Download FATTY ACID METABOLISM

FATTY ACID
METABOLISM
1
FATTY ACID METABOLISM
 A fatty acid is a carboxylic acid.
often with a long unbranched aliphatic chain, which
is either saturated or unsaturated.
Carboxylic acids as short as butyric acid (4 carbon
atoms) are considered to be fatty acids,
Fatty acids are produced by the hydrolysis of the
ester linkages in a fat or biological oil (both of
which are triglycerides), with the removal of
glycerol.
The most abundant natural fatty acids have an even
number of carbon atoms.
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Major Physiological Roles oF Fatty Acids
1. Fatty Acids (FAs) are building blocks of phospholipids and
glycolipids.
2. Many proteins are modified by the covalent attachment
of FAs, which targets them to membrane locations.
3. FAs are fuel molecules that are stored as triacylglycerols
(or triglycerides) (TGs).
4. FAs derivatives serve as hormones and intracellular
messengers.
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In mammals, the major site of
accumulation of triacylglycerols
is the cytoplasm of adipose cells
(fat cells)
4
• Fatty acid
degradation and
synthesis are
relatively simple
processes that are
essentially the
reverse of each
other.
5
TGs Are Highly Concentrated
Energy Stores
• 9 Kcal/g VS 4 Kcal/g for protein/carbohydrates.
• Anhydrous storage in adipose VS hydrated glycogen in
liver/muscle
• A typical 70kg man would have fuel reserves:
100,000 kcal in TGs, 25,000 kcal in protein (mostly in muscle),
600 kcal in glycogen 40 kcal in glucose.
• TGs constitute about 11 kg of his total body weight.
• If the 11kg TGs to be stored in Glycogen form, his total
body weight would be 55kg greater (11+55 kg) + 59.
• Storing energy in TGs saves 55Kg of body weight.
• Glycogen & Glucose provide energy for short-term (a day
or so), while TGs support the body with energy for
several weeks.
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Dietary Lipids Are Digested by
Pancreatic Lipases
• TGs in the intestinal lumen are incorporated into micelles
formed with the aid of bile salts (e.g. glycocholate)
• Amphipathic molecules synthesized from cholesterol in the
liver and secreted from the gall bladder.
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• Incorporation of TGs into micelles orients the ester bonds of
the TGs toward the surface of the micelle.
• This renders the bonds more susceptible to digestion by
pancreatic lipases that are in aqueous solution.
• The digestion products are carried in micelles to the
intestinal epithelium where they are absorbed across the
plasma membrane.
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Dietary Lipids Are Transported in
Chylomicrons
• In the intestinal mucosal cells, the TGs are re-synthesized
from FAs and monoacylglycerols.
• Then packaged into chylomicrons.
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Bile salt
Dietary
TGs
Other lipids &
proteins
micelles
H2 O
Pancreatic
Lipase
Chylomicrons
Lymph
system
FAs
TGs
Monoglyceroles
Intestinal
lumen
Intestinal
mucosa
Storage in adipose
tissue
Monoglyceroles
TGs
FAs
lipoprotein
lipases
b-oxidation in
muscle
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The Utilization of Fatty Acids as Fuel
Requires Three Stages of Processing
1. The lipids must be mobilized:
–
–
TGs are degraded to FAs and glycerol
They are released from the adipose tissue and transported
to the energy-requiring tissues.
2. At these tissues, the fatty acids must be activated and
transported into mitochondria for degradation.
3. The fatty acids are broken down step-by-step into acetyl
CoA, which is then processed in the citric acid cycle.
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1. Triacylglycerols Are Hydrolyzed by
Cyclic AMP-Regulated Lipases
epinephrine, norepinephrine, glucagon,
adrenocorticotropic hormone
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Glycerol formed by lipolysis is absorbed by the liver
where it enters glycolysis or gluconeogenesis.
phosphatase
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2. Fatty Acids Are Linked to CoA Before
They Are Oxidized
• Fatty acids are oxidized in mitochondria.
• They are activated before they enter the mitochondrial
matrix.
• ATP drives the formation of a thioester linkage between
the carboxyl group of a FA and the sulfhydryl group of
CoA.
• This activation reaction takes place on the outer
mitochondrial membrane, where it is catalyzed by acyl
CoA synthase (also called fatty acid thiokinase).
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The activation of a fatty acid is
accomplished in two steps:
1.
2.
The FA reacts with ATP to form an acyl adenylate.
The sulfhydryl group of CoA then attacks the acyl
adenylate, which is tightly bound to the enzyme, to
form acyl CoA and AMP.
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• These partial reactions are freely reversible
• One high-transfer-potential compound is cleaved (between
PPi and AMP) and one is formed (the thioester acyl CoA).
• How is the overall reaction driven forward?
• The answer is that PPi is rapidly hydrolyzed by a
pyrophosphatase
• The equivalent of two molecules of ATP are consumed.
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Carnitine Carries Long-Chain Activated
Fatty Acids into the Mitochondrial Matrix
• Activated long-chain FAs are transported across the
membrane by conjugating them to carnitine, a zwitterionic
alcohol.
• This reaction is catalyzed by carnitine acyltransferase I (also
called carnitine palmitoyl transferase I), bound to the outer
mitochondrial membrane.
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• Acyl carnitine is shuttled across
the inner mitochondrial
membrane by a translocase
• The acyl group is transferred back
to CoA on the matrix side of the
membrane.
• This reaction, which is catalyzed by
carnitine acyltransferase II
(carnitine palmitoyl transferase II),
is simply the reverse of the
reaction that takes place in the
cytosol.
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• Normally, the transfer of an acyl group from an alcohol to a
sulfhydryl group is thermodynamically unfavorable.
• However equilibrium constant for this reaction for carnitine
is near 1.
• Apparently because carnatine and its esters are solvated
differently from most other alcohols and their esters due to
the zwitterionic nature of carnitine.
• As a result, the O-acyl link in carnitine has a high grouptransfer potential.
• Finally, the translocase returns carnitine to the cytosolic side
in exchange for an incoming acyl carnitine.
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3. Fatty Acid Oxidation
•
Through a sequence of 4 reactions:
1.
2.
3.
4.
Oxidation by FAD
Hydration by H2O
Oxidation by NAD+
Thiolysis by CoA
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1. oxidation of acyl CoA by an acyl CoA
dehydrogenase to give an enoyl CoA
with a trans C2=C3.
electron-transferring
flavoprotein
ETF:ubiquinone reductase, an
iron-sulfur protein
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2. Stereospecific hydration of the
C2=C3 by enoyl CoA hydratase.
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3. A second oxidation reaction converts
the hydroxyl group at C3 into a keto
group and generates NADH.
This oxidation is catalyzed by L-3hydroxyacyl CoA dehydrogenase,
which is specific for the L isomer of the
hydroxyacyl substrate
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4. Thiolysis of 3-ketoacyl CoA by the
thiol group of a second molecule of
CoA yields acetyl CoA and an acyl
CoA shortened by two carbon atoms.
This thiolytic cleavage is catalyzed by bketothiolase.
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• The shortened acyl CoA then
undergoes another cycle of
oxidation, starting with the
reaction catalyzed by acyl CoA
dehydrogenase
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Step
1
Reaction
Enzyme
Fatty acid + CoA + ATP
Acyl CoA synthetase [also called fatty acid
thiokinase and fatty acid:CoA ligase (AMP)]
acyl CoA + AMP + PPi
2
Carnitine + acyl CoA
3
Acyl CoA + E-FAD
trans-
Carnitine acyltransferase (also called carnitine
acyl carnitine + CoA palmitoyl transferase)
D2
-enoyl CoA + E-FADH2
Acyl CoA dehydrogenases (several isozymes having
different chain-length specificity)
4
trans-D2 -Enoyl CoA +H2O
5
L-3-Hydroxyacyl CoA + NAD+
L-3-Hydroxyacyl CoA dehydrogenase
3-ketoacyl CoA + NADH + H+
6
3-ketoacyl CoA + CoA
b-Ketothiolase (also called thiolase)
acetyl CoA + acyl CoA (shortened by C2)
Enoyl CoA hydratase (also called crotonase or 3L-3-hydroxyacyl CoA hydroxyacyl CoA hydrolyase)
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The Complete Oxidation of Palmitate
Yields 106 Molecules of ATP
• The degradation of palmitoyl CoA (C16-acyl CoA) requires
seven reaction cycles.
• In seventh cycle, C4-ketoacyl CoA is thiolyzed to two
molecules of acetyl CoA.
• Hence, the stoichiometry of oxidation of palmitoyl CoA is:
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• Approximately 2.5 of ATP are generated per each of the 7
molecules of NADH.
• 1.5 of ATP are formed for each of the 7 molecules of FADH2.
• Recall that the oxidation of acetyl CoA by the citric acid cycle
yields 10 molecules of ATP.
• Hence, the number of ATP molecules formed in the oxidation
of palmitoyl CoA is:
–
–
–
–
10.5 from the 7 molecules of FADH2
17.5 from the 7 molecules of NADH
80 from the 8 molecules of acetyl CoA
~2 ATP are consumed in the activation of palmitate
• The total is 106 ATP.
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Certain Fatty Acids Require Additional
Steps for Degradation
1.The oxidation of unsaturated fatty acids
requires additional steps.
2.Fatty acids containing an odd number of
carbon atoms yield a propionyl CoA at
the final thiolysis step.
3.Must be converted into an easily usable
form by additional enzyme reactions.
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1. Unsaturated fatty acids
• Palmitoleate, C16 unsaturated fatty acid has one double
bond between C-9 and C-10, (D9).
• It is activated and transported across the inner mitochondrial
membrane in the same way as saturated fatty acids.
• Palmitoleoyl CoA then undergoes 3 cycles of degradation;
carried out by the same enzymes as in saturated ones.
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• The cis-D3-enoyl CoA formed in
the third round is not a
substrate for acyl CoA
dehydrogenase.
• The presence of a C3=C4
prevents the formation of
another double bond between
C2 and C3.
• An isomerase changes the
position and configuration of
this double bond.
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The polyunsaturated fatty
acid: (linoleate, C18) with
cis-D9 and cis-D12 double
bonds.
The cis-D3 formed after three
rounds of b-oxidation is
converted into a trans-D2 by
the isomerase.
The acyl CoA produced by
another round of b-oxidation
contains a cis-D4 double bond.
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Dehydrogenation of this species by acyl
CoA dehydrogenase yields a 2,4-dienoyl
intermediate, which is not a substrate for
the next enzyme in the b-oxidation pathway.
2,4-dienoyl CoA reductase, uses NADPH
to reduce the 2,4-dienoyl intermediate to
trans-D3-enoyl CoA.
Cis-D3-Enoyl CoA isomerase then converts
trans-D3-enoyl CoA into the trans-D2 form, a
customary intermediate in the b-oxidation
pathway
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• Only two extra enzymes needed for oxidation
of any polyunsaturated fatty acid:
– Odd-numbered double bonds are handled by the
isomerase.
– Even-numbered ones by both reductase and
isomerase.
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Odd-Chain Fatty Acids Yield Propionyl
Coenzyme A in the Final Thiolysis
Step
• Fatty acids having an odd number of carbon
atoms are minor species.
• They’re oxidized in same way as those having
an even number of C.
• Propionyl CoA and acetyl CoA, rather than two
molecules of acetyl CoA, are produced in the
final round of degradation.
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• Propionyl CoA is carboxylated by propionyl CoA carboxylase
yielding D-isomer of methylmalonyl CoA at the expense of ATP
hydrolysis.
– This enzyme is a biotin enzyme that is homologous to
and has a catalytic mechanism like that of pyruvate
carboxylase.
• The D-isomer is racemized into the L-isomer.
• The L-isomer of methylmalonyl CoA is converted by a mutase into
succinyl CoA
– The (-CO-S-CoA) group migrates from C2 to C3 in
exchange for a hydrogen atom.
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Fatty Acids Are Also Oxidized in
Peroxisomes
• Some oxidation takes place in cellular
organelles called peroxisomes that have high
concentrations of catalase.
• Fatty acid oxidation in these organelles, stops
at octanyl CoA.
• It may serve to shorten long chains to make
them better substrates of b-oxidation in
mitochondria.
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• Peroxisomal oxidation differs from b-oxidation in the initial
dehydrogenation reaction.
– In peroxisomes, a flavoprotein dehydrogenase transfers electrons to O2 to
yield H2O2
– Catalase is needed to convert the H2O2 produced in the initial reaction into
H2O and O2 .
• Subsequent steps are identical with their mitochondrial counterparts,
although they are carried out by different isoforms of the enzymes.
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Fats burn in the flame of carbohydrates
• The entry of acetyl CoA into the citric acid
cycle depends on the availability of
oxaloacetate for the formation of citrate.
• Oxaloacetate is normally formed from
pyruvate, the product of glycolysis, by
pyruvate carboxylase
• The concentration of oxaloacetate is lowered
if carbohydrate is unavailable or improperly
utilized.
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In fasting or diabetes
• Oxaloacetate is consumed to form glucose by the
gluconeogenesis.
• Under these conditions, acetyl CoA is diverted to the
formation of acetoacetate and D-3-hydroxybutyrate.
• Acetoacetate, D-3-hydroxybutyrate, and acetone are
often referred to as ketone bodies.
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D-3-Hydroxybutyrate
dehydrogenase
Thiolase
Hydroxymethylglutaryl
CoA synthase
Hydroxymethylglutaryl
CoA cleavage enzyme
41
Ketone Bodies Are a Major Fuel in
Some Tissues
• Acetoacetate and 3-hydroxybutyrate are produced
mainly in the liver mitochondria.
• Acetoacetate and 3-hydroxybutyrate are normal fuels of
respiration and are quantitatively important as sources of
energy.
– Heart muscle and the renal cortex use acetoacetate in
preference to glucose.
– In contrast, glucose is the major fuel for the brain and red
blood cells in normal conditions.
– However, the brain adapts to the utilization of acetoacetate
during starvation and diabetes
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• Acetoacetate can be activated by the transfer of
CoA from succinyl CoA in a reaction catalyzed by a
specific CoA transferase.
• Acetoacetyl CoA is then cleaved by thiolase to
yield two molecules of acetyl CoA, which can then
enter the citric acid cycle.
• The liver has acetoacetate available to supply
other organs because it lacks this particular CoA
transferase.
• Acetoacetate also has a regulatory role:
– High levels of acetoacetate in the blood lead to a
decrease in the rate of lipolysis in adipose tissue.
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Animals Cannot Convert Fatty Acids into
Glucose
• Acetyl CoA cannot be converted into pyruvate or
oxaloacetate in animals.
• Oxaloacetate is regenerated, but it is not formed de novo
when the acetyl unit of acetyl CoA is oxidized by the citric
acid cycle.
• In contrast, plants have two additional enzymes enabling
them to convert the carbon atoms of acetyl CoA into
oxaloacetate.
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Fatty Acids Synthesis
45
Fatty Acids Are Synthesized and
Degraded by Different Pathways
•
Some important differences between the pathways are:
1. Synthesis takes place in the cytosol, in contrast with
degradation, which takes place primarily in the
mitochondrial matrix.
2. Intermediates in fatty acid synthesis are covalently linked to
the sulfhydryl groups of an acyl carrier protein (ACP),
whereas intermediates in fatty acid breakdown are
covalently attached to the sulfhydryl group of coenzyme A.
3. The enzymes of fatty acid synthesis in higher organisms are
joined in a single polypeptide chain called fatty acid
synthase. In contrast, the degradative enzymes do not seem
to be associated.
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4.
The growing fatty acid chain is elongated by the sequential
addition of two-carbon units derived from acetyl CoA.
 The activated donor of two carbon units in the
elongation step is malonyl ACP. The elongation reaction
is driven by the release of CO2.
5. The reductant in fatty acid synthesis is NADPH, whereas the
oxidants in fatty acid degradation are NAD+ and FAD.
6. Elongation by the fatty acid synthase complex stops on
formation of palmitate (C16).
 Further elongation and the insertion of double bonds
are carried out by other enzyme systems.
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The Formation of Malonyl Coenzyme A
Is the Committed Step in Fatty Acid
Synthesis
• Fatty acid synthesis starts with the carboxylation of acetyl
CoA to malonyl CoA by acetyl CoA carboxylase, which
contains a biotin prosthetic group.
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• The carboxyl group of biotin is covalently attached to the eamino group of a lysine residue, as in pyruvate carboxylase.
• A carboxybiotin intermediate is formed at the expense of the
hydrolysis of a molecule of ATP.
• The activated CO2 group in this intermediate is then
transferred to acetyl CoA to form malonyl CoA.
• Acetyl CoA carboxylase is also the essential regulatory
enzyme for fatty acid metabolism.
49
Intermediates in Fatty Acid Synthesis
Are Attached to an Acyl Carrier
Protein
• They are linked to the sulfhydryl terminus of a phosphopantetheine
group, which is, in turn, attached to a serine residue of the acyl carrier
protein.
• Acyl carrier protein (ACP) a single polypeptide chain of 77 residues, can
be regarded as a giant prosthetic group, a "macro CoA."
50
The Elongation Cycle in Fatty Acid
Synthesis
• The enzyme system that catalyzes the synthesis of
saturated long-chain fatty acids from acetyl CoA, malonyl
CoA, and NADPH is called the fatty acid synthase.
• The elongation phase of fatty acid synthesis starts with
the formation of acetyl ACP and malonyl ACP.
• Acetyl transacylase (nonspecific) and malonyl
transacylase (specific) catalyze these reactions.
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 The decarboxylation of malonyl ACP
contributes a substantial decrease in free
energy.
• In effect, ATP drives the condensation
reaction:
1
– ATP is used to carboxylate acetyl CoA to malonyl
CoA.
– The free energy stored in malonyl CoA is
released in the decarboxylation accompanying
the formation of acetoacetyl ACP.
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Acetoacetyl ACP is reduced to D-3hydroxybutyryl ACP.
• This reaction differs from the
corresponding one in fatty acid
degradation in two respects:
– The D rather than the L isomer is formed.
2
– NADPH is the reducing agent, whereas
NAD+ is the oxidizing agent in boxidation.
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 Then D-3-hydroxybutyryl ACP is dehydrated to
form crotonyl ACP, which is a trans-D2-enoyl
3
ACP.
 Crotonyl ACP is reduced to butyryl ACP.
• NADPH is again the reductant, whereas FAD is
the oxidant in the corresponding reaction in boxidation.
4
• The enzyme that catalyzes this step is enoyl
ACP reductase.
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 In the second round of FA synthesis, butyryl ACP
condenses with malonyl ACP to form a C6-b-ketoacyl
ACP.
• The elongation cycles continue until C16-acyl ACP is
formed.
 This intermediate is a good substrate for a thioesterase
that hydrolyzes C16-acyl ACP to yield palmitate and ACP.
FAs with an odd number of carbon atoms are
synthesized starting with propionyl ACP, which is
formed from propionyl CoA by acetyl transacylase.
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Fatty Acids Are Synthesized by a
Multifunctional Enzyme Complex in Eukaryotes
•
Many eukaryotic multienzyme complexes are covalently
linked enzymes in multifunctional proteins:
1. An advantage of this arrangement is that the
synthetic activity of different enzymes is coordinated.
2. A multienzyme complex consisting of covalently
joined enzymes is more stable than one formed by
noncovalent attractions.
3. Intermediates can be efficiently handed from one
active site to another without leaving the assembly.
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Schematic Representation of Animal Fatty Acid Synthase:
Each of the identical chains in the dimer contains three domains
Malonyl
transferase
Thioesterase
2
3
acyl carrier
protein
b-ketoacyl
reductase
dehydratase
enoyl
reductase
1
Condensing enzyme
b-ketoacyl synthase
Acetyl
transferase
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Condensation
Reduction
Dehydration
Reduction
Translocation
New Malonyl unit
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• Five more rounds of condensation and reduction
produce a palmitoyl (C16) chain on the condensing
enzyme.
• Palmitoyl is hydrolyzed to palmitate by the thioesterase
on domain 3 of the opposite chain.
• The flexibility and 20-Å maximal length of the
phosphopantetheinyl moiety are critical for the function
of this multienzyme complex.
– The enzyme subunits need not undergo large structural
rearrangements to interact with the substrate.
– Instead, the substrate is on a long, flexible arm that can reach
each of the numerous active sites.
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The Stoichiometry of Fatty Acid Synthesis:
The stoichiometry of the synthesis of palmitate is:
The equation for the synthesis of the malonyl CoA
used in the preceding reaction is:
Hence, the overall stoichiometry for the synthesis
of palmitate is:
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Citrate Carries Acetyl Groups from
Mitochondria to the Cytosol for Fatty Acid
Synthesis
• Fatty acids are synthesized in the cytosol, whereas acetyl CoA
is formed from pyruvate in mitochondria.
• Carnitine carries only long-chain fatty acids.
• The barrier to acetyl CoA is bypassed by citrate, which carries
acetyl groups across the inner mitochondrial membrane.
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When present
at high conc.
ATP-citrate lyase
Malate dehydrogenase
First TCA
reaction
Malic enzyme
Pyruvate carboxylase
The sum
62
• One molecule of NADPH is generated for each molecule
of acetyl CoA that is transferred from mitochondria to
the cytosol.
• Hence, eight molecules of NADPH are formed when eight
molecules of acetyl CoA are transferred to the cytosol for
the synthesis of palmitate (C16).
• The additional six molecules of NADPH required for this
process come from the pentose phosphate pathway.
63
Acetyl Coenzyme A Carboxylase Plays a Key
Role in Controlling Fatty Acid Metabolism
1. Global Regulation.
–
–
Global regulation is carried out by means of
reversible phosphorylation.
Acetyl CoA carboxylase is switched off by
phosphorylation and activated by
dephosphorylation.
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AMP
(+)
ATP
(-)
Inactivated at low energy
charge
Phosphatases ?
P
Activated by insulin
And inactivated by catablic
hormons (Epinephrin and
Glucagon)
Epinephrine
Glucagon
(-)
PKA
Insulin
65
2. Local Regulation.
–
–
Acetyl CoA carboxylase is allosterically
stimulated by citrate.
Citrate partly reverses the inhibition produced
by phosphorylation.
66
Isocitrate
Citrate
Acetyl CoA
Citrate facilitates the
polymerization of the
inactive octamers into
active filaments.
ATP
(-)
Cytosolic
Citrate
Palmitoyl CoA
Translocase
Mitochondrial
Citrate
Fatty Acids
67
Elongation of Fatty Acids
• The major product of fatty acid synthase is palmitate.
• In eukaryotes, longer fatty acids are formed by reactions catalyzed by
enzymes on the cytosolic face of the endoplasmic reticulum
membrane.
• These reactions add two-carbon units sequentially to the carboxyl ends
of both saturated and unsaturated fatty acyl CoA substrates.
• Malonyl CoA is the two-carbon donor in the elongation of fatty acyl
CoAs.
• Condensation is driven by the decarboxylation of malonyl CoA.
68
Membrane-Bound Enzymes Generate
Unsaturated Fatty Acids
• Double bonds are introduced into long-chain acyl CoAs in
endoplasmic reticulum systems.
• For example: in the conversion of stearoyl CoA into oleoyl
CoA, a cis-D9 double bond is inserted by an oxidase.
69
• Mammals lack the enzymes to introduce double bonds at
carbon atoms beyond C-9 in the fatty acid chain.
• Hence, mammals cannot synthesize linoleate (18:2 cis-D9,
D12) and linolenate (18:3 cis-D9, D12, D15).
• The two essential fatty acids linoleate and linolenate
furnished by the diet are the starting points for the
synthesis of a variety of other unsaturated fatty acids.
70