Lipid Biosynthesis I, II

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 Learning Outcomes:

At the end of this lecture, students should be able to:

1. Describe fatty acids biosynthesis

2. Explain fatty acid stoichiometry
3. Describe polyunsaturated fatty acids and eicosanoids
synthesis
4. Describe triacylglycerols synthesis
5. Describe phospholipids and sphingolipids synthesis

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 Catabolism and anabolism of fatty acids
proceed via different pathways
• Catabolism of fatty acids
– produces acetyl-CoA
– produces reducing power (NADH, FADH2)
– takes place in the mitochondria
• Anabolism of fatty acids
– requires acetyl-CoA and malonyl-CoA
– requires reducing power from NADPH
– takes place in cytosol in animals, chloroplast in plants
Anabolism is not simply the reverse of catabolism.
Biosynthetic pathways typically diverge from breakdown
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pathways to overcome irreversible steps in catabolism
 Fatty Acid Synthesis Occurs in Cell
Compartments Where NADPH Levels Are High
• Cytosol of animal cells have
high NADPH levels

• Sources of NADPH:
– In adipocytes: pentose
phosphate pathway and
malic enzyme NADPH made
as malate converts to pyruvate
+ CO2

– In hepatocytes and
mammary gland: pentose
phosphate pathway
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 Fatty Acid Synthesis Occurs in Cell
Compartments Where NADPH Levels Are High

• 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.

• The additional six molecules of NADPH required for this process

come from the pentose phosphate pathway.

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Subcellular Localization of
Lipid Metabolism

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 Citrate is Shuttled out of Mitochondria

Intermediates of the
citric acid cycle are
drawn off as
precursors in many
biosynthetic
pathways.

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 Acetyl CoA is converted to citrate inside
Mitochondria
• In most eukaryotes, the acetyl-CoA for
lipid synthesis is made in the
mitochondria
– Lipid synthesis occurs in the cytosol
– Acetyl-CoA cannot cross
mitochondrial inner membrane to
the cytosol
• Acetyl-CoA is converted to citrate
– Acetyl-CoA + oxaloacetate  citrate
• First reaction of CAC
• Catalyzed by citrate synthase
• Citrate passes through citrate
transporter 8
 Citrate is cleaved to regenerate acetyl-CoA
• Citrate (now in cytosol) is cleaved
by citrate lyase
– Regenerates acetyl-CoA and
oxaloacetate
– Requires ATP
– Acetyl-CoA can now be used
for lipid synthesis

• What happens to the

oxaloacetate because there is no
oxaloacetate transporter either?

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 Shuttle for Transfer of Acetyl Groups from
Mitochondria to Cytosol
• The net of
transporting one
acetyl-CoA molecule
from mitochondrial
matrix:
– Cost two ATP:
citrate lyase and
pyruvate
carboxylase
– Generate one
NADPH

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 Oxaloacetatecyt is converted to malate
Malate dehydrogenase in cytosol reduces oxaloacetate to malate
Two potential fates for malate:
• First: malate can be converted to pyruvatecyt and NADPHcyt via
the malic enzyme
• NADPH used for lipid synthesis (another major way to
generate NADPH in addition to PPP)
• Pyruvatecyt sent back to mitochondria via pyruvate
transporter
• Pyruvatemt is converted back to oxaloacetatemt by
pyruvate carboxylase, requires ATP
• Second: malate can be transported back to mitochondria via
malate --ketoglutarate transporter
• Malatemt is reoxidized to oxaloacetatemt by malate
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dehydrogenase
 Malonyl-CoA is formed from acetyl-CoA and
bicarbonate in cytoplasm
• Carboxylation of acetyl CoA forming Malonyl-CoA irreversibly
• Catalyzed by acetyl-CoA carboxylase (ACC)
• Reaction occurs in cytoplasm
• Enzyme has three subunits:
– biotin carboxylase: attaches CO2 to a nitrogen biotin ring attached to
biotin carrier protein. ATP is required. HCO3− (bicarbonate) is the
source of CO2
– biotin carrier protein: carries the attached CO2 from the biotin
carboxylase region to the transcarboxylase active site
– Transcarboxylase: transfers CO2 from biotin to acetyl-CoA producing
malonyl-CoA

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 The Acetyl-CoA Carboxylase Reaction

• This reaction is similar to other

biotin-dependent carboxylation
reactions

• The carboxyl group from HCO3-

is transferred to biotin in an
ATP-dependent reaction

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 The Acetyl-CoA Carboxylase Reaction

• The carboxyl group is carried by

the biotin to a different active
site, where the CO2 is
transferred to acetyl-CoA to
yield malonyl-CoA
– functions to make
subsequent steps more
thermodynamically
favorable
Bacterial acetyl-CoA
carboxylase has three separate
polypeptide subunits
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 Fatty Acid Synthesis
• Catalyzed by Fatty Acid Synthase (FAS I)
- Catalyzes a repeating four-step sequence that elongates the fatty acyl
chain by two carbons at each step
• FAS I in mammals composed of seven active sites are in
separate domains within a single multifunctional
polypeptide chain
• Overall goal: attach two-C acetate unit from malonyl-CoA to a
growing chain and then reduce it
• Reaction involves cycles of four enzyme-catalyzed steps:
– Condensation of the growing chain with activated acetate
– Reduction of carbonyl to hydroxyl
– Dehydration of alcohol to trans-alkene
– Reduction of alkene to alkane 15
 Acyl Carrier Protein (ACP) serves as a shuttle
in fatty acid synthesis
• Contains a covalently attached
prosthetic group 4’-phosphopantetheine
– Pantothenic acid is vitamin B5

• Delivers acetate (in the first step) or

malonate (in all the next steps) to the
fatty acid synthase

• Shuttles the growing chain from one

active site to another during the four-
step reaction
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 Charging ACP and FAS I with Acyl Groups
Activates Them
• Two thiols must be charged with the correct acyl groups
before the condensation reaction can begin.
– thiol from 4-phosphopantethine in ACP
– thiol from Cys in fatty acid synthase

The acetyl group of acetyl-CoA is transferred to ACP.

Catalyzed by malonyl/aceyl-CoA transferase (MAT) that catalysis 2
rxns:
1- The transfer of the acetyl group of acetyl-CoA to ACP (which
is then transferred to β-ketoacyl-ACP synthase (KS))
2- The transfer of the malonyl group from malonyl-CoA to the —
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SH group of ACP
Step 1 of FAS I: Condensation (Elongation)
Step2 of FAS 1: Reduction

Note that malonyl-CoA and acetyl-

CoA have already been attached to
complex via thioester linkages

1- a β-keto product
- CO2 from the malonyl group is
eliminated
- the acyl chain is extended by
two carbons

2- the β-keto product is

reduced to an alcohol
– requires NADPH 18
Step 3-4 of FAS I: Dehydration and Reduction

3- H2O is eliminated to
form a double bond

4- The double bond is

reduced to form the
corresponding saturated
fatty acyl group
– requires NADPH

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 Overall Palmitate (16C) Synthesis

• Thioesterase (TE) = hydrolyzes the thioester linkage

between palmitate and ACP to release free palmitate 20
Stoichiometry of Synthesis of Palmitate (16:0)
1) 7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs… using 7
ATP
7 Acetyl-CoA + 7 CO2 + 7 ATP  7 malonly-CoA + 7 ADP + 7 Pi

The seven cycles of condensation, reduction, dehydration, and

reduction:
Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ 
Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O
2) In eukaryotes: acetyl-CoA is transported into the cytosol with a
cost of 2 ATP used by acetyl shuttle system
-Therefore, cost of FA synthesis is 3 ATPs per 2-C unit except the
first acetyl-CoA (it only cost 2 ATP)
• Net = ATP for acetyl-CoA transport + ATP for malonyl-CoA
synthesis
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= (8 ×2) ATP + 7 ATP = 23 ATP
Biosynthesis of Fatty Acids Requires Acetyl-CoA and
Two Forms of Chemical Energy

• Biosynthesis of fatty acids requires:

– Acetyl-CoA
– The group transfer potential of ATP to make malonyl-
CoA
– The reducing power of NADPH to reduce the β-keto
group and the double bond

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 Long-Chain Saturated Fatty Acids Are
Synthesized from Palmitate
• Palmitate can be lengthened to longer-
chain fatty acids (precursor)
- Elongation systems in the smooth
endoplasmic reticulum and mitochondria
- Each elongation step adds units of 2 carbons
- Stearate (18:0) is the most common product

• Palmitate and stearate can be

desaturated
- Palmitate(16:0)palmitoleate(16:1; 9)
- Stearate (18:0)oleate (18:1; 9)
- Catalyzed by fatty acyl-CoA desaturase in
animals
- It is a 9-desaturase: introduces the double
bond between C-9 and C-10 23
 Desaturation of Fatty Acids Requires a Mixed-
Function Oxidase
• Fatty acyl-CoA desaturase = catalyzes an oxidative reaction that
introduces a double bond into a fatty acid chain
– is a mixed-function oxidase
– requires NADPH, cytochrome b5, and cytochrome b5 reductase
• O2 accepts four electrons from two substrates:
– two electrons come from the saturated fatty acid.
– two electrons come from ferrous state of cytochrome b5

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 Plants can desaturate positions beyond C-9

• Humans have 4, 5, 6, and 9 desaturases but cannot

desaturate beyond 9
• Plants can produce:
– 6: linoleate 18:2(9,12): may convert to arachidonate
– 3: -linolenate 18:3 (9,12,15): may convert to
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

• These fatty acids are “essential” to humans.

– Polyunsaturated fatty acids (PUFAs) help control membrane fluidity
– Essential fatty acids = must be obtained from dietary plant
material

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Biosynthesis of Triacylglycerols

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 Fatty Acids Have Two Fates
• Fatty acids synthesized or ingested have one of two fates:
– incorporation into triacylglycerols for the storage of
metabolic energy
– incorporation into the phospholipid components of
membranes

• Both pathways begin with the formation of fatty acyl esters

of glycerol

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 Fat (Triacylglycerol) and Phospholipids Synthesis
• Triacylglycerols and glycerophospholipids
share two precursors:
- Glycerol 3-phosphate; the
precursor for the backbone of fat
and phospholipids
- fatty acyl-CoA
• Sources of glycerol 3-phosphate:
First: most glycerol 3-phosphate comes
from drawing off dihydroxyacetone
phosphate (DHAP) from glycolysis by
glycerol 3-phosphate dehydrogenase
Second: some glycerol 3-phosphate is
directly made from glycerol by glycerol
kinase (in liver and kidney) 28
 Acyl transferases attach two fatty acids to
glycerol 3-phosphate
• Phosphatidic acid is the precursor to
TAGs and phospholipids
• Made of glycerol 3-phosphate + 2 fatty
acyl CoA
• Acyl transferase = catalyzes the
acylation of the two free hydroxyl
groups of L-glycerol 3-phosphate by
two molecules of fatty acyl–CoA to
yield diacylglycerol 3-phosphate
(phosphatidic acid)

• Ester linkage forms with L-glycerol 3-

phosphate and CoA is released 29
 To make TAG, phosphatidic acid is
dephosphorylated and acylated
• To make TAG:
First: phosphatidic acid is
dephosphorylated by
Phosphatidic acid phosphatase
producing 1,2-diacylglycerol

Second: 1,2-diacylglycerol is
acylated with a third fatty acid
by acyl transferase producing
triacylglycerol

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 What is the source of the glycerol 3-phosphate
needed for fatty acid reesterification?
• During lipolysis (stimulated by glucagon
or epinephrine), glycolysis is inhibited
– So DHAP is not readily available to make
glycerol 3-phosphate

• And adipose cells don’t have glycerol

kinase to make glycerol 3-phosphate
on-site
• So adipose cells make DHAP from
pyruvate via glyceroneogenesis
(shortened version of gluconeogenesis)
– Explains why adipose cells express pyruvate
carboxylase and PEPCK even though fat
cells don’t make glucose 31
 Roles of Glyceroneogenesis
• Glyceroneogenesis functions to:
– help control the rate of fatty acid release to the blood
– control the rate at which free fatty acids are delivered to
mitochondria for use in thermogenesis
– support the synthesis of glycerol 3-phosphate in fasting
humans

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The Triacylglycerol Cycle During Starvation

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 Biosynthesis of Membrane Phospholipids
• Occurs in Smooth ER, inner
mitochondrial membrane
and Golgi complex ???????

• Begin with A polar head group

is attached to C-3 OH group of
phosphatidic acid or
diacylglycerol by
phosphodiester bond and two
water molecules eliminated
(condensation)
• After synthesis, phospholipids
are transported from ER to
membranes by vesicles
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 Attaching the Phospholipid Head Group Requires
Activation by CDP
• One of the hydroxyls is first activated by attachment of a cytidine
diphosphate (CDP) nucleotide
• CMP is then displaced in a nucleophilic attack by the other hydroxyl

Strategies for Forming the

Phosphodiester Bond:
• Strategy 1 = CDP is attached to
diacylglycerol, forming the
activated phosphatidic acid
CDP-diacylglycerol

• Strategy 2 = CDP is attached to

the hydroxyl of the head
group 35
 Pathways for Phospholipid Biosynthesis Are
Interrelated

• many pathways begin with CDP-diacylglycerol, including

synthesis of:
– phosphatidylglycerol
– cardiolipin
– phosphatidylinositol
• conversion to its phosphorylated derivatives requires
specific phosphatidylinositol kinases
– phosphatidylserine  phosphatidylethanolamine 
phosphatidylcholine

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Phospholipid and Triacylglycerol Biosynthetic
Pathways

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 Lipid Biosynthesis II

Learning Outcomes:
At the end of this lecture, students should be able to:

1. Explain fatty acid biosynthesis regulation

2. Explain the regulation triacylglycerol synthesis and
breakdown cycle
3. Explain the role of gluconeogenesis enzymes in
adipose tissues

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 Fatty acid synthesis is tightly regulated via ACC
• Acetyl CoA carboxylase (ACC) catalyzes
the rate-limiting step of fatty acid
synthesis

• Allosteric regulation:
– ACC is feedback-inhibited by
palmitoyl-CoA
– ACC is activated by citrate

• Hormonal Regulation
– Glucagon and epinephrine:
1. Reduce sensitivity of citrate
activation
2. Lead to phosphorylation and 39
inactivation of ACC
Regulation of Acetyl-CoA Carboxylase By Covalent
Modification
• Phosphorylation inactivates the enzyme
– triggered by the hormones glucagon and epinephrine or by
high [AMP]
– reduces sensitivity of citrate activation and slows fatty acid
synthesis
– causes polymerization of ACC into long, inactive filaments

Additional Regulation of Fatty

Acid Pathways
- Pathways are also regulated at the level of gene expression
example: fatty acids bind PPARs (nuclear receptor proteins).
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FA synthesis and breakdown are reciprocally regulated

Malonyl-CoA inhibits fatty acid import into mitochondria

- Ensure that fat synthesis and oxidation don’t occur simultaneously

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 Hormonal regulation of triacylglycerol
synthesis
• Insulin results in stimulation of
triacylglycerol synthesis
– Lack of insulin (diabetes mellitus)
decreases fatty acid synthesis. This will
cause acetyl-CoA accumulation,
therefore, thus ketone bodies
synthesis increases

• Glucagon and epinephrine result in

stimulation of triacylglycerol
breakdown (mobilization of fatty
acids)

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 Thiazolidinediones Treat Type 2 Diabetes by
Increasing Glyceroneogenesis

• Thiazolidinediones = class of drugs that reduces the

levels of fatty acids circulating in the blood and increases
sensitivity to insulin
– promotes the increased expression of PEP
carboxykinase in adipose tissue

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 Regulation of Glyceroneogenesis
• Glucocorticoids increase the expression of the gene encoding
PEP carboxykinase in the liver to increase gluconeogenesis
and glyceroneogenesis

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