Fatty Acid Synthesis

Molecular Biochemistry II

Fatty Acid Synthesis

Contents of this page:
Synthesis of malonyl-CoA via Acetyl-CoA Carboxylase
Fatty Acid Synthase
Fatty acid elongation and desaturation

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

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

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

As with other carboxylation reactions (e.g., Pyruvate Carboxylase), 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).

The overall reaction, which is is spontaneous, may be summarized as:
HCO₃^- + ATP + acetyl-CoA à ADP + P_i + malonyl-CoA

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.

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.

Regulation of Acetyl-CoA Carboxylase:

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, and there is allosteric control via local metabolites. Conformational changes associated with regulation:

When in the active conformation, Acetyl-CoA Carboxylase self-associates to form multimeric filamentous complexes. See electron micrograph p. 932 of Biochemistry, 3rd Edition, by Voet & Voet.
Transition to the inactive conformation is associated with dissociation to yield the monomeric form of the enzyme (protomer).

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

AMP-Activated Kinase catalyzes phosphorylation of Acetyl-CoA Carboxylase causing inhibition of the ATP-utilizing production of 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 (see section fatty acid oxidation ).

A cyclic-AMP cascade, activated by the hormones glucagon and 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. The antagonistic effect of insulin, produced when blood glucose is high, is attributed to activation of Protein Phosphatase.

Regulation of Acetyl-CoA Carboxylase by local metabolites:

Palmitoyl-CoA, the product of Fatty Acid Synthase, promotes the inactive conformation of Acetyl-CoA Carboxylase (diagram above), diminishing production of malonyl-CoA, the precursor of fatty acid synthesis. This is an example of feedback inhibition.
Citrate allosterically activates Acetyl-CoA Carboxylase. Citrate concentration 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.

Fatty acid synthesis, from acetyl-CoA and malonyl-CoA, occurs by a series of reactions that are:

in bacteria catalyzed by six different enzymes plus a separate acyl carrier protein.
in mammals catalyzed by individual domains of a very large polypeptide that includes an acyl carrier protein 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. The NADPH is produced mainly by the Pentose Phosphate Pathway.

Prosthetic groups of Fatty Acid Synthase include:

the thiol of the side-chain of a cysteine residue in the Condensing Enzyme domain of the complex.
the thiol of phosphopantetheine, which is equivalent in structure to part of coenzyme A (structures at right & below).

Phosphopantetheine (Pant) is covalently linked via a phosphate ester to a serine hydroxyl of the acyl carrier protein domain of Fatty Acid Synthase. The long flexible arm of phosphopantetheine helps its thiol to move from one active site to another within the complex.

Individual steps of the reaction pathway are catalyzed by the catalytic domains of the mammalian Fatty Acid Synthase, listed in the diagrams below.

As each of the substrates acetyl-CoA and malonyl-CoA bind to the complex (designated steps 1 & 2), 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.

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.

In steps 4-6, the b-ketone is reduced to an alcohol, by electron transfer from NADPH.

Dehydration yields a trans double bond.

Reduction at the double bond by NADPH yields a saturated chain.

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

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.

The primary structure of the mammalian Fatty Acid Synthase protein is summarized at right.

Fatty Acid Synthase in mammals is a homo-dimer.

X-Ray crystallographic analysis at 3.2 Å resolution shows the dimeric Fatty Acid Synthase to have an X-shape. The two copies of the protein are displayed at right in different colors.

The domain arrangement is shown at right. Each copy of the dimeric protein has an S shape, with the N-terminal KS (Condensing Enzyme / b-Ketoacyl Synthase) domain folded back to form part of the central interaction domain

The X-ray analysis does not resolve the C-terminal ACP (acyl carrier protein) and Thioesterase domains, which are predicted from the primary structure to be near the b-Ketoacyl Reductase (KR) domains. These domains may be too flexible to be resolved by crystallography.

The Fatty Acid Synthase complex is somewhat asymmetric. There is evidence for conformational changes relating to catalysis. Protein flexibility may facilitate transfer of ACP-attached reaction intermediates among the several active sites in each half of the complex.

For images see:

website at ETH Zurich
website of the Asturias lab at Scripps
article by Maier, Leibundgut & Ban, 2008 (requires subscription to Science).

KR = b-Ketoacyl Reductase;
ER = Enoyl Reductase; DH = Dehydratase;
KS = b-Ketoacyl Synthase (Condensing Enzyme); MAT = Malonyl/Acetyl-CoA Transacylase.

Explore at right the structure of the mammalian Fatty Acid Synthase.

Mammalian Fatty Acid Synthase

Summary of fatty acid synthesis (ignoring H⁺ and water):
acetyl-CoA + 7 malonyl-CoA + 14 NADPH à palmitate + 7 CO₂ + 14 NADP⁺ + 8 CoA

Summary taking into account ATP-dependent synthesis of malonate:
8 acetyl-CoA + 14 NADPH + 7 ATP à palmitate + 14 NADP⁺ + 8 CoA + 7 ADP + 7 P_i

Fatty acid synthesis occurs in the cytosol. Acetyl-CoA generated in the mitochondria is transported to the cytosol via a shuttle mechanism involving citrate (p. 937).

Fatty acid synthesis and b-oxidation pathways may be compared (see also diagram p. 931):

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

Fatty Acid Synthase is transcriptionally regulated.

In liver:
- Fatty Acid Synthase expression is stimulated by insulin, a hormone produced when blood glucose is high. 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 role in regulating 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 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. 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. A family of enzymes designated Fatty Acid Elongases or ELOVL (elongation of very long chain fatty acid) catalyze the initial condensation step.

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., D¹². Thus some polyunsaturated fatty acids are dietary essentials, e.g., linoleic acid, 18:2 cis D^9,12 (18 carbon atoms long, with cis double bonds at carbons 9-10 & 12-13).

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 to form 2 H₂O as a fatty acid is oxidized to form a double bond.

Two electrons pass from NADH to the desaturase via the FAD-containing reductase and cytochrome b₅, the order of electron transfer being:
NADH à FAD à cyt b₅ à desaturase
Two electrons are extracted from the fatty acid as the double bond is formed.

For example the overall reaction for desaturation of stearate (18:0) to form oleate (18:1 cis D⁹) is:

stearate + NADH + H⁺ + O₂ à oleate + NAD⁺ + 2H₂O

Additional material on Fatty Acid Synthesis:
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