Lipid Metabolism 2: Acetyl-CoA carboxylase, fatty acid synthase reaction, and regulation of fatty acid synthesis

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Lipid Metabolism 2Acetyl-CoA carboxylase, fatty
acid synthase reaction, and regulation of fatty
acid synthesis
Bioc 460 Spring 2008 - Lecture 36 (Miesfeld)
The fatty acid synthase enzyme in eukaryotes is
dimer of two very large polypeptide chains, each
encoding seven functional units
C247 is a fatty acid synthase inhibitor that
reduces breast cancer incidence in mice
AMP-activated kinase (AMPK) is a regulator of
acetyl-CoA carboxylase
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Key Concepts in Lipid Metabolism

• Fatty acid synthesis and degradation have several
  similarities and many differences. Both require
  carrier molecules and the enzymology of adding or
  subtracting acetate units to a hydrocarbon chain
  are similar. However, synthesis takes place in
  the cytosol, uses NADPH as coenzyme in redox
  reactions, and the building block is malonyl-CoA.
  
• Acetyl-CoA carboxylase is the key regulated
  enzyme in fatty acid synthesis and is responsible
  for generating malonyl-CoA in a carboxylation
  reaction using acetyl-CoA. Acetyl-CoA
  carboxylase activity is regulated by both
  allostery (metabolic signaling) and
  phosphorylation (hormonal signaling).
• The fatty acid synthase protein complex consists
  of six enzymatic activities and the acyl carrier
  protein (ACP). Seven reaction cycles are
  required to synthesize palmitate (C16) from 1
  acetyl-CoA and 7 malonyl-CoA at cost of 14 NADPH.
  
• The citrate shuttle is responsible for moving
  acetyl-CoA equivalents from the mitochondrial
  matrix to the cytosol when glucose levels are
  high and the citrate cycle is feedback inhibited
  by a high energy charge in the cell.

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• Comparison of fatty acid synthesis and degradation

While the chemistry of the four core reactions
required for the removal or addition of C2 acetyl
groups to the hydrocarbon chain are similar
between fatty acid degradation and synthesis, the
two pathways are in fact quite distinct in terms
of the required enzymes, subcellular location and
source of redox energy. Fatty acid degradation
occurs in the mitochondrial matrix and utilizes
FAD and NAD as the oxidants in two oxidation
reactions, whereas, fatty acid synthesis occurs
in the cytosol and is dependent on NADPH serving
as the reductant in the two corresponding
reduction reactions. Other differences are listed
below.
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Review of Pathway Questions

• 1. What purpose does fatty acid synthesis serve
  in animals?
• Fatty acid oxidation in mitochondria is
  responsible for providing energy to cells when
  glucose levels are low. Triacylglycerols stored
  in adipose tissue of most humans can supply
  energy to the body for 3 months during
  starvation.
• Fatty acid synthesis reactions in the cytosol of
  liver and adipose cells convert excess acetyl CoA
  that builds up in the mitochondrial matrix when
  glucose levels are high into fatty acids that can
  be stored or exported as triacylglycerols.

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Review of Pathway Questions

• 2. What is the net reaction in the synthesis C16
  palmitate?
• Fatty acid oxidation
• Palmitate 7 NAD 7 FAD 8 CoA 7 H2O ATP
  ? 8 acetyl CoA 7 NADH 7 FADH2 AMP 2
  Pi 7 H
• Fatty acid synthesis8 Acetyl CoA 7 ATP 14
  NADPH 14 H ? Palmitate 8 CoA 7 ADP
  7 Pi 14 NADP 6 H2O

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Review of Pathway Questions

• 3. What are the key enzymes in fatty acid
  synthesis?Acetyl CoA carboxylase - catalyzes
  the commitment step in fatty acid synthesis using
  a biotin-mediated reaction mechanism that
  carboxylates acetyl-CoA to form the C3 compound
  malonyl-CoA. The activity of acetyl CoA
  carboxylase is regulated by both reversible
  phosphorylation (the active conformation is
  dephosphorylated) and allosteric mechanisms
  (citrate binding stimulates activity,
  palmitoyl-CoA inhibits activity). Fatty acid
  synthase - this large multi-functional enzyme is
  responsible for catalyzing a series of reactions
  that sequentially adds C2 units to a growing
  fatty acid chain covalently attached to the
  enzyme complex. The mechanism involves the
  linking malonyl-CoA to an acyl carrier protein,
  followed by a decarboxylation and condensation
  reaction that extends the hydrocarbon chain.

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Acetyl-CoA carboxylase catalyzes the commitment
step in fatty acid synthesis which converts
acetyl-CoA to malonyl-CoA
Malonyl-CoA serves as the donor of C2 acetyl
groups during each round of the fatty acid
synthesis reaction cycle. The E. coli acetyl CoA
carboxylase enzyme consists of three subunits
which encode a biotin carboxylase, a biotin
carrier protein and a transcarboxylase.
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Acetyl-CoA carboxylase catalyzes the commitment
step in fatty acid synthesis which converts
acetyl-CoA to malonyl-CoA
In the first step, the biotin carboxylase subunit
of the enzyme uses ATP to form carboxyphosphate
which is then dephosphorylated to drive the
formation of carboxybiotin. The carboxybiotin
arm then swings across the enzyme complex and
positions the carboxyl group in a second active
site where the transcarboxylase subunit transfers
the carboxyl group from carboxybiotin to acetyl
CoA to form the reaction product malonyl
CoA.This same carboxyl group used to form
malonyl CoA from acetyl CoA is removed by
decarboxylation in step 4 of the fatty acid
synthesis reaction cycle (decarboxylation is a
highly exergonic reaction). Therefore, malonyl
CoA essentially serves as the "activated"
carboxylated form of acetyl CoA.
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The swinging arm mechanism of acetyl-CoA
carboxylase
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The fatty synthesis reaction cycle
The four core reactions of fatty acid degradation
and fatty acid synthesis are chemically similar
although different enzymes are utilized and the
two pathways are physically separated
(degradation takes place in the mitochondrial
matrix and fatty synthesis is a cytosolic
pathway). Acetyl CoA enters the reaction cycle
through malonyl CoA which is covalently linked to
acyl carrier protein (ACP) through a thioester.
Following decarboxylation of the malonyl group,
and condensation with the enzyme-bound fatty acyl
group, the extended hydrocarbon chain is
chemically modified and then translocated from
ACP back to the fatty acid synthase enzyme.
The reduced ACP thiol is then ready to accept
another malonyl group and start the cycle over
again.
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The fatty synthesis reaction cycle
Acetyl-CoA is the priming group only in the first
cycle, after that, only malonyl-CoA is added to
the ACP carrier protein each time. There are
four reaction steps required each cycle to result
in the net addition two carbons to the growing
fatty acid chain.
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The fatty synthesis reaction cycle
Each cycle of the fatty acid synthase reaction
requires the input of one malonyl-CoA and the
oxidation of 2 NADPH molecules (4 e- total). The
synthesis of C16 palmitate therefore requires 14
NADPH.
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The fatty synthesis reaction cycle
In the final step, the enzyme palmitoyl
thioesterase catalyzes a hydrolysis reaction to
release palmitate.
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The fatty synthesis reaction cycle
Let us take a closer look at these reaction steps
to see just how cool this fat making protein
machine really is. In the first step of
palmitate synthesis, an acetyl-CoA is used as a
primer before the addition of the first
malonyl-CoA. This "priming" reaction is mediated
by the enzyme malonyl/acetyl CoA-ACP
transacetylase (MAT) and only occurs during the
first cycle of the reaction pathway. The sulfur
atom in ACP is located at the end of a
phosphopantetheine prosthetic group which is
linked to a serine residue in the ACP protein.
In step 2, the acetyl group attached to ACP is
translocated to the thiol group of a cysteine
residue in the ?-ketoacyl-ACP synthase (KS)
subunit This translocation reaction is catalyzed
by the KS enzyme itself and is required during
each turn of the cycle.
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The fatty synthesis reaction cycle
Acetyl-CoA is added first to the ACP and then
transferred to the KS subunit (not shown here).
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The fatty synthesis reaction cycle
The condensation reaction between the acetyl
group on the KS subunit and the malonyl group on
the ACP carrier protein is catalyzed by the
?-ketoacyl-ACP synthase (KS) subunit in which the
acetyl group is transferred to malonyl-ACP in a
decarboxylation reaction leading to the formation
of acetoacetyl-ACP. Note that in subsequent
cycles of the reaction, the growing fatty acyl
chain is linked to the KS subunit and used in the
condensation reaction with the malonyl group on
ACP. In the next reaction, acetoacetyl-ACP is
then converted to D-3-hydroxybutyryl-ACP through
a reduction reaction catalyzed by
?-ketoacyl-ACP-reductase (KR) and NADPH
oxidation. This is followed by a dehydration
reaction catalyzed by ?-hydroxyacyl-ACP-dehydratas
e (DH) to form ?,?-trans-butenoyl-ACP, and a
second NADPH-dependent reduction reaction
catalyzed by the enzyme enoyl-ACP-reductase (ER)
leading to the formation of butyryl-ACP.
Lastly, the butyryl group is translocated to
Cys163 of the KS subunit to regenerate ACP-SH
which is then ready to accept another malonyl
group in the next cycle.
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Malonyl-CoA is always the incoming group and the
condensation reaction, and subsequent
modification reactions take place on the ACP
carrier protein. In the last step (step 5 here),
the extended chain is translocated to the KS
subunit to make room for then next malonyl group.
In the final reaction, palmitate is release from
ACP by the enzyme palmitoyl thioesterase (TE)
which hydrolyzes the fatty acid and regenerates
the SH group on ACP.
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Summary of the fatty synthesis pathway
We can now put the entire fatty acid synthesis
pathway together by looking at the ATP and NADPH
requirements for synthesizing one molecule of the
C16 fatty acid palmitate from eight molecules of
the C2 metabolite acetyl CoA. We begin by
forming seven molecules of malonyl CoA using the
acetyl CoA carboxylase reaction 7 Acetyl CoA
7 CO2 7 ATP --gt 7 malonyl 7 ADP 7 Pi We
then use these seven malonyl CoA molecules for
seven turns of the reaction cycle beginning with
the priming of fatty acid synthase by one
molecule of acetyl CoA 1 Acetyl CoA 7 malonyl
CoA 14 NADPH 14 H --gt
palmitate 7 CO2 8 CoA 14 NADP 6 H2O
There are 7 dehydration steps required for
palmitate, why only 6 net H2O?
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Summary of the fatty synthesis pathway
The net fatty acid synthesis reaction for
palmitate (C18) can then be written as 8 Acetyl
CoA 7 ATP 14 NADPH 14 H --gt
palmitate 8 CoA 7 ADP 7 Pi 14 NADP 6
H2O The 14 NADPH molecules required to
synthesize one molecule of palmitate comes
primarily from the pentose phosphate pathway
(lecture 34), although some NADPH is also
generated by reactions in the citrate shuttle as
described in the next couple of slides.
Write the net reaction for the synthesis of C18
stearate.
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The citrate shuttle transports acetyl-CoA
equivalents from the matrix to the cytosol and
generates NADPH
The majority of acetyl CoA used for fatty acid
synthesis in the cytosol is derived from
reactions that take place in the mitochondrial
matrix. However, mitochondria do not contain an
acetyl CoA transporter, therefore a shuttle
system, called the citrate shuttle, is required
to move the C2 units across the membrane.
Citrate transport out of the mitochondria
provides a mechanism to stimulate fatty acid
synthesis in the cytosol when acetyl CoA
accumulates in the mitochondrial matrix. This
build-up of acetyl CoA occurs when high glucose
levels stimulate the conversion of pyruvate to
acetyl CoA resulting in a high energy charge in
the cell and feedback inhibition of the citrate
cycle reactions. Under these conditions,
citrate synthase produces citrate from acetyl CoA
and oxaloacetate which is then transported to the
cytosol rather than being converted to isocitrate
by the enzyme aconitase.
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The citrate shuttle transports acetyl-CoA
equivalents from the matrix to the cytosol and
generates NADPH
Once in the cytosol, the citrate is cleaved by
the enzyme citrate lyase to generate cytosolic
acetyl CoA and oxaloacetate. The acetyl CoA is
used for fatty acid synthesis and the
oxaloacetate is converted to malate by cytosolic
malate dehydrogenase.
The production of cytosolic NADPH by malic enzyme
provides additional reducing equivalents for
fatty acid synthesis and supplements the NADPH
generated by the pentose phosphate pathway.
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Regulation of fatty acid synthesis
The primary control point for regulating flux
through the fatty acid biosynthetic pathway is
the modulating the activity of acetyl CoA
carboxylase. The activity of acetyl CoA
carboxylase is controlled by both allosteric
mechanisms (metabolic control) and covalent
modification (hormonal control). Acetyl CoA
carboxylase is most active when it is in a
homopolymeric form. Citrate and palmitoyl CoA
are metabolites that bind to an allosteric site
on the enzyme stimulating polymerization or
depolymerization, respectively.
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Metabolic regulation of acetyl-CoA carboxylase
Allosteric regulation of acetyl CoA carboxylase
activity makes sense because when cytosolic
citrate levels are high it means that the citrate
shuttle is active and fatty acid synthesis is
favored.
However, when palmitoyl-CoA levels in the cytosol
are high, it serves as a feedback inhibitor to
decrease flux through the fatty acid synthesis
pathway.
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Hormonal regulation of acetyl-CoA carboxylase
Hormone signaling also regulates the activity of
acetyl CoA carboxylase. Insulin signaling leads
to dephosphorylation and enzyme activation
(polymerization), whereas, glucagon signaling
results in phosphorylation and enzyme
inactivation (monomeric form).
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Regulation of acetyl-CoA carboxylase activity
Insulin activates acetyl CoA carboxylase activity
by stimulating dephosphorylation through protein
phosphatase 2A (PP2A). In contrast, glucagon and
epinephrine signaling activate the enzyme
AMP-activated protein kinase (AMPK) which
phosphorylates acetyl CoA carboxylase and shifts
the equilibrium to the inactive monomeric form.
Insulin signaling is activated by high serum
glucose levels, and therefore activation of
acetyl CoA carboxylase activity ensures that
excess glucose will be rapidly converted to fatty
acid for long term energy storage. Similarly,
glucagon or epinephrine signaling is activated by
low serum glucose levels, or neuronal input,
respectively, and they lead to inhibition of
acetyl CoA carboxylase activity to spare glucose
for other purposes. Importantly, citrate binding
to phosphorylated acetyl CoA carboxylase can
result in partial enzyme activation by shifting
the equilibrium in favor of polymer formation.
This mechanism provides a way for the cell to
respond to short term metabolic changes (excess
citrate) by stimulating fatty acid synthesis even
before long term hormone signaling is activated.
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AMPK is an important metabolic sensor
The regulatory protein AMPK is activated by low
energy charge in the cell (high levels of AMP).
The activity of AMPK is regulated by both AMP
binding and by phosphorylation at a highly
conserved threonine residue. The enzyme that
phosphorylates AMP kinase is functionally
referred to as AMP kinase kinase (AMPKK). When
the energy charge in the cell is low, then AMPKK
activity is stimulated by AMP binding, leading to
activation of AMPK and inhibition of acetyl CoA
carboxylase. However, when glucose levels are
high, insulin signaling stimulates the activity
of protein phosphatase 2C (PP2C), resulting in
dephosphorylation of AMPK and accumulation of
active acetyl-CoA carboxylase. The net result
is an increase in fatty acid acid synthesis which
makes sense because when glucose levels are high,
it is important to store stimulate fatty acid
synthesis.
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AMPK is an important metabolic sensor
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Three Metabolic Control Points of FA Synthesis

• There are three metabolic control mechanisms that
  regulate flux through the fatty acid synthesis
  pathway.
• Excess acetyl CoA in the mitochondria results in
  citrate export to the cytosol which activates
  acetyl CoA carboxylase activity (stimulates
  enzyme polymerization), thereby producing malonyl
  CoA.
• Malonyl CoA inhibits carnitine acyltransferase I
  activity to prevent mitochondrial import and
  degradation of newly synthesized fatty acyl CoA
  molecules.
• When palmitoyl CoA levels exceed the metabolic
  needs of the cell, feedback inhibition of acetyl
  CoA carboxylase activity by palmitoyl CoA
  (stimulates enzyme depolymerization) decreases
  flux through the fatty acid synthesis pathway.

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Three Metabolic Control Points of FA Synthesis
What is the likely metabolic fate of the
palmitoyl-CoA if this were a liver cell? What
if it were a fat cell?