18. Fatty Acid Synthesis

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Fatty Acid Synthesis

Molecular Biochemistry II Rohit

Jhawer

I have reviewed

this document

2006.10.14

19:03:40 +05'30'


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ATP-dependent carboxylation provides energy input.

The CO2 is lost later during condensation with thegrowing fatty acid.

The spontaneous decarboxylation drives the

condensation reaction.

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

The input to fatty acid

synthesis is acetyl-CoA,

which is carboxylated to

malonyl-CoA.


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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 carboxylgroup to acetyl-CoA at a second active site 2 .

Acetyl-CoA

Carboxylasecatalyzes the

2-step reaction

by whichacetyl-CoA is

carboxylated

to formmalonyl-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


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The overall reaction, which is spontaneous, may be

summarized as:

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

ll

Enzyme-biotin

HCO3-

+ ATP ADP + Pi Enzyme-biotin-CO2

-

OCH3-C-SCoA

acetyl-CoA

O

-O2C-CH2-C-SCoA

malonyl-CoA

ll

Enzyme-biotin

1

2


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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 translocatebetween the 2 active sites.

CHCH

H2C

S

CH

NH

C

N

O

(CH2)4 C NH (CH2)4 CH

CO

NH

O

CO

O

Carboxybiotin lysine

residue


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Acetyl-CoA Carboxylase, which converts acetyl-CoAto malonyl-CoA, is the committed step of the fatty acid

synthesis pathway.

The mammalian enzyme is regulated, by

phosphorylation

allosteric regulation by local metabolites.

The active conformation of the enzyme associates in

multimeric filamentous complexes.The inactive conformation of the enzyme exists as

individual protomers.


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dissociate into inactive monomers.

This prevents energy-utilizing fatty acid synthesis when

cellular energy stores are depleted (when ATP has beendephosphorylated all the way to AMP).

AMP-Activated

Kinase catalyzesphosphorylation

of Acetyl-CoA

Carboxylase,causing

inhibition.

Phosphorylationcauses the

filamentous

enzyme to

Phosphorylated protomer of

Acetyl-CoA Carboxylase (inactive)

Dephosphorylated Polymer ofAcetyl-CoA Carboxylase (active)

Citrate

Dephosphorylated,

e.g., by insulin-

activated Protein

Phosphatase

Palmitoyl-CoA

Phosphorylated, e.g., via

AMP-activated Kinasewhen cellular stress or

exercise depletes ATP.

Regulation of Acetyl-CoA Carboxylase


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In such tissues malonyl-CoA, produced via one isoform

of Acetyl-CoA Carboxylase, functions mainly as aninhibitor of fatty acid oxidation.

When AMP is high (ATP low), malonyl-CoA production isdiminished, releasing fatty acid oxidation from inhibition.

The role of AMP-Activated Kinase is

significant even in

tissues (e.g., cardiacmuscle) that do not

significantly synthesize

fatty acids.

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA

ATP + HCO3

ADP + Pi

Acetyl-CoACarboxylase

(inhibited by

AMP-ActivatedKinase)


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A cAMP cascade, activated by glucagon & epinephrinewhen 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, thealternative metabolic fuel used when blood glucose is low.

H3C C SCoA

O

CH2 C SCoA

O

OOC

acetyl-CoA

malonyl-CoA


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The antagonistic effect of insulin, produced when

blood glucose is high, is attributed to activation ofProtein Phosphatase.




Citrate

Dephosphorylated,

e.g., by insulin-activated Protein

Phosphatase

Palmitoyl-CoA

Phosphorylated, e.g., via

AMP-activated Kinasewhen cellular stress or




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Citrate activates Acetyl-CoA Carboxylase, promoting

activation & enzyme polymerization. [Citrate] is high

when there is adequate acetyl-CoA entering Krebs Cycle.Excess acetyl-CoA is converted to fatty acids for storage.

Regulation by

local metabolites:

Palmitoyl-CoA

(product of Fatty

Acid Synthase)promotes the

inactiveprotomer

state of Acetyl-CoA Carboxylase

(feedback

inhibition).




Citrate

Dephosphorylated,

e.g., by insulin-

activated Protein

Phosphatase

Palmitoyl-CoA

Phosphorylated, e.g., viaAMP-activated Kinase

when cellular stress or




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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 asingle large polypeptide.

Evolution of the mammalian Fatty Acid Synthaseapparently 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.

SHH


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Fatty Acid

Synthaseprosthetic groups:

the thiol of the side-

chain of a cysteineresidue 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


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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 movefrom 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

serineresidue

phosphopantetheine

of acyl carrier protein

phosphate


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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 domainsalong the primary sequence of the protein is

summarized below.

There is still debate over the arrangement of domains in3D within the complex. An atomic resolution structure

of the entire complex has not yet been achieved.

Condensing Malonyl/acetyl-CoA Dehydratase Enoyl -Ketoacyl ACP ThioesteraseEnzyme (Cys) Transacylase (Ser) Reductase Reductase (Pant)

N- -C

Order of domains in primary structure of mammalian Fatty Acid Synthase


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As each of the substrates acetyl-CoA & malonyl-CoAbind 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 phosphopantetheinethiol of the acyl carrier protein (ACP) domain.

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


N- -C



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The condensation reaction (step 3) involvesdecarboxylation 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

CH3

Pant

S

Cys

S

CH3CH2

COO

Pant

S

Cys

SH

C

CH2

C

O

CH3

O

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

C O C OC O

1 2 3

1 Malonyl/acetyl-CoA-ACP Transacylase

2 Malonyl/acetyl-CoA-ACP Transacylase

3 Condensing Enzyme (-Ketoacyl Synthase)

NADPH NADP+NADPH NADP+ H O


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4. The -ketone is reduced to an alcohol by e transferfrom 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 NADPNADPH NADP

C

CH

HC

O

CH3

C

CH2

CH2

O

CH3

OH

H2O

4 5 6

4 -Ketoacyl-ACP Reductase5 -Hydroxyacyl-ACP Dehydratase

6 Enoyl-ACP Reductase


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Following transfer of the growing fatty acid from

phosphopantetheine to the Condensing Enzyme's

cysteine sulfhydryl, the cycle begins again, with anothermalonyl-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).


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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 acidpalmitate is the final

product of the Fatty Acid Synthase complex.


N- -C



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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 substratetransfers also occur.

Pant-SH HS-Cys

Cys-SH HS-Pant

Fatty Acid Synthase dimer


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Explore with Chime the structure of theE. coli

-Ketoacyl-ACP Synthase III, equivalent to the

domains of the mammalian Fatty Acid Synthasethat catalyze the initial acetylation and

condensation reactions.


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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 + 14NADP+ + 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.

O id i & F A id S h i


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-Oxidation & Fatty Acid Synthesis

Compared Oxidation Pathway Fatty Acid Synthesispathwaylocation mitochondrial matrix cytosol

acyl carriers

(thiols) Coenzyme-A

phosphopantetheine

(ACP) & cysteine

e acceptors/donor FAD & NAD+ NADPH

-OHintermediate L D

2-C product/donor acetyl-CoAmalonyl-CoA

(& acetyl-CoA)


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In fat cells:

Expression of SREBP-1 and of Fatty Acid Synthase is

inhibitedby leptin, a hormone that has a role inregulating 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 acidsynthesis.


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Elongationbeyond 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, exceptthat NADPH serves as electron donor for the final

reduction step.

Polyunsaturated fatty acids esterified to coenzyme Aare substrates for the ER elongation machinery,

which uses malonyl-CoA as donor of 2-carbon units.


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O


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Formation of a double bond in a fatty acid involves thefollowing 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 ora 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


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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.

2epass from NADH to the desaturase via theFAD-containing reductase & cytochrome b5, the

order of electron transfer being:

NADH FAD cyt b5 desaturase

2e are extracted from the fatty acid as the doublebond 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

18. Fatty Acid Synthesis

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