

NH2

O

OHO

HH

H

CH2

H

OPOPOH2C

O

O O

O

P

O

O O

C

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

-mercaptoethylamine

pantothenate

ADP-3'- phosphate

Coenzyme A

phosphopantetheine

H3N+ C COO

CH2

SH

H

cysteine

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

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

OPOH2C

O

OC

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

CH2 CH

NH

C O

-mercaptoethylamine

pantothenate

serine residue

phosphopantetheine of acyl carrier protein

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.

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

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

Order of domains in primary structure of mammalian Fatty Acid 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.

Pant

SH

Cys

SH

Pant

SH

Cys

S

C

CH3

O

Pant

S

Cys

S

C

CH3

OC

CH2

COO

O

Pant

S

Cys

SH

C

CH2

C

O

CH3

O

acetyl-S-CoA HS-CoA malonyl-S-CoA HS-CoA CO2

1 2 3

1 Malonyl/acetyl-CoA-ACP Transacylase 2 Malonyl/acetyl-CoA-ACP Transacylase

3 Condensing Enzyme (-Ketoacyl Synthase)

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.

Pant

S

Cys

SH

C

CH2

C

O

CH3

O

Pant

S

Cys

SH

C

CH2

HC

O

CH3

Pant

S

Cys

SH

Pant

S

Cys

SH

NADPH NADP+NADPH NADP+

C

CH

HC

O

CH3

C

CH2

CH2

O

CH3

OH

H2O

4 5 6

4 -Ketoacyl-ACP Reductase

5 -Hydroxyacyl-ACP Dehydratase 6 Enoyl-ACP Reductase

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

Pant

S

Cys

SH

C

CH2

CH2

O

CH3

Pant

SH

Cys

S

C

CH2

O

CH2

CH3

Pant

S

Cys

S

C

CH2

O

CH2

CH3

C

CH2

COO

O

Malonyl-S-CoA HS-CoA

7 2

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

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.

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

Order of domains in primary structure of mammalian Fatty Acid Synthase

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

Explore with Chime the structure of the E. coli -Ketoacyl-ACP Synthase III, equivalent to the domains of the mammalian Fatty Acid Synthase that catalyze the initial acetylation and condensation reactions.

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. Acetyl-CoA generated in mitochondria is transported to the cytosol via a shuttle mechanism involving citrate.

-Oxidation & Fatty Acid SynthesisCompared

Oxidation Pathway Fatty Acid Synthesis

pathway location mitochondrial matrix cytosol

acyl carriers (thiols)

Coenzyme-A phosphopantetheine (ACP) & cysteine

e acceptors/donor FAD & NAD+ NADPH

-OH intermediate L D

2-C product/donor acetyl-CoA malonyl-CoA

(& acetyl-CoA)

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.

In fat cells:

Expression of SREBP-1 and of Fatty Acid Synthase is inhibited by leptin, a hormone that has a role in regulating food intake and fat metabolism.

Leptin is produced by fat cells in response to excess fat storage.

Leptin regulates body weight by decreasing food intake, increasing energy expenditure, and inhibiting fatty acid synthesis.

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 malonyl-CoA as donor of 2-carbon units.

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).

C

O

OH

910

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.

C

O

OH

910

oleate 18:1 cis 9

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