Download Lipid Synthesis 1. Fatty acid synthesis

5-1
Lipid Synthesis
Fatty acids are a more efficient form of energy storage than carbohydrates because they are less
hydrated, as result of fewer hydroxyl groups being available for hydrogen bonding. The energy
content of fat tissue is 38 kJ/gm compared to 17 kJ/gm for carbohydrates.
The processes of fatty acid degradation had been worked out prior to fatty acid synthesis, and there
was some conjecture that perhaps synthesis might simply be the reverse of degradation. Indeed, it
was realized early on that [14C] acetate was a direct precursor for fatty acids providing some
substance to the conjecture (since acetate was the product of β-oxidation).
However, as work progressed a number of significant differences between synthesis and
degradation were noted including: 1. β-oxidation occurs in the mitochondria and synthesis occurs in
the cytoplasm; 2. citrate is required for synthesis as an activator; and 3. CO2 is required for
synthesis but not incorporated.
Ultimately, the principal enzyme fatty acid synthase was isolated and characterized. From
eucaryotes it was found to be a single large protein with several activites whereas in bacteria, it was
a complex of several proteins each with a different activity. Ultimately, the individual activities in the
larger single protein were correlated with those of the separate enzymes, and it was realized that
the overall processes were the same. The simpler bacterial system allowed for a dissection of the
system.
1. Fatty acid synthesis
As with all processes there is a "preparatory" phase in which substrates are prepared to enter the
synthase complex. For lipids, that step involves the carboxylation of acetate in a process that
explains the roles of both citrate and CO2.
1
H 3C
O
ATP
C
SCoA
acetyl CoA
biotin
ADP + Pi
H2
C
-OOC
CO2
AcetylCoA carboxylase
O
C
SCoA
malonyl CoA
1. CO2 is incorporated explaining where it is involved in the process (we will see its release shortly).
2. The carboxylase step is the slowest or rate determining reaction of the synthesis pathway and
the enzyme requires citrate for activity through its involvement in stabilizing the multimeric form of
the enzyme. By controling the slowest reaction, citrate controls fatty acid synthesis.
5-2
monomer
(inactive)
2
multimer - citrate
(active)
Acyl carrier protein
While not specifically part of a preparatory phase, it is necessary to introduce an unusual protein,
acyl carrier protein. In bacteria, it is a small ~10 kDa protein with a 4-phosphopantetheine chain
attached to a specific serine. The 4-phosphopantetheine chain is also a component of CoASH and
its active portion is the -SH. In eucaryotic enzymes, the 4-phosphopantetheine chain is attached to
the larger protein. It is best viewed as a flexible arm that "carries" the growing fatty acid chain from
enzyme to enzyme (bacteria) or active site to active site (eucaryotes).
O
Ser
O
P
O
H2
C
O
CH3
OH
O
C
C
H
C
O
H2
C
H
N
H2
C
C
H2
C
H
N
H2
C
SH
CH3
As with the SH of CoASH, its main role is to form thiol esters with growing fatty acid chains. This
gives rise to the nomenclature, ACP-SH and fatty acyl-ACP.
At this point we can get into the actual series of reactions involved in the synthesis.
3
H 3C
O
ACP-SH
4
CoA-SH
H 3C
C
β-Ketoacyl-ACP
synthase
O
C
Acetyl-CoA-ACP
transacetylase
(once per fatty acid)
SCoA
acetyl-CoA
SACP
Syn-SH
H 2C
O
malonyl-CoA
S-Syn
6
O
β-Ketoacyl-ACP
synthase
C
CoA-SH
H 2C
C
SCoA
ACP-SH
O
CO2 +
Syn-SH
C
Malonyl-CoA-ACP
transacetylase
H+
acetyl-Synthase
-O
ACP-SH
O
C
acetyl-ACP
COO-
5
H 3C
SACP
malonyl-ACP
H 3C
O
C
H 2C
O
C
SACP
acetoacetyl-ACP
(β-ketoacyl-ACP)
5-3
H3C
7
O
NADP+
NADPH + H+
C
H2C
H3C
O
H
C
8
OH
H2C
C
β-Ketoacyl-ACP
reductase
HC
butenoyl-ACP
(trans-Δ2-enoyl-ACP)
β-hydroxybutyryl-ACP
(β-hydroxyacyl-ACP)
C
CO2
NADPH + H+
Enoyl-ACP
reductase
Steps 6 to 9
2NADP+
S-ACP
2NADPH + 2H+
MalonylACP
hexanoyl-ACP
(C6)
β-Ketoacyl-ACP
synthase
S-Syn
CoASH
C
O
SACP
Step 5
butyryl-ACP
(acyl-ACP)
MalonylCoA
Once palmityl-ACP is formed the last step in this series of reactions
involves the cleavage of palmitic acid. Longer chains are produced
usually by a different series of reactions.
(CH2)14CH3
C16
H2C
ACP-SH
C12
C14
CH2
O
C8
C10
CH3
Step 4
C
9
NADP+
(CH2)2CH3
O
C
SACP
The butyryl-ACP is now "equivalent" to acetyl-CoA and returns to step #4
where it is transfered onto the β-ketoacyl-ACP synthase. Then steps #5
to #9 are repeated to generate a C6 hexanoyl-ACP, and so on through
five more rounds (7 in total) until C16 palmitoyl-ACP is formed.
(CH2)4CH3
O
β-Hydroxyacyl-ACP
dehydratase
SACP
acetoacetyl-ACP
(β-ketoacyl-ACP)
CH
O
C
SACP
H3C
H2O
C
H2O
(CH2)14CH3
ACP-SH
O
C
Palmitoyl-ACP
thioesterase
S-ACP
O
O
palmitate
(palmitic acid
if protonated)
palmitoyl-ACP
Overall
7ATP
7 acetyl-CoA
7 ADP + 7Pi
14 NADPH + 14 H+
14 NADP+
7 malonyl-CoA
acetyl-CoA
palmitate
8 CoASH
5-4
or 8 AcCoA + 7 ATP + 14 NADPH
palmitate
CH3-CH2-CH2-CH2- - - - - - - - - - - - - - - - - - - - CH2-COOacetate
malonate
malonate
synthesis
degradation
2. Sources of NADPH
1. from the Pentose Phosphate Pathway in liver cells
2. from malic enzyme in fat cells
malate
pyruvate + CO2
NADP+
NADPH + H+
3. Sources of AcCoA
ethanol
pyruvate
amino acids
acetate
citrate
lipids
AcCoA
CO2 + energy (TCA cycle)
glyoxalate shunt (plants and bacteria)
Citrate?
Citrate synthase is considered irreversible ( a very large negative ∆G'o) in vivo and an alternate
path is required to generate AcCoA from citrate. This involves ATP-citrate lyase and proceeds
because of the involvement of ATP hydrolysis which provides the necessary energy.
ATP-citrate lyase
citrate + CoASH
Acetyl-CoA + OAA
ATP
ADP + Pi
5-5
4. Unsaturated fatty acid synthesis
Unsaturations can be introduced into fatty acids at two different stages: 1. after the saturated fatty
acid is completed and 2. at a step during synthesis.
1. Into the completed fatty acid:
Fattyacyl-CoA monoxygenase
O2
2 H2O
palmitoyl-CoA
palmitoleyl-CoA
(cis ∆9-)
NADH + H+
NAD+
There are 4 such enzymes that introduce the unsaturations at C4, C5, C6 and C9. The enzymes
are also sometimes called terminal desaturases and are part of an electron transport chain that
includes cytochrome b5.
2. At an intermediate step of synthesis:
Essentially, the unsaturation is introduced in a configuration that cannot be reduced during
subsequent steps (bacteria).
OH
H3C(H2C)5
CH
C
H2
H2O
O
O
H
C
C
C
H2
SACP
β-hydroxy decanoylACP dehydratase
HC
C
C
H2
SACP
H3C(H2C)5
β-Hydroxyacyl-ACP
dehydratase
Not a substrate for the
enoyl-ACP reductase
and is taken directly to
step 4 (transferred to the
synthase.
H2O
O
H
C
H3C(H2C)5
C
H2
C
C
H
SACP
cis ∆3-decenoyl-ACP
Syn-SH
β-Ketoacyl-ACP
synthase
ACP-SH
O
normal pathway
H
C
O
HC
H2
C
H3C(H2C)11
C
H2
C
C
H2
palmitoyl-ACP
SACP
H3C(H2C)5
C
C
H2
S-Syn
three rounds of
synthesis adding 6
carbons
5-6
O
H
C
HC
C
(CH2)7
S-ACP
H3C(H2C)5
palmitoleyl-ACP
Lec #16
5. Control of fatty acid synthesis
Fatty acid synthesis is controlled at several levels including enzyme activity regulation,
transcriptional control and hormonal control.
In the case of hormonal control, adrenalin activates protein kinase via adenylate cyclase and
cAMP, the protein kinase, in turn, activates pancreatic lipases and β-oxidation. Insulin formed
under conditions of high glucose, activates synthesis as a means of storing energy in the form of fat.
glycogen (energy storage)
glc6P
-
+
PEP
pyr
malonylCoA
AcCoA
+
fatty acids
(energy storage)
+
OAA
citrate
isocitrate
(energy release)
ATP
This diagram illustrates the central role of citrate in the control of energy metabolism. Not only
does it activate gluconeogenesis for energy storage in carbohydrates, but it activates energy
storage in lipids and inhibits energy release in glycolysis. Under energy rich conditions it can also
act as a source of AcCoA for additional lipid synthesis. Also in the diagram, the activation of
pyruvate carboxylase by AcCoA is noted.
In the following diagram, the complication of the mitochondrial barrier is imposed on the larger
picture of lipid/carbohydrate synthesis and degradation. The main underlying point is that βoxidation occurs in the mitochondria and synthesis occurs in the cytoplasm
Mitochondria
5-7
Cytoplasm
energy rich conditions
TCA Cycle
citrate
citrate
AcCoA
AcCoA
OAA
fatty acyl
CoA
OAA
malate
malate
fatty acids
pyruvate
pyruvate
fatty acyl
carnitine
fatty acyl
carnitine
fatty acyl
CoA
complex lipids
energy poor conditions
It is important to remember that free fatty acids do not exist in large amounts in free form but are
rapidly assimilated into complex lipids, particularly triglycerides and phospholipids.
6. Synthesis of triacylglycerides and phospholipids
CH2OH
ATP
ADP
CH2OH
CHOH
NAD+
NADH + H+
C
CHOH
2
glycerol
Glycerol phosphate
acyl transferases
O
Glycerol-3-phosphate
2
CH2OPO3
dehydrogenase
CH2OPO3
(intestines)
dihydroxyacetone
glycerol
phosphate
phosphate
Glycerol kinase
(liver)
CH2OH
CH2OH
O
2 C
R
SCoA
O
O
CH2O
CR1
CHO
C
R2
O
O
2 CoASH
C
R
O
2
CH2OPO3
diacyl phosphatidate
H2O
Pi
Phosphatidate
phosphatase
CH2O
CR1
CHO
C
R2
CH2OH
diacyl glyceride
Route A to phospholipids
SCoA
CoASH
CH2O
CR1
CHO
C
R2
O
Diacylglyceride
acyl transferase
CH2O
C
R3
O
O
triacyl glyceride
Route B to phospholipids
5-8
(a) Route A to phospholipids
2 Pi
IPPase
O
CR1
CH2O
CHO
H2O
CTP
O
O
NH2
CR1
CH2O
PPi
C
R2
CHO
2
CH2OPO3
diacyl phosphatidate
O
N
CR2
N
2
Phosphatidate cytidylyl
transferase
CH2OPO3PO2
O
O
CDP diacyl glycerol
H
H
H
OH
H
OH
serine
CDP diacyl glycerol
serine-O-phosphatidyl
transferase
O
CR1
CH2O
CHO
CH2O
CR1
NH3
CHO
C
R2
CH2OPO2
O
CO2
O
Phosphatidyl serine
decarboxylase
CH2
OH2C
O
CMP
O
NH3
C
R2
CH2OPO2
OH2C
COO
phosphatidyl serine
phosphatidyl
ethanolamine
(b) Route B to phospholipids
ATP
H2
C
(H3C)3N
ADP
C
H2
(H3C)3N
Choline kinase
choline
2
H2
C
OH
OPO3
C
H2
phosphocholine
CTP
Phosphocholine
CR1
cytidylyl
O
transferase
IPPase
PPi
2 Pi
C
R2
H2O
O
CH2O
O
CH2O
CR1
CHO
C
R2
CH2OPO2
CHO
O
N(CH3)3
CMP
CH2OH
CH2
OH2C
phosphatidyl
choline
(H3C)3N
Phosphocholine
transferase
2
H2
C
OCDP
C
H2
CDP-choline
This route can also generate phosphatidyl ethanolamine.
5-9
7. Synthesis of terpenes and steroids.
acetyl-CoA
O
CoASH
C
H 3C
β-Ketoacyl-CoA
thiolase
SCoA
acetyl-CoA
O
O
C
C
H 3C
C
H2
SCoA
acetoacetyl-CoA
acetyl-CoA
HMG-CoA
synthase
2 NADP+
OH
H 3C
C
CoASH
H2
C
OH
H2
C
H 3C
OH
H 2C
COO-
mevalonate
3 ATP
CoASH
2 NADPH + 2 H+
C
O
H2
C
C
HMG-CoA
H 2C
reductase
COOImportant site of regulation:
(a) inhibited by a cholesterol metabolite hydroxymethyl
(b) activated by insulin
glutaryl-CoA
(c) inhibited by glucagon
(HMG-CoA)
SCoA
3 enzymes
3ADP +Pi
+ CO2
3
H 3C
C
H2
C
H2
C
H 2C
H 3C
3
C
OPO3PO3
CH
OPO3PO3
H 2C
∆3-isopentenyl pyrophosphate
Isopentenyl
pyrophosphate
isomerase
H 3C
∆2-isopentenyl pyrophosphate
The isopentenyl pyrophosphates (isoprene units) are the building blocks for the larger terpenes and
steroids. The steps on the next page outline generally how the larger molecules are generated.
The overall process is as follows:
AcCoA
HMGCoA
isopentenylPP
terpenes
steroids
5-10
monoterpenes (C10)
2 Pi
PPi
OPP
IPPase
OPP
OPP
PPO
Prenyl transferase
geranyl pyrophosphate
sesquiterpenes (C15)
ubiquinones
Prenyl
transferase
carotenoids (C40)
OPP
dimerize
diterpenes (C20)
farnesyl pyrophosphate
Squalene
synthase
(dimerize)
hormones
23 steps
squalene
cholesterol
HO
triterpenes (C30)
bile acids
5-11
Acetyl-CoA
Cholesterol
HMG-CoA
reductase
HDL
(high density
lipoprotein)
Fatty acylcholesterol
acylCoA-cholesterol
acyl transferase
LDL receptors
(uptake into
tissue)
LDL
(low density
lipoprotein)
excretion as
bile acids
Regulation
1. HMG-CoA reductase
(a) insulin activates through dephosphorylation
(b) glucagon inactivates through phosphorylation
(c) cholesterol metabolites inhibit through stimulation of proteolysis
(d) transcriptional regulation
2. AcylCoA cholesterol acyl transferase is activated by cholesterol
3. LDL receptor synthesis is repressed by intracellular cholesterol.
A common cause of high blood cholesterol possibly leading to atherosclerosis is a genetic
predisposition to low LDL receptor levels that result in diminished cholesterol uptake.
Some control of this is possible through compactin or lovostatin, drugs which inhibit HMG-CoA
reductase, or resins which bind to bile acids removing them from the metabolic system ,
thereby displacing the pathway to convert more cholesterol into bile acids.
8. Summary
1. Fatty acid synthesis
2. Unsaturated fatty acid synthesis
3. Regulation by and importance of citrate
4. Triglyceride and phospholipid synthesis
5. Terpene and steroid synthesis.
Lec #17