Transcript Lecture 33 - University of Arizona

Lipid Metabolism 2:
Acetyl-CoA carboxylase, fatty acid synthase
reaction, and regulation of fatty acid synthesis
Bioc 460 Spring 2008 - Lecture 36 (Miesfeld)
C247
C247 is a fatty acid
synthase inhibitor that
reduces breast cancer
incidence in mice
The fatty acid synthase enzyme
in eukaryotes is dimer of two
very large polypeptide chains,
each encoding seven functional
units
AMP-activated kinase
(AMPK) is a regulator of
acetyl-CoA carboxylase
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.
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.
Difference
subcellular location
carrier protein
enzymes
redox
building block
FA Synthesis
cytosol
acyl carrier protein (ACP)
all activities on a single polypeptide chain
reductant is NADPH
malonyl CoA (formed from Acetyl CoA)
FA Degra da tion
mitochondrial matrix
Coenzyme A (CoA)
multiple enzymes required
oxidants are NAD+ and FAD
acetyl CoA
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.
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 synthesis:
8 Acetyl CoA + 7 ATP + 14 NADPH + 14 H+ →
Palmitate + 8 CoA + 7 ADP + 7 Pi + 14 NADP+ + 6 H2O
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.
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.
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.
The swinging arm mechanism of acetyl-CoA carboxylase
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.
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.
The fatty synthesis reaction cycle
Each cycle of the fatty acid synthase
reaction requires the input of one malonylCoA and the oxidation of 2 NADPH
molecules (4 e- total). The synthesis of C16
palmitate therefore requires 14 NADPH.
The fatty synthesis reaction cycle
In the final step, the enzyme
palmitoyl thioesterase
catalyzes a hydrolysis reaction
to release palmitate.
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.
The fatty synthesis reaction cycle
Acetyl-CoA is added first to the
ACP and then transferred to the KS
subunit (not shown here).
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-hydroxybutyrylACP through a reduction reaction catalyzed by -ketoacyl-ACP-reductase
(KR) and NADPH oxidation. This is followed by a dehydration reaction
catalyzed by -hydroxyacyl-ACP-dehydratase (DH) to form ,-transbutenoyl-ACP, and a second NADPH-dependent reduction reaction catalyzed
by the enzyme enoyl-ACP-reductase (ER) leading to the formation of butyrylACP.
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.
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.
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 --> 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+ -->
palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O
There are 7 dehydration steps required for palmitate, why only 6 net H2O?
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+ -->
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.
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.
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.
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.
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.
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).
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.
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.
AMPK is an important metabolic sensor
Three Metabolic Control Points of FA Synthesis
There are three metabolic control mechanisms that regulate flux
through the fatty acid synthesis pathway.
1. 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.
2. Malonyl CoA inhibits carnitine acyltransferase I activity to
prevent mitochondrial import and degradation of newly
synthesized fatty acyl CoA molecules.
3. 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.
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?