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8/13/2019 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'
8/13/2019 18. Fatty Acid Synthesis
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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.
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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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).
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., viaAMP-activated Kinase
when cellular stress or
exercise depletes ATP.
Regulation of Acetyl-CoA Carboxylase
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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.
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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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.
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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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