biosynthesis fatty acids
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BIOSYNTHESIS
OF FATTY ACIDS
doc. Ing. Zenbia Chavkov, CSc.
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The pathway for the of FAs is
not the reversal of the oxidation pathwayBoth pathways are separated within
different cellular compartments
In humansthe pathway for FA synthesis occurs primarily
in the cytoplasmof the liver and adipose tissue,to a lesser extend in lactating mammary glands,
brain, lungs,
and kidneys
whereas,
oxidation occurs in the mitochondria
-
The other major difference
is the use of nucleotide co-factors
Oxidation of fats involves the reduction of FAD, NAD+
Synthesis of fats
involves the oxidation of NADPH
Both oxidation and synthesis of fatsutilize an activated 2C intermediate,
acetyl-CoA
-
Acetyl-CoAmust be first transported
out of mitochondria
using
citrate shuttle transport system
The total energy requirementfor converting mitochondrial acetyl-CoA
into cytoplasmic acetyl-CoA is
1 ATP
-
Origin of Cytoplasmic Acetyl-CoA
Acetyl-CoAis generated in the mitochondria primarily from the sources:
The pyruvate dehydrogenase (PDH) reaction(glycolysis glucose pyruvic acid acetyl-CoA)
Fatty acid oxidation AAs degradation and ketone bodies
In order to be utilized for fatty acid synthesis
they must be present in the cytoplasm
The shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energy diminishes
-
Source of reducing equivalents
The origin of NADPH for FA biosynthesis
depends on cell type
In liver, the 2 NADPHs come from the pentose phosphate pathway
In adipose tissue,NADPH is generated by malic enzyme
-OOC-CH2-CH-COO- CH3-C-COO
-
| ||
OH O
The pentose phosphate pathway
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Another source for NADPH
for these reactions isthe isocitrate shuttle
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The synthesis of malonyl-CoAis the first committed step of FAs synthesis
Acetyl-CoA carboxylase (ACC),is the major site of regulation of FAs synthesis
ACC requires a biotin co-factor
-
First, CO2 is covalently bound to biotin using energy from hydrolysis of ATP
Then, the CO2 is transferred to acetyl-CoA producing malonyl-CoA
The biotinyl group serves as a temporary carrier of CO2
-
The carboxylation of
acetyl-CoAto form
malonyl-CoA
catalyzed by
acetyl-CoA
carboxylase
is the rate-limiting step of FA biosynthesis
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 may be summarized as:
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Acetyl-CoA Carboxylase activity,
in the mammals 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
-
The rate of fatty acid synthesis is controlled by the equilibrium between
monomeric and polymeric acetyl-CoA carboxylase
(ACC)The activity of ACC requires polymerization
This conformational change is controlled
by local metabolites(citrate, palmitoyl-CoA and other long-chain
fatty acyl-CoAs)
-
Regulation by local metabolites
The equilibrium between monomeric and polymeric
acetyl-CoA carboxylase is
inhibited bypalmitoyl- CoA
(product of FA synthase) other long-chain
fatty acyl-CoAs
enhanced by citrate
(promoting enzyme polymerization)
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Regulation of Acetyl-CoA carboxylase activitythrough
hormone mediated phosphorylation
Glucagon and epinephrinepromote phosphorylation
anddecrease the enzymatic
activity ( )
Insulin promotes dephosphorylation and increases the activity ( )
-
With Acetyl-CoA Carboxylase inhibited,
acetyl-CoA remains available
for ketone bodies synthesis
the alternative metabolic fuelused when blood glucose is low
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Changes in dietaffect the amount of fatty acid biosynthesis
by affecting the amount of acetyl-CoA carboxylase
A dietrich in carbohydrate or low in fatincreases the biosynthesis of the enzyme
by affecting the rate of transcription
Starvation or diet high in fathas the opposite effect and
reduces the rate of synthesis of acetyl-CoA carboxylase
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Synthesis of the Acyl chain
The reactions of FA biosynthesis take place on
a multifunctional protein,called fatty acid synthase (fatty acid synthase complex)
A polyprotein is a single protein with more then 1 activity,
and fatty acid synthase is formed from 2 chains of this protein
The active enzyme is a dimer of identical subunits
-
There is some evidence
that the 2 copies of the multi-domain enzyme
are aligned antiparallel, as below
Pant-SH HS-Cys
Cys-SH HS-Pant
Fatty Acid Synthase dimer
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Fatty AcidSynthase
prosthetic 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
H3N+ C COO
CH2
SH
H
cysteine
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The fatty acid synthase complexcontains 2 types thiol groups
The central thiol,made up of
4phosphopantetheinea derivative of coenzyme A, covalently linked
by a phosphodiester bond to
serine residue of acyl carrier protein, or ACP
The peripheral thiol,belongs to a cysteinyl residue
on ketoacyl-ACP synthase
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Like fat oxidation,
fat synthesis involves 4 enzymatic activities
-keto-ACP synthase,
-keto-ACP reductase,
3-OH acyl-ACP dehydratase
Enoyl-CoA reductase
The two reduction reactions
require NADPH oxidation to NADP+
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The acetyl-CoA are transferred to ACP+ malonyl CoA by the action of:
Acetyl-CoA transacylase Malonyl-CoA transacylase
The attachment of these carbon atoms to ACP
allows them to enter the fatty acid synthesis cycle
During the sequence of reaction, the growing FA takesthe form of a thioester attached to the:
Peripheral SH group of a cysteine residue of the protein
or to the central SH groupof a protein-bound phosphopantetheine
-
The biosynthetic intermediatesdo not diffuse awayfrom the polyprotein
but are passed
from one enzyme active site to the next active site
by acyl carrier protein (ACP)
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Individual steps
of the Fatty Acid Synthase
reaction pathway
In the first reaction, (to initiate biosynthesis)
acetyl-CoA is transferred:
From CoA
To the central SH (thiol) groupof phosphopantetheine
to form a covalent bond with release of CoA
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Then,
the acetyl groupis transferred
to the peripheral SH(thiol) group of a cysteine
Next,
the malonyl group
is transferred to the pantetheine central SH group of ACP, just vacated by the acetyl group
Now the reactants are poised for the first condensation reaction
Peripheral thiol
Central thiol
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In the first step,
the acetyl group and
malonyl groupsare condensed,
with the release of CO2
This forms CONDENSATIONacetoacetate attachedto the pantetheine (central) SH group
The condensation reaction of fatty acid biosynthesis is
catalyzed by -ketoacyl-ACP synthase
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REDUCTION, step 2.
Using NADPH,
acetoacetyl-ACP undergoes a reduction,
yielding -hydroxybutyryl-ACP and NADP+ in reaction
The ketone is reduced to a hydroxyl group,
mediated
by -ketoacyl-ACP reductase
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DEHYDRATION, step 3.
Then the compound is dehydrated
to 2,3-trans-butenoyl-ACP(crotonyl-ACP) catalyzed by
-hydroxyacyl-ACP-dehydratase
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REDUCTION, step 4.
The double bond is reduced by NADPH + H+
in reaction catalyzed by
2,3 trans-enoyl-ACP reductase
to form butyryl-S-ACP
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HLengthened fatty acid chain is then translocated
to the peripheral SH (thiol) group of a cysteineketoacyl ACP synthase
Another malonyl group is added to the central SH (thiol) group
of ACP
This series of reactions form
a 4-carbon acyl group still attached to the phosphopantetheine (central -SH)
-
In the next reaction:
The growing fatty acyl chain is transferred to the
cysteine (peripheral thiol),
Another malonyl group is added to the pantetheine
-SH (central thiol) and cycle begins again
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This cycle of condensation,
reduction, dehydration, reduction
and transfer of the acyl group continues
until chain of 16 carbons has been created
The resulting palmitoyl group - palmitate is released from
the fatty acid synthase complexby an exergonic hydrolysis reaction
Palmitate,a 16-C saturated fatty acid,
is the final product of the FA synthase reactions
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Therefore:
Acetate group is added at the beginning
Then need 1 malonate to extend the chain by 2 carbons
3C = malonate
2C = acetate
1C = CO2
3C2C
1C
FAS FAS
3C
1C
FAS2C
2C
2C
2C
2C
3C
1C
2C
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That fatty acid synthesis by multienzyme complex
stops at palmitate is probably
due to limitation in the size of an active siteof fatty acid synthase
Palmitatecan then undergo separate
elongation and/or unsaturationto yield other fatty acid molecules
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Fatty acid biosynthesis
is energetically expensive
however, occurs when is
abundant precursor
to provide both
the massthe energy
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BIOENERGETICS OF FA BIOSYNTHESIS
1 mole ATP is required for the generation of 1 mole of acetyl-CoA from citrate
7 moles of ATP are required for the transport of acetyl-CoA from mitochondria into cytosol, as a substrate
for the synthesis of malonyl-CoA
7 additional moles of ATP are required for the synthesis of 7 moles of malonyl-CoA
from acetyl-CoA and CO2
A total of 15 ATPequivalents are required
for the synthesis of palmitate from citrate
14 moles of NADPHare required for the biosynthesis of 1 mole of palmitate
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THE REGULATION OF FAT METABOLISM
Occurs via two distinct mechanisms
One is short term regulation which is regulation effected by events such as
substrate availability, allosteric effectors and/or enzyme modification
Control of a given pathways' regulatory enzymes can
also occur by
alteration of enzyme synthesis and turn-over rates of synthesis
These changes are long term regulatory effects
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Insulin stimulates lipogenesis
by several mechanisms
It increases the transport of glucose into cell(in adipose tissue)
and thereby increases the availability of both: pyruvate for FAs synthesis glycerol-3-P for esterification of the newly formed FAs
Insulis converts the inactive form of pyruvatedehydrogenase to the active form(in adipose tissue but not in liver!)
Insulin activates acetylCo-A carboxylase. It involves dephosphorylation by a protein phosphatase
accompanied by change in aggregation of monomers to a more polymeric state
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Insulin by its ability
to depress the level of intracellular cAMP, inhibits lipolysis in adipose tissue
and thereby reduces the concentrationof plasma free FAs and long-chain acyl-CoA,
an inhibitor of lipogenesis
By this same mechanism
insulin antagonizes the action of glucagon and epinephrine,
which inhibit acetyl-CoA carboxylase and therefore lipogenesis,
by increasing cAMP, allowing cAMP dependent protein kinase to inactivate the enzyme by phosphorylation
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Regulation of fat metabolism also occurs through
malonyl-CoA induced inhibition of carnitine acyltransferase I.
This functions
to prevent the newly synthesized FAsfrom entering the mitochondria
and being oxidized
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ELONGATION AND DESATURATION
Stearic are major constituent of FAs
Oleic acids found in human cells
The fatty acid product released
from fatty acid synthase (FAS)
is palmitate
which is a 16:0 fatty acid,
(16 carbons and no sites of unsaturation)
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Although
the FA synthase complex stops at 16 C atoms,
human cells:
Can extend the length of the FA chainPosseses the machinery for converting
saturated to unsaturated FAs
2-carbon units can be added: To endogenously synthesized or dietary fatty acids
by elongation reactions
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ELONGATION AND UNSATURATIONof fatty acids occurs after palmitate (16C)
in both the mitochondriaand endoplasmic reticulum
(microsomal membranes)
The endoplasmic reticulum pathway
is quantitatively more important
This strategy agrees with the role of mitochondria
functioning as a catabolic organell
The substrate for the elongation reactionsis fatty acyl-CoA
and not fatty acyl-ACP
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The endoplasmic reticulum
contains the enzyme activities found in the FA synthase
complex, that succesively
reduce, dehydrate, and reducethe compound
to produce fatty acyl-CoA containing 2 additional carbon atoms
The reactions are analogous to the condensation reactions
that occurs during conventional FA biosynthesis
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The resultant product is 2C longer
The fatty acyl-CoA substratefor the elongation reaction
is malonyl-CoA
More then 1 elongation reaction can occur,
and fatty acids up to 26 C atoms can be synthesized
The reduction reactions of elongation
require NADPH as co-factorjust as for the similar reactions
catalyzed by FAS
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Mitochondrial elongation
Involves acetyl-CoA unitsis a reversal of oxidation
Except that the final reductionutilizes NADPH instead of FADH2
as co-factor
Acetyl-CoA, not malonyl-CoAdonates the 2C units in mitochondria
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C-C-C-C-C-C=C-C-C=C-C-C=C-C-C=C-C-C-C-COOH
C-C-C-C-C-C=C-C-C=C-C-C-C-C-C-C-C-COOH
125818
Animals cannot
put double
bonds in this
part of the
molecule,
plants can!
Essential fatty acids:
Linoleate 18:2(9,12)
Arachidonate
20:4(5,8,11,14)
n : = ( x,y..)1114
191218
-
CH3
COOH
17 15 12 9 7 5 3
(cis 9,12 octadecadienoic acid)Linoleic acid
Since these enzymes cannot introduce sites of unsaturation beyond C9
they cannot synthesize either
linoleate (18:2D9, 12 )linolenate (18:3D9, 12, 15)
These fatty acids must be acquired from the diet and are, therefore, referred to as
essential fatty acids
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These essential FAs are
necessary for normal membrane structure
Linoleic acidespecially important, serves as a precursor
for the synthesis of arachidonic acid,from which
the eicosanoids(the prostaglandins, thromboxanes)
are formed
is also a constituent of epidermal cell
sphingolipidsthat function as
the skins water permeability barrier
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It is this role of FAs in eicosanoid synthesis that leads to
poor growth, wound healingdermatitis
in persons on fat free diets
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Desaturation
occurs in the ER membranes
in mammalian cells involves
4 broad specificity fatty acyl-CoA desaturases
(non-heme iron containing enzymes)
These mixed-function oxidase requireNADPH and molecular oxygen
to add a hydroxyl group to the fatty acid
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Arachidonic acidis produced by
elongation and the addition of 2 double bondsas shown in Fig.
desaturation
Common inhealthy diet
Some availablefrom meat and eggs
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-6 Pathway
Anti-inflammatorymetabolites
Pro-inflammatorymetabolites
Linoleic acid (18:2)
-Linolenic acid (18:3)
Dihomo- -linolenic acid (20:3)
Arachidonic acid (20:4)
Released from stores
(Slow)
Meat andeggs
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Acetyl CoA carboxylase
Irreversible two-step reaction
CO2
BIOTIN
Biotin carrier protein
-O C
O
Lys
C=O
H-N
Biotin carboxylase
C=O
H-N Lys
BIOTIN
TranscarboxylaseO = C
-O
-
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
The primary phosphorylation of ACC occurs through the action of AMP-activated protein kinase, AMPK
This is not the same as cAMP- dependent protein kinase, PKA!
Phosphorylation
causes the filamentous enzyme to dissociate into inactive mononomers