14 - Biosynthesis of Fatty Acids

TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Metabolic Biochemistry::14 - Biosynthesis of Fatty Acids

Biosynthesis of Fatty Acids

Key Principle

Fatty acid biosynthesis and breakdown occur via different pathways, catalyzed by different enzymes, in different cellular compartments. Biosynthesis uniquely requires the three-carbon intermediate malonyl-CoA.

---

Acetyl-CoA Carboxylase — The Committed Step

The acetyl-CoA carboxylase reaction converts acetyl-CoA to malonyl-CoA. The enzyme has three functional regions:

Domain	Function
Biotin carrier protein	Carries activated CO₂ via a long flexible arm
Biotin carboxylase	Activates CO₂ by attaching it to biotin nitrogen (ATP-dependent)
Transcarboxylase	Transfers activated CO₂ from biotin to acetyl-CoA → malonyl-CoA

$\text{Acetyl-CoA}+{\text{HCO}}_3^{-}+\text{ATP}\stackrel{\text{acetyl-CoA carboxylase}}{\to }\text{Malonyl-CoA}+\text{ADP}+{\text{P}}_i$

Regulators of acetyl-CoA carboxylase:

Activators	Inhibitors
Citrate	Palmitoyl-CoA
Insulin	Epinephrine (via phosphorylation)
AMP	Glucagon (via PKA → phosphorylation)

Anki cloze

The committed step of fatty acid biosynthesis is catalyzed by {1:acetyl-CoA carboxylase}, which converts acetyl-CoA to {2:malonyl-CoA} in an {3:ATP}-dependent reaction using {4:biotin} as a cofactor.

Anki cloze

Acetyl-CoA carboxylase is allosterically {1:activated} by citrate and {2:inhibited} by palmitoyl-CoA.

---

Acyl Carrier Protein (ACP)

• The prosthetic group is 4′-phosphopantetheine, covalently attached to the hydroxyl group of a Ser residue in ACP.
• Phosphopantetheine contains the B vitamin pantothenic acid, also found in CoA.
• Its –SH group is the site of entry of malonyl groups during fatty acid synthesis.

SCCNC(=O)CCNC(=O)[C@@H](O)C(C)(C)COP(=O)(O)OC[C@@H]1OC(n2cnc3c(N)ncnc23)[C@H](O)[C@@H]1OP(=O)(O)O

Anki cloze

The prosthetic group of ACP is {1:4′-phosphopantetheine}, attached to a {2:Ser} residue; its {3:–SH} group accepts malonyl groups during fatty acid synthesis.

---

Fatty Acid Synthase (FAS) in Vertebrates

• A single large polypeptide (type I FAS).
• Contains six catalytic domains plus ACP at the center, with the phosphopantetheine arm ending in a –SH group.
• The enzyme acting in the next step is conventionally highlighted.

Charging the Synthase Complex

Before elongation begins, two thiol groups must be loaded:

1. Acetyl-CoA–ACP transacetylase (AT) transfers the acetyl group of acetyl-CoA → Cys–SH of β-ketoacyl-ACP synthase (KS).
2. Malonyl-CoA–ACP transferase (MT) transfers the malonyl group from malonyl-CoA → phosphopantetheine–SH of ACP.

Anki cloze

In the fatty acid synthase complex, the acetyl starter group is loaded onto the {1:Cys–SH} of β-ketoacyl-ACP synthase, while the malonyl group is loaded onto the {2:phosphopantetheine–SH} of ACP.

---

The Four-Step Elongation Cycle

Each cycle adds two carbons to the growing acyl chain. The cycle is repeated seven times to produce palmitate (C16).

Step 1 — Condensation (β-ketoacyl-ACP synthase, KS)

The acetyl group (on Cys–SH of KS) condenses with the malonyl group (on ACP–SH), releasing CO₂ and forming acetoacetyl-ACP (a β-keto product).

$\text{Acetyl-[KS]}+\text{Malonyl-ACP}\stackrel{\text{KS}}{\to }\text{Acetoacetyl-ACP}+{\text{CO}}_2$

Why malonyl-CoA and not acetyl-CoA for elongation?

The decarboxylation of the malonyl group drives condensation forward thermodynamically, making each elongation step energetically favorable.

Anki cloze

During condensation in fatty acid synthesis, the decarboxylation of the {1:malonyl} group releases {2:CO₂} and drives the reaction forward thermodynamically.

---

Step 2 — Reduction of the Carbonyl Group (β-ketoacyl-ACP reductase, KR)

The β-keto group at C-3 is reduced to form D-β-hydroxybutyryl-ACP.

$\text{Acetoacetyl-ACP}+\text{NADPH}+{\text{H}}^{+}\stackrel{\text{KR}}{\to }\text{D-}\beta \text{-hydroxybutyryl-ACP}+{\text{NADP}}^{+}$

Stereochemistry

The product is the D stereoisomer — opposite to the L stereoisomer produced in fatty acid β-oxidation.

Anki cloze

The β-keto group in fatty acid synthesis is reduced by {1:NADPH} to give the {2:D}-β-hydroxy stereoisomer, whereas β-oxidation produces the {3:L} stereoisomer.

---

Step 3 — Dehydration (β-hydroxyacyl-ACP dehydratase, HD)

Water is eliminated from C-2 and C-3 of D-β-hydroxybutyryl-ACP, creating a trans double bond → trans-2-butenoyl-ACP.

$\text{D-}\beta \text{-hydroxybutyryl-ACP}\stackrel{\text{HD}}{\to }\text{trans-2-butenoyl-ACP}+{\text{H}}_2\text{O}$

Anki cloze

The dehydration step of fatty acid synthesis removes water from C-{1:2} and C-{2:3}, producing {3:trans-2-butenoyl}-ACP.

---

Step 4 — Reduction of the Double Bond (enoyl-ACP reductase, ER)

The trans double bond of trans-2-butenoyl-ACP is reduced (saturated) → butyryl-ACP.

$\text{trans-2-butenoyl-ACP}+\text{NADPH}+{\text{H}}^{+}\stackrel{\text{ER}}{\to }\text{butyryl-ACP}+{\text{NADP}}^{+}$

Anki cloze

The enzyme {1:enoyl-ACP reductase (ER)} uses {2:NADPH} to reduce the double bond of trans-2-butenoyl-ACP, yielding {3:butyryl}-ACP.

---

Completing the Cycle

1. The butyryl group is transferred from phosphopantetheine–SH of ACP → Cys–SH of KS.
2. A new malonyl group is loaded onto the now-free phosphopantetheine–SH of ACP.
3. The four-step cycle repeats.

Anki cloze

After each elongation cycle, the acyl chain is transferred from {1:ACP} to the {2:Cys–SH of KS (β-ketoacyl-ACP synthase)}, freeing ACP to accept the next {3:malonyl} group.

---

Synthesis vs. Degradation — Comparison

Feature	Synthesis	Degradation (β-oxidation)
Location	Cytosol	Mitochondrial matrix
Acyl carrier	ACP (phosphopantetheine)	CoA
Electron carrier	NADPH (reduction)	FAD, NAD⁺ (oxidation)
C-unit donor	Malonyl-CoA (3C)	—
Intermediate stereochemistry	D-β-hydroxy	L-β-hydroxy
Key reaction directions	Condensation → reduction → dehydration → reduction	Oxidation → hydration → oxidation → thiolysis

Anki cloze

Fatty acid synthesis uses {1:NADPH} as electron donor, whereas β-oxidation generates {2:FADH₂} and {3:NADH}.

Anki cloze

Fatty acid synthesis occurs in the {1:cytosol}, while β-oxidation occurs in the {2:mitochondrial matrix}.

---

Final Product: Palmitate (16:0)

• FAS is structurally designed to halt elongation at 16 carbons.
• The thioesterase (TE) domain specifically recognizes the 16-carbon saturated chain and hydrolyzes the thioester bond between the palmitoyl group and ACP → releasing free palmitate.

$\text{Palmitoyl-ACP}+{\text{H}}_2\text{O}\stackrel{\text{Thioesterase}}{\to }\text{Palmitate}+\text{ACP-SH}$

CCCCCCCCCCCCCCCC(=O)O

After release, palmitate can be:

• Activated to palmitoyl-CoA (by acyl-CoA synthetase)
• Used in lipid synthesis (triglycerides, phospholipids)
• Elongated or desaturated
• Stored in lipid droplets

Anki cloze

Palmitate is released from FAS by the {1:thioesterase (TE)} domain, which hydrolyzes the thioester bond between the {2:palmitoyl} group and {3:ACP}.

Anki cloze

The final product of cytosolic fatty acid synthase is {1:palmitate} (C{2:16}), because the thioesterase domain specifically recognizes {3:16}-carbon saturated chains.

---

Cellular Location of Fatty Acid Synthesis

Fatty acid synthesis occurs where NADPH is abundant (high $\frac{[\text{NADPH}]}{[{\text{NADP}}^{+}]}$ ratio):

Organism/Cell type	Location
Animal cells, yeast	Cytosol
Mitochondria	Fatty acid elongation only
ER	Elongation + desaturation

---

Sources of Cytosolic NADPH

(a) Malic Enzyme

$\text{Malate}+{\text{NADP}}^{+}\stackrel{\text{malic enzyme}}{\to }\text{Pyruvate}+{\text{CO}}_2+\text{NADPH}$

(b) Pentose Phosphate Pathway (PPP)

$\text{Glucose-6-phosphate}+{\text{NADP}}^{+}\stackrel{\text{G6PD}}{\to }\text{6-phosphogluconolactone}+\text{NADPH}+{\text{H}}^{+}$

• The PPP is parallel to glycolysis and generates NADPH and ribose-5-phosphate (nucleotide precursor).
• The rate-limiting enzyme is glucose-6-phosphate dehydrogenase (G6PD).
• Insulin increases G6PD activity (via de novo enzyme biosynthesis, requiring new RNA production).

Anki cloze

The two main sources of cytosolic NADPH for fatty acid synthesis are the {1:pentose phosphate pathway} and {2:malic enzyme}.

Anki cloze

The rate-limiting enzyme of the pentose phosphate pathway is {1:glucose-6-phosphate dehydrogenase (G6PD)}, which is induced by {2:insulin}.

---

Shuttle for Transfer of Acetyl Groups: Mitochondria → Cytosol

Acetyl-CoA cannot cross the inner mitochondrial membrane directly. It exits as citrate:

$\text{Acetyl-CoA}+\text{OAA}\stackrel{\text{citrate synthase}}{\to }\text{Citrate}(\text{in matrix})$

In the cytosol, citrate is cleaved by citrate lyase (ATP-citrate lyase):

$\text{Citrate}+\text{CoA}+\text{ATP}\stackrel{\text{citrate lyase}}{\to }\text{Acetyl-CoA}+\text{OAA}+\text{ADP}+{\text{P}}_i$

Oxaloacetate (OAA) is then reduced → malate → returns to the mitochondrion, or is oxidized by malic enzyme → pyruvate + NADPH.

The outer mitochondrial membrane is freely permeable to all these compounds. The shuttle operates across the inner membrane.

Anki cloze

Acetyl groups exit the mitochondrion as {1:citrate} and are regenerated in the cytosol by {2:citrate lyase (ATP-citrate lyase)}, which consumes {3:ATP}.

Anki cloze

The acetate export cycle from mitochondria consumes {1:2} ATP molecules per acetyl-CoA that enters fatty acid synthesis (via {2:citrate lyase} and {3:pyruvate carboxylase}).

---

Energy Expenditure for Fatty Acid Synthesis

• 1 ATP per malonyl-CoA formed (acetyl-CoA carboxylase step).
• 2 ATP per acetyl-CoA exported from mitochondria (citrate lyase + pyruvate carboxylase).

Total ATP cost

3 ATP are consumed per two-carbon unit incorporated into the growing fatty acid chain.

$\text{Total for palmitate synthesis (7 elongations):}7\times 3=21\text{ ATP}$

Anki cloze

For each two-carbon block incorporated during fatty acid synthesis, a total of {1:3} ATP molecules are consumed.

---

Regulation of Fatty Acid Synthesis

Allosteric Regulation of Acetyl-CoA Carboxylase

• Citrate → polymerizes and activates the enzyme.
• Palmitoyl-CoA (end product) → inhibits (feedback inhibition).

Hormonal (Covalent) Regulation

Signal	Effect	Mechanism
Insulin	↑ synthesis	Activates phosphatase → dephosphorylates (activates) ACC
Glucagon / Epinephrine	↓ synthesis	PKA → phosphorylates (inactivates) ACC

Malonyl-CoA and β-Oxidation Cross-regulation

• High carbohydrate intake → ↑ malonyl-CoA.
• Malonyl-CoA inhibits carnitine acyltransferase I (CAT I) → blocks fatty acid entry into mitochondria for β-oxidation.
• This ensures synthesis and degradation are not simultaneously active.

Anki cloze

Malonyl-CoA inhibits {1:carnitine acyltransferase I (CAT I)}, thereby blocking {2:β-oxidation} and preventing simultaneous synthesis and degradation of fatty acids.

Anki cloze

Acetyl-CoA carboxylase is covalently inactivated by {1:phosphorylation} via {2:PKA}, triggered by glucagon or epinephrine, and reactivated by {3:phosphatase} under insulin signaling.

---

Routes of Synthesis of Other Fatty Acids

Palmitate is the central precursor:

Palmitate (16:0)
├─ elongation → Stearate (18:0)
│                └─ desaturation → Oleate (18:1Δ9)
│                                   └─ elongation → Longer saturated FAs
└─ desaturation → Palmitoleate (16:1Δ9)

Essential Fatty Acids

Mammals cannot introduce double bonds beyond C-9 (i.e., cannot synthesize Δ12 or Δ15 desaturases). Therefore:

• Linoleate (18:2 Δ9,12) — dietary essential
• α-Linolenate (18:3 Δ9,12,15) — dietary essential

From essential fatty acids, mammals can synthesize:

Precursor	Product	Function
Linoleate (ω-6)	Arachidonate (20:4 Δ5,8,11,14)	Prostaglandins (PG2), Thromboxanes (TX2), Leukotrienes (LT4), Lipoxins
α-Linolenate (ω-3)	EPA (20:5 Δ5,8,11,14,17) → DHA (22:6 Δ4,7,10,13,16,19)	PG3, TG3, LT5, Resolvins, Protectins, Maresins

Anki cloze

Mammals cannot synthesize {1:linoleate} (18:2Δ9,12) and {2:α-linolenate} (18:3Δ9,12,15) because they lack {3:Δ12 and Δ15 desaturases}; these must be obtained from the {4:diet}.

Anki cloze

Arachidonate is derived from {1:linoleate} (ω-{2:6}) and is the precursor for {3:prostaglandins}, {4:thromboxanes}, leukotrienes, and lipoxins.

---

Location of Elongation and Desaturation

Process	Location
Fatty acid elongation	Mitochondria and ER
Fatty acid desaturation	ER only

Elongation in the ER — ELOVL Enzymes

• The ELOVL (Elongation of Very Long-Chain Fatty Acids) enzyme family: ELOVL1–ELOVL7.
• Each isoform has distinct substrate specificity for chain length and saturation.
• Catalyzes: condensation of acyl-CoA + malonyl-CoA → reduction → dehydration → reduction (analogous to cytosolic FAS but in the ER).

Anki cloze

Fatty acid desaturation occurs exclusively in the {1:endoplasmic reticulum (ER)}, while elongation occurs in both the {2:ER} and {3:mitochondria}.

Anki cloze

The ELOVL enzyme family (ELOVL{1:1–7}) catalyzes fatty acid elongation in the {2:ER} by condensing an acyl-CoA with {3:malonyl-CoA}.

---

Desaturation Mechanism (ER)

Desaturation uses a mixed-function oxidase system on the lumenal face of the smooth ER:

$\text{Fatty acyl-CoA}+\text{NADPH}+{\text{H}}^{+}+{\text{O}}_2\stackrel{\text{fatty acyl-CoA desaturase}}{\to }\text{Monounsaturated acyl-CoA}+{\text{NADP}}^{+}+2{\text{H}}_2\text{O}$

Electron flow:
$\text{NADPH}\to \text{Cyt }{b}_5\text{ reductase (FAD)}\to \text{Cyt }{b}_5{\text{ (Fe}}^{3+}/{\text{Fe}}^{2+})\to \text{fatty acyl-CoA desaturase}\to {\text{O}}_2$

Anki cloze

Fatty acid desaturation requires {1:O₂} and {2:NADPH} and occurs on the {3:lumenal face of the smooth ER}, with electrons transferred via {4:cytochrome b₅}.

---

Eicosanoid Synthesis from Arachidonate

Cyclic Pathway → Prostaglandins & Thromboxanes

1. Phospholipase A₂ releases arachidonate from phospholipids.
2. COX (cyclooxygenase activity of prostaglandin H₂ synthase) → PGG₂.
3. Peroxidase activity of COX → PGH₂ → prostaglandins + thromboxanes.

Drug target

Aspirin irreversibly inhibits COX by acetylating an essential Ser residue.
Ibuprofen and naproxen inhibit the same step (competitive/structural mimicry), but reversibly.

CC(=O)Oc1ccccc1C(=O)O

(Aspirin / acetylsalicylate)

Anki cloze

Aspirin inhibits {1:COX (prostaglandin H₂ synthase)} by irreversibly {2:acetylating} an essential {3:Ser} residue, blocking prostaglandin and thromboxane synthesis.

Linear Pathway → Leukotrienes

• 5-Lipoxygenase converts arachidonate → 5-HPETE → leukotrienes.

Anki cloze

Leukotrienes are synthesized from arachidonate via the {1:linear (lipoxygenase)} pathway, whereas prostaglandins arise via the {2:cyclic (COX)} pathway.

---

🧠 Mnemonic

Mnemonic — FAS elongation cycle steps: "Can Kangaroos Do Everything?"

• Condensation (KS)
• Keto-reduction (KR) — NADPH
• Dehydration (HD)
• Enoyl-reduction (ER) — NADPH

Mnemonic — Essential fatty acids: "Lions And Lambs"

• Linoleate (18:2, ω-6)
• Alpha-Linolenate (18:3, ω-3)
  Both must come from the diet.

---

TLDR

• Malonyl-CoA is the unique three-carbon intermediate of fatty acid biosynthesis, formed from acetyl-CoA by acetyl-CoA carboxylase (biotin-dependent, ATP-consuming; activated by citrate, inhibited by palmitoyl-CoA and phosphorylation).
• FAS in vertebrates is a single multifunctional polypeptide; the central prosthetic arm is ACP with a 4′-phosphopantetheine–SH group.
• Each elongation cycle adds 2 carbons in four steps: condensation (KS) → carbonyl reduction by NADPH (KR) → dehydration (HD) → double bond reduction by NADPH (ER), yielding a D-β-hydroxy intermediate (opposite stereochemistry to β-oxidation).
• Synthesis and β-oxidation differ in location (cytosol vs. mitochondria), acyl carrier (ACP vs. CoA), electron carrier (NADPH vs. FAD/NAD⁺), and intermediate stereochemistry (D vs. L).
• The thioesterase (TE) domain of FAS terminates elongation at C16, releasing palmitate.
• NADPH for synthesis comes from the pentose phosphate pathway (G6PD, induced by insulin) and malic enzyme.
• Acetyl-CoA exits mitochondria as citrate, regenerated in the cytosol by citrate lyase (costing 1 ATP); total cost = 3 ATP per two-carbon unit.
• Malonyl-CoA inhibits CAT I, preventing simultaneous β-oxidation — a key metabolic switch.
• Palmitate → stearate (elongation) → oleate/palmitoleate (desaturation in ER); mammals lack Δ12/Δ15 desaturases, making linoleate and α-linolenate dietary essentials.
• Arachidonate (from linoleate) → prostaglandins/thromboxanes via COX (inhibited by aspirin/ibuprofen) or → leukotrienes via lipoxygenase.
• Desaturation uses O₂, NADPH, cytochrome b₅, and occurs on the lumenal face of the smooth ER; elongation occurs in ER (ELOVL1–7) and mitochondria.