Fatty Acid Synthesis & Beta-Oxidation
Biochemistry · Lipids · lean revision notes
Fatty Acid Synthesis & Beta-Oxidation
Fatty acid metabolism is a perennial NEET PG high-scorer because it links enzymology, compartmentalisation, vitamin cofactors, regulation and inborn errors in a single, tightly examined web. Mastering the two opposing pathways — cytosolic synthesis (lipogenesis) and mitochondrial β-oxidation — together with the carnitine shuttle and MCAD deficiency, reliably fetches 2–3 marks every cycle.
Big-picture orientation
The two pathways are deliberately separated so they never run simultaneously (futile cycling is prevented):
| Feature | Fatty acid synthesis | Fatty acid β-oxidation |
|---|---|---|
| Location | Cytosol | Mitochondrial matrix |
| Carrier | Acyl carrier protein (ACP) of FAS | Coenzyme A |
| Carbon unit added/removed | 2-C as malonyl-CoA (3-C donor, loses CO₂) | 2-C as acetyl-CoA |
| Reducing/oxidising equivalent | NADPH (uses) | FAD + NAD⁺ (generates FADH₂, NADH) |
| Key vitamin | Biotin (B7) for ACC; pantothenate in ACP | Riboflavin (FAD), niacin (NAD⁺) |
| Activated/inhibited state | Well-fed, high insulin | Fasting, high glucagon |
| Committed/regulatory step | Acetyl-CoA carboxylase (ACC) | Carnitine CPT-1 (entry) |
| Inhibitor signal | Palmitoyl-CoA, glucagon (↓) | Malonyl-CoA inhibits CPT-1 |
High-yield: The single most-tested reciprocal control fact — malonyl-CoA, the first committed product of synthesis, inhibits CPT-1 and thus blocks β-oxidation. When you are making fat, you cannot burn it.
Part 1 — De novo fatty acid synthesis (lipogenesis)
Where the substrate comes from: the citrate shuttle
Acetyl-CoA is generated in the mitochondrion but FAS is cytosolic, and acetyl-CoA cannot cross the inner mitochondrial membrane. It is exported as citrate.
Flow: Acetyl-CoA + OAA → citrate (citrate synthase) → citrate exits via tricarboxylate carrier → in cytosol, ATP-citrate lyase cleaves citrate → acetyl-CoA + OAA → OAA → malate (MDH) → malate → pyruvate by malic enzyme, generating NADPH.
High-yield: The citrate shuttle simultaneously delivers acetyl-CoA and contributes NADPH (via malic enzyme). The other major NADPH source for lipogenesis is the HMP (pentose phosphate) shunt.
Step 1 — Acetyl-CoA carboxylase (ACC): the committed, rate-limiting step
Acetyl-CoA + CO₂ + ATP → malonyl-CoA (3 carbons)
- Cofactor: biotin (carboxylation enzyme — remember pyruvate carboxylase, propionyl-CoA carboxylase, ACC all use biotin).
- ACC exists as inactive protomers and active polymers.
Regulation of ACC (the regulatory heart of the pathway):
| Activator | Inhibitor |
|---|---|
| Citrate (allosteric → polymerisation) | Palmitoyl-CoA (end-product, depolymerises) |
| Insulin (dephosphorylation → active) | Glucagon/adrenaline (phosphorylation → inactive) |
| AMPK phosphorylates and inactivates ACC |
High-yield: Insulin activates ACC by dephosphorylation; glucagon and AMPK inactivate it by phosphorylation. Citrate is the allosteric activator; palmitoyl-CoA is the feedback inhibitor.
Step 2 — The fatty acid synthase (FAS) complex
In humans, FAS is a single multifunctional dimeric polypeptide with 7 catalytic activities + ACP. Each monomer carries a phosphopantetheine arm (derived from pantothenic acid / vitamin B5) on ACP, plus a cysteine –SH on the condensing enzyme (ketoacyl synthase).
The repeating elongation cycle adds 2 carbons each turn — mnemonic "Can Anyone Really Drive A Car" is not standard; use the functional sequence:
Condensation → Reduction → Dehydration → Reduction
- Condensation (β-ketoacyl synthase): acetyl (on Cys-SH) + malonyl (on ACP) → 4-C β-ketoacyl-ACP + CO₂ released.
- Reduction (β-ketoacyl reductase): uses NADPH → β-hydroxyacyl-ACP.
- Dehydration (dehydratase): → enoyl-ACP (trans double bond).
- Reduction (enoyl reductase): uses NADPH → saturated acyl-ACP.
After 7 cycles, palmitate (C16, saturated) is released by thioesterase.
Stoichiometry for palmitate:
Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 14 H⁺ → Palmitate + 7 CO₂ + 8 CoA + 14 NADP⁺ + 6 H₂O
Including the 7 ACC reactions: 8 acetyl-CoA + 7 ATP + 14 NADPH → 1 palmitate.
High-yield: Palmitate (16C) is the sole product of the cytosolic FAS complex. Elongation beyond C16 and desaturation occur in the smooth endoplasmic reticulum (microsomal system, uses malonyl-CoA + NADPH). Mitochondria perform minor elongation using acetyl-CoA + NADPH/NADH.
Desaturation
Microsomal Δ⁹, Δ⁶, Δ⁵, Δ⁴ desaturases introduce double bonds, requiring O₂, NADH, cytochrome b₅. Humans cannot desaturate beyond C9 toward the methyl end → cannot make linoleic (ω-6) and α-linolenic (ω-3) acids → these are essential fatty acids. Arachidonic acid becomes essential only if linoleic acid is deficient.
Part 2 — The carnitine shuttle (entry into β-oxidation)
Long-chain fatty acyl-CoA cannot cross the inner mitochondrial membrane. The carnitine shuttle is the gateway and a favourite exam target.
Flow:
- FA activated in cytosol → acyl-CoA by acyl-CoA synthetase (thiokinase) on outer mitochondrial membrane; costs 2 high-energy bonds (ATP → AMP + 2 Pi).
- CPT-1 (carnitine palmitoyltransferase-1) on outer membrane swaps CoA for carnitine → acylcarnitine. Rate-limiting step; inhibited by malonyl-CoA.
- Carnitine-acylcarnitine translocase (CACT) carries acylcarnitine in, carnitine out.
- CPT-2 on inner membrane regenerates acyl-CoA in the matrix.
High-yield: Carnitine is synthesised from lysine + methionine in liver/kidney (needs vitamin C). CPT-1 deficiency → hypoketotic hypoglycaemia, hepatomegaly, normal/low muscle carnitine. CPT-2 deficiency (commonest) → adult exercise-induced myopathy, rhabdomyolysis, myoglobinuria. Primary carnitine deficiency = defect in OCTN2 carnitine transporter → cardiomyopathy.
Note: Short- and medium-chain fatty acids enter mitochondria independently of carnitine.
Part 3 — β-Oxidation
Occurs in the mitochondrial matrix. Each cycle shortens the chain by 2 carbons and follows four steps — mnemonic "Oxidation, Hydration, Oxidation, Cleavage" or the enzyme order:
Acyl-CoA → (1) FAD oxidation → (2) hydration → (3) NAD⁺ oxidation → (4) thiolytic cleavage → Acetyl-CoA + acyl-CoA (n–2)
| Step | Enzyme | Reaction | Product / equivalent |
|---|---|---|---|
| 1 | Acyl-CoA dehydrogenase (chain-length specific: VLCAD, LCAD, MCAD, SCAD) | Oxidation | FADH₂ (→1.5 ATP) |
| 2 | Enoyl-CoA hydratase | Hydration of trans double bond | 3-hydroxyacyl-CoA |
| 3 | 3-hydroxyacyl-CoA dehydrogenase | Oxidation | NADH (→2.5 ATP) |
| 4 | β-ketothiolase (thiolase) | Thiolytic cleavage | Acetyl-CoA + shortened acyl-CoA |
The first step's FADH₂ feeds electrons via ETF (electron-transferring flavoprotein) → ETF-QO → ubiquinone.
ATP yield — the calculation you must be able to do fast
For palmitate (C16, fully saturated), modern P/O ratios (NADH = 2.5, FADH₂ = 1.5):
- Cycles = (16/2) − 1 = 7 cycles → 7 FADH₂ + 7 NADH
- Acetyl-CoA produced = 16/2 = 8, each via TCA = 10 ATP → 80
- From β-oxidation: 7 × (1.5 + 2.5) = 28
- Gross = 80 + 28 = 108
- Subtract 2 for activation (ATP→AMP) → Net = 106 ATP
High-yield: Palmitate net = 106 ATP (new values) or 129 ATP (old values: NADH=3, FADH₂=2). Know the formula: for a saturated FA with n carbons, acetyl-CoA = n/2, cycles = (n/2 − 1). Always subtract 2 for activation.
Odd-chain fatty acid oxidation
Final cycle yields acetyl-CoA + propionyl-CoA (3C).
Flow: Propionyl-CoA → (propionyl-CoA carboxylase, biotin) → D-methylmalonyl-CoA → (racemase) → L-methylmalonyl-CoA → (methylmalonyl-CoA mutase, vitamin B12 / adenosylcobalamin) → succinyl-CoA → TCA / gluconeogenesis.
High-yield: This is the only route by which fatty acids contribute to gluconeogenesis (via the propionyl-CoA → succinyl-CoA anaplerotic carbon). Vitamin B12 deficiency → ↑ methylmalonic acid (methylmalonic aciduria); a classic distinguishing lab from folate deficiency. Branched-chain (phytanic acid) and isoleucine/valine/methionine catabolism also produce propionyl-CoA.
Unsaturated fatty acid oxidation
Requires two extra enzymes:
- Enoyl-CoA isomerase — converts cis-Δ³ to trans-Δ² double bonds (for mono/poly-unsaturated).
- 2,4-dienoyl-CoA reductase (uses NADPH) — for polyunsaturated FAs.
Each pre-existing double bond means one fewer FADH₂-generating step → slightly less ATP than the equivalent saturated FA.
Other oxidation routes (rapid)
- α-oxidation (peroxisomes/ER): for branched-chain phytanic acid (β-carbon is blocked by methyl). Defect → Refsum disease (retinitis pigmentosa, cerebellar ataxia, peripheral neuropathy, ichthyosis).
- ω-oxidation (microsomal): produces dicarboxylic acids; minor, increases in MCAD deficiency.
- Peroxisomal β-oxidation: handles very-long-chain FA (VLCFA); uses FAD → H₂O₂ (not ATP). Defect in transporter → X-linked adrenoleukodystrophy (ALD) with raised VLCFA; Zellweger syndrome = absent peroxisomes.
Part 4 — Ketone bodies (linked downstream)
Excess acetyl-CoA from hepatic β-oxidation during fasting/diabetes → ketogenesis in liver mitochondria.
Flow: 2 acetyl-CoA → acetoacetyl-CoA → (HMG-CoA synthase, rate-limiting) HMG-CoA → (HMG-CoA lyase) acetoacetate → β-hydroxybutyrate (major in blood) and spontaneous → acetone.
High-yield: Liver makes but cannot use ketone bodies — it lacks thiophorase (succinyl-CoA acetoacetyl-CoA transferase / SCOT). Extrahepatic tissues (brain, muscle, heart) use them. β-hydroxybutyrate:acetoacetate ratio rises in severe ketoacidosis; nitroprusside dipsticks detect acetoacetate/acetone, NOT β-hydroxybutyrate — hence ketosis may be underestimated early in DKA.
Inborn errors — MCAD and friends
MCAD deficiency (medium-chain acyl-CoA dehydrogenase)
The commonest inherited fatty-acid oxidation disorder; autosomal recessive; common ACADM gene mutation K304E (985A>G).
- Presents at 3 months–2 years after a fasting/illness trigger (prolonged fast, intercurrent infection).
- Hypoketotic hypoglycaemia (cannot oxidise FA → no acetyl-CoA → no ketones, and gluconeogenesis substrate falls), lethargy, vomiting, seizures, coma; may mimic SIDS / Reye-like picture.
- Lab: dicarboxylic aciduria (C6–C10), low/absent ketones, raised C8 (octanoyl) acylcarnitine on tandem mass spectrometry (newborn screening marker).
- Management: avoid fasting, frequent feeds, IV dextrose during illness; generally good prognosis once recognised.
High-yield: Hypoketotic hypoglycaemia + dicarboxylic aciduria + raised C8-acylcarnitine = MCAD deficiency. Contrast with hyperketotic hypoglycaemia of glycogen storage / ketotic states. Treatment is dietary: never fast.
| Disorder | Enzyme/defect | Hallmark |
|---|---|---|
| MCAD deficiency | Medium-chain acyl-CoA DH (ACADM) | Hypoketotic hypoglycaemia, ↑C8, dicarboxylic aciduria |
| CPT-1 deficiency | Carnitine palmitoyltransferase-1 | Hypoketotic hypoglycaemia, hepatomegaly, ↑ carnitine |
| CPT-2 deficiency | CPT-2 (commonest carnitine defect) | Adult exertional myopathy, rhabdomyolysis |
| Primary carnitine deficiency | OCTN2 transporter | Cardiomyopathy, low plasma carnitine |
| Jamaican vomiting sickness | Hypoglycin A (unripe ackee fruit) inhibits acyl-CoA DH | Hypoglycaemia, vomiting |
| Refsum disease | Phytanoyl-CoA hydroxylase (α-oxidation) | Phytanic acid ↑, retinitis pigmentosa |
| X-linked ALD | Peroxisomal VLCFA transporter (ABCD1) | ↑ VLCFA, adrenal + CNS demyelination |
Key differentials & cross-links
- Hypoglycaemia + low ketones → fatty-acid oxidation defect (MCAD, CPT) or hyperinsulinism. Hypoglycaemia + high ketones → glycogen storage / ketotic hypoglycaemia / GH-cortisol deficiency.
- Methylmalonic aciduria → B12 deficiency or mutase defect (odd-chain FA / propionate pathway).
- Biotin is shared by ACC, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase — multiple carboxylase deficiency presents with combined metabolic acidosis, alopecia, rash.
Recently asked / exam angle
- "Which inhibits CPT-1?" → Malonyl-CoA (most repeated single fact).
- "Rate-limiting enzyme of fatty acid synthesis?" → Acetyl-CoA carboxylase; cofactor biotin.
- "NADPH for lipogenesis comes from?" → HMP shunt + malic enzyme.
- "Number of NADPH used to make palmitate?" → 14.
- "Site of fatty acid synthesis vs oxidation?" → cytosol vs mitochondrial matrix.
- "Net ATP from complete oxidation of palmitate?" → 106 (or 129 old).
- "Enzyme needing B12 in odd-chain oxidation?" → methylmalonyl-CoA mutase.
- "Commonest FA oxidation disorder / hypoketotic hypoglycaemia in infant?" → MCAD deficiency (↑C8).
- "Enzyme liver lacks for ketone utilisation?" → thiophorase (SCOT).
- "Unripe ackee fruit toxin?" → hypoglycin A inhibiting acyl-CoA dehydrogenase.
- Image/diagram-based: identifying the 4-step β-oxidation spiral or citrate shuttle.
Rapid revision
- Synthesis = cytosol, NADPH, ACP, makes palmitate (C16) only; oxidation = matrix, FAD/NAD⁺, CoA.
- ACC (biotin) is rate-limiting for synthesis: citrate activates, palmitoyl-CoA + glucagon + AMPK inhibit; insulin activates by dephosphorylation.
- Malonyl-CoA is the 2-C donor for synthesis AND the inhibitor of CPT-1 — the reciprocal switch.
- Human FAS = one multifunctional dimer, 7 activities + ACP (phosphopantetheine from vitamin B5).
- Citrate shuttle exports acetyl-CoA and yields NADPH via malic enzyme; HMP shunt is the other NADPH source.
- 14 NADPH + 7 ATP + 8 acetyl-CoA → palmitate.
- Carnitine shuttle: CPT-1 (outer, rate-limiting) → CACT → CPT-2 (inner); short/medium-chain FA bypass carnitine.
- β-oxidation spiral: oxidation (FADH₂) → hydration → oxidation (NADH) → thiolysis (acetyl-CoA).
- Palmitate complete oxidation = net 106 ATP (subtract 2 for activation).
- Odd-chain → propionyl-CoA → (B12 mutase) → succinyl-CoA: only FA route feeding gluconeogenesis; B12 deficiency → methylmalonic aciduria.
- MCAD deficiency: hypoketotic hypoglycaemia + dicarboxylic aciduria + ↑C8-acylcarnitine; treat by avoiding fasting.
- Liver makes ketones but cannot use them (no thiophorase); β-hydroxybutyrate is the major blood ketone but is missed by nitroprusside sticks.