β-Oxidation & Carnitine Shuttle – Comprehensive Video Notes

Overall Context

• Topic: β-Oxidation of fatty acids & the carnitine shuttle – how cells mobilize fat and convert it to acetyl-CoA for the TCA cycle.
• Course emphasis (Exam 3):
  • Recognize pathway logic rather than memorize every enzyme/substrate name.
  • Be able to describe: activation step, transport into mitochondria, 4-step spiral of β-oxidation, products & energetic yield.
  • Carnitine shuttle is specifically testable.
• Deeper studies/MCAT: expect detailed enzyme memorization, stoichiometric bookkeeping, integration with glycolysis & TCA.

Why Fatty Acids Require Special Handling

• Fatty acids are amphipathic but largely hydrophobic → poorly soluble in aqueous cytosol.
• Must be chemically activated & shuttled into the mitochondrial matrix (site of the TCA cycle & oxidative phosphorylation).
• Long-chain fatty acids usually have an even number of carbons; β-oxidation removes 2-carbon units, and fatty-acid synthesis adds 2-carbon units → explains this even-number prevalence.

Step 1 – Chemical Activation (Cytosol)

• Enzyme: acyl-CoA synthetase (a “fatty-acyl-CoA ligase”).
• Reaction: (\text{Fatty acid} + \text{ATP} + \text{CoA} \;\xrightarrow{\text{acyl-CoA synthetase}}\; \text{Fatty acyl-CoA} + \text{AMP} + \text{PP_i}) \qquad (\Delta G^\circ' \approx -15\;\text{kJ·mol}^{-1})
  • ATP is cleaved at the α–β bond (pyrophosphate cleavage), not the usual γ-phosphate removal.
  • Generates a high-energy thioester (fatty-acyl-CoA) analogous to the acetyl-CoA created in the pyruvate “bridge” reaction.
• Pyrophosphate (PPᵢ) is rapidly hydrolyzed by inorganic pyrophosphatase, further driving the reaction forward.

Step 2 – Carnitine Shuttle (Crossing the Inner Mitochondrial Membrane)

• Outer membrane is permeable to acyl-CoA; inner membrane is not.
• Transfer the acyl group from CoA to carnitine:
  1. Carnitine acyltransferase I (CAT I) on the outer membrane forms acyl-carnitine.
  2. Acyl-carnitine is translocated through an acyl-carnitine/carnitine antiporter.
  3. Carnitine acyltransferase II (CAT II) on the matrix side regenerates fatty-acyl-CoA + free carnitine.
• The swap is energetically near-neutral, acting purely as a carrier mechanism.

Step 3 – β-Oxidation Spiral (Matrix)

Each “turn” shortens the fatty-acyl-CoA by two carbons and produces one acetyl-CoA plus reduced electron carriers. Four core reactions:

Terminology & concepts:

• β carbon (C3) is the locus of oxidation → eponymous β-oxidation.
• The pathway is often drawn as a spiral (not straight linear, not a true cycle) because the substrate shortens with each round.
• Repeat until only a two-carbon fragment remains → final acetyl-CoA.

Hydration vs. Hydrolysis (Exam Favorite!)

• Hydration: adds H and OH from water without breaking the substrate into two separate molecules.
• Hydrolysis: water is used to cleave a bond, generating two products (e.g., peptide bond hydrolysis).

Energetic Yield & ATP Equivalents

• Rule of thumb (oxidative phosphorylation):
  • 1\,\text{NADH} \approx 2.5\,\text{ATP}
  • 1\,\text{FADH}_2 \approx 1.5\,\text{ATP}
• Per turn of β-oxidation (up to but excluding acetyl-CoA oxidation in the TCA):
  • 1 FADH₂ → ~1.5 ATP
  • 1 NADH → ~2.5 ATP
  • Plus the downstream TCA/ETC yield of the produced acetyl-CoA ((~10\,\text{ATP})).
• Example global stoichiometry for palmitoyl-CoA (C16): (\text{Palmitoyl-CoA} + 7\,\text{FAD} + 7\,\text{NAD}^+ + 7\,\text{H}2\text{O} + 7\,\text{CoA} \to 8\,\text{Acetyl-CoA} + 7\,\text{FADH}2 + 7\,\text{NADH} + 7\,\text{H}^+)
  • 8 Acetyl-CoA → 8 turns of the TCA cycle.
  • When you sum ATP equivalents from all FADH₂, NADH, and the TCA cycles, fat far out-performs glucose per carbon because its carbons are more reduced (few/no hydroxyls attached).

Structural/Mechanistic Highlights

• Thioester chemistry (C=O–SCoA) is a recurring motif (fatty-acyl-CoA, acetyl-CoA) due to its high-energy nature and ability to undergo nucleophilic attack.
• FAD as a prosthetic flavin stays locked in the enzyme, unlike NAD⁺/NADH which freely diffuse.
• The β-ketoacyl intermediate is electrophilic, enabling nucleophilic attack by CoA in the thiolysis step.

Relation to Other Pathways & Physiology

• Bridge: Pyruvate → acetyl-CoA uses a different CoA-dependent thioester formation (pyruvate dehydrogenase) but illustrates the centrality of acetyl-CoA.
• β-Oxidation supplies acetyl-CoA to the TCA cycle and electrons directly to the electron-transport chain (ETC) via NADH/FADH₂.
• In fasting or carbohydrate-limited states, rapid β-oxidation → excess acetyl-CoA → ketone-body formation.
• Clinical: defects in carnitine transport or CAT I/CAT II enzymes lead to hypoketotic hypoglycemia, muscle weakness.

Ethical/Practical/Real-World Notes

• Dietary implications: High-fat caloric density; understanding β-oxidation helps explain fad diets, ketosis, metabolic disorders.
• Pharmacology/toxicity: Some herbicides hinder β-oxidation; clinical carnitine supplementation is used in deficiencies.
• Exercise physiology: During prolonged endurance exercise, reliance shifts from glycogen to fatty-acid β-oxidation.

Key Take-Home Messages (Exam-Oriented Bullets)

• Activation: ATP → AMP + PPᵢ; fatty-acyl-CoA formed via thioester bond ((\Delta G^\circ' < 0)).
• Carnitine shuttle: required to pass the inner mitochondrial membrane; reversible acyl transfers between CoA and carnitine.
• Four-reaction spiral (dehydrogenation–hydration–dehydrogenation–thiolysis); β-carbon is oxidized.
• Each round yields 1 FADH₂, 1 NADH, 1 acetyl-CoA, and a fatty-acyl-CoA shortened by two carbons.
• Even-numbered carbon chains & spiral depiction = hallmark of β-oxidation.
• Hydration ≠ hydrolysis: know the conceptual difference.
• Electron carriers generated feed the ETC; NADH ~2.5 ATP, FADH₂ ~1.5 ATP.
• Fatty acids release more ATP per carbon than glucose due to their more reduced state.