Unit 3: Lipid Metabolism

1. β-Oxidation of Saturated Fatty Acids

1.1 Introduction and Subcellular Localization

β-Oxidation is the catabolic pathway by which fatty acid molecules are broken down in the mitochondria to generate acetyl-CoA, which enters the citric acid cycle, and reduced coenzymes (NADH and FADH₂), which feed into the electron transport chain. The process is termed β-oxidation because the cleavage of the fatty acid chain occurs at the β-carbon atom (C-3 position), removing two-carbon acetyl units sequentially from the carboxyl end.

Subcellular Site: The enzymes responsible for β-oxidation are localized in the mitochondrial matrix. However, fatty acid activation occurs in the outer mitochondrial membrane or cytosol.

1.2 Fatty Acid Activation and Carnitine Shuttle

Before fatty acids can enter the mitochondrial matrix for oxidation, they must undergo a two-phase transport preparation process:

1. Activation of Fatty Acids: Free fatty acids in the cytosol are activated to fatty acyl-CoA by the enzyme acyl-CoA synthetase (thiokinase) located on the outer mitochondrial membrane. This reaction consumes one ATP molecule and hydrolyzes it to AMP and inorganic pyrophosphate (PP_i). High pyrophosphatase activity subsequently hydrolyzes PP_i into two inorganic phosphates (2 P_i), driving the reaction irreversibly forward.

   Fatty Acid + ATP + CoASH → Acyl-CoA + AMP + PP_i
   PP_i + H₂O → 2 P_i

2. Carnitine Shuttle System: The inner mitochondrial membrane is impermeable to acyl-CoA derivatives. Long-chain fatty acyl-CoA compounds require a specialized carnitine carrier system to enter the matrix:
   • Carnitine Palmitoyltransferase-I (CPT-I): Located on the outer mitochondrial membrane, CPT-I transfers the acyl group from acyl-CoA to carnitine, forming acylcarnitine and releasing free CoASH.
   • Carnitine-Acylcarnitine Translocase: An inner membrane antiporter that transports acylcarnitine into the mitochondrial matrix in exchange for free carnitine moving out.
   • Carnitine Palmitoyltransferase-II (CPT-II): Located on the inner surface of the inner mitochondrial membrane, CPT-II converts acylcarnitine back to acyl-CoA and free carnitine within the matrix.

1.3 Four Sequential Steps of β-Oxidation (Even-Chain Fatty Acids)

Once inside the mitochondrial matrix, acyl-CoA undergoes a repeating four-step cyclic reaction sequence:

Each round of β-oxidation shortens the fatty acyl-CoA chain by two carbons, producing 1 Acetyl-CoA, 1 FADH₂, and 1 NADH.

1.4 Energetics and ATP Yield of Palmitate Oxidation

Palmitate is a 16-carbon saturated fatty acid (Palmitoyl-CoA). Its complete oxidation requires 7 cycles of β-oxidation.

• Products Generated:
  • 8 molecules of Acetyl-CoA (via 7 cleavage cycles)
  • 7 molecules of FADH₂
  • 7 molecules of NADH

Note: Under traditional calculations (3 ATP/NADH, 2 ATP/FADH₂, 12 ATP/Acetyl-CoA), the net yield is calculated as 129 ATP. Modern biochemistry uses P/O ratios yielding 106 ATP.

1.5 β-Oxidation of Odd-Chain Fatty Acids

Fatty acids with an odd number of carbon atoms undergo standard β-oxidation until the final cycle produces one molecule of Acetyl-CoA (2 carbons) and one molecule of Propionyl-CoA (3 carbons).

Conversion of Propionyl-CoA to Succinyl-CoA:

1. Carboxylation: Propionyl-CoA is carboxylated by Propionyl-CoA carboxylase to D-methylmalonyl-CoA. This enzyme requires biotin and 1 ATP.

   Propionyl-CoA + HCO₃^- + ATP → D-Methylmalonyl-CoA + ADP + P_i

2. Racemization: Methylmalonyl-CoA racemase converts D-methylmalonyl-CoA to L-methylmalonyl-CoA.
3. Isomerization: Methylmalonyl-CoA mutase rearranges L-methylmalonyl-CoA into Succinyl-CoA. This enzyme requires Vitamin B₁₂ (Deoxyadenosylcobalamin) as a coenzyme.

   L-Methylmalonyl-CoA → Succinyl-CoA

Succinyl-CoA directly enters the citric acid cycle and can serve as a substrate for gluconeogenesis, making odd-chain fatty acids weakly gluconeogenic.

1.6 Comparison: Even-Chain vs. Odd-Chain Fatty Acid Oxidation

2. Biosynthesis of Palmitic Acid

2.1 Subcellular Localization and Citrate-Malate Shuttle

Biosynthesis of palmitic acid (de novo lipogenesis) occurs in the cytosol of lipogenic tissues such as the liver, lactating mammary glands, and adipose tissue.

Citrate Shuttle:

Acetyl-CoA is produced in the mitochondrial matrix but cannot cross the inner mitochondrial membrane. Acetyl-CoA is combined with oxaloacetate by citrate synthase to form citrate, which crosses into the cytosol via the citrate translocase.

Citrate + ATP + CoASH + H₂O → Acetyl-CoA + Oxaloacetate + ADP + P_i (catalyzed by ATP-Citrate Lyase in cytosol)

2.2 Production of Malonyl-CoA (Committed Step)

The rate-limiting and committed step of fatty acid synthesis is the carboxylation of acetyl-CoA to form malonyl-CoA catalyzed by Acetyl-CoA Carboxylase (ACC).

Acetyl-CoA + HCO₃^- + ATP → Malonyl-CoA + ADP + P_i

• Coenzyme Required: Biotin (covalently attached to ACC).
• Regulation: ACC is activated by citrate and insulin; it is inhibited by palmitoyl-CoA, glucagon, and epinephrine.

2.3 Fatty Acid Synthase (FAS) Multienzyme Complex

In eukaryotes, fatty acid synthesis is carried out by Fatty Acid Synthase (FAS), a homodimeric enzyme complex consisting of two identical polypeptide subunits arranged head-to-tail. Each monomer contains seven catalytic domains and an Acyl Carrier Protein (ACP) domain containing a 4'-phosphopantetheine prosthetic group.

2.4 Reaction Sequence of Palmitate Synthesis

The synthesis loop adds two-carbon units iteratively using malonyl-CoA as the donor:

1. Loading / Priming: Acetyl-CoA is loaded onto the Cys-SH group of β-ketoacyl-ACP synthase (KS), and Malonyl-CoA is loaded onto the ACP-SH group.
2. Condensation: The acetyl group is transferred onto the malonyl unit on ACP, accompanied by decarboxylation (release of CO₂), yielding β-ketobutyryl-ACP (catalyzed by β-ketoacyl-ACP synthase).
3. Reduction 1: The β-keto group is reduced to a β-hydroxy group using NADPH (catalyzed by β-ketoacyl-ACP reductase).
4. Dehydration: Water is removed to introduce a trans double bond, yielding trans-Δ²-enoyl-ACP (catalyzed by β-hydroxyacyl-ACP dehydratase).
5. Reduction 2: The double bond is reduced by NADPH to yield Butyryl-ACP (catalyzed by enoyl-ACP reductase).

The butyryl group is transferred back to the Cys-SH of KS, a new malonyl group attaches to ACP, and the 4-step sequence repeats. After 7 total cycles, 16-carbon Palmitoyl-ACP is formed.

Chain Termination: Thioesterase cleaves Palmitoyl-ACP to release free Palmitate and ACP.

2.5 Stoichiometry and Energy Requirement

The overall balanced equation for the biosynthesis of one molecule of palmitate is:

8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H⁺ → Palmitate + 8 CoASH + 7 ADP + 7 P_i + 14 NADP⁺ + 6 H₂O

• NADPH Sources: Hexose Monophosphate (HMP) Shunt (Pentose Phosphate Pathway) and Cytosolic Malic Enzyme.

2.6 Comparison: β-Oxidation vs. Fatty Acid Biosynthesis

3. Ketogenesis

3.1 Definition and Subcellular Site

Ketogenesis is the metabolic pathway by which excess acetyl-CoA derived from fatty acid breakdown is converted into water-soluble ketone bodies. This pathway occurs during conditions of carbohydrate limitation, such as prolonged starvation, low-carbohydrate intake, or uncontrolled diabetes mellitus.

Site of Synthesis: Exclusively in the mitochondrial matrix of hepatocytes (liver cells). Although the liver produces ketone bodies, it cannot utilize them because it lacks the enzyme β-ketoacyl-CoA transferase (thiophorase).

3.2 Enzymatic Pathway of Ketogenesis

The three recognized ketone bodies are Acetoacetate, β-Hydroxybutyrate, and Acetone.

1. Condensation of Acetyl-CoA Units: Two acetyl-CoA molecules condense to form acetoacetyl-CoA, catalyzed by mitochondrial thiolase.

   2 Acetyl-CoA ⇌ Acetoacetyl-CoA + CoASH

2. Synthesis of HMG-CoA: Acetoacetyl-CoA condenses with a third acetyl-CoA to form β-hydroxy-β-methylglutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase.

   Acetoacetyl-CoA + Acetyl-CoA + H₂O → HMG-CoA + CoASH

   Rate-Limiting Step: Mitochondrial HMG-CoA synthase is the key rate-limiting enzyme of ketogenesis.

3. Cleavage to Acetoacetate: HMG-CoA lyase cleaves HMG-CoA into acetoacetate and acetyl-CoA.

   HMG-CoA → Acetoacetate + Acetyl-CoA

4. Interconversion and Byproduct Formation:
   • β-Hydroxybutyrate Formation: Acetoacetate is reversibly reduced to β-hydroxybutyrate by β-hydroxybutyrate dehydrogenase using NADH.

     Acetoacetate + NADH + H⁺ ⇌ β-Hydroxybutyrate + NAD⁺

   • Acetone Formation: Acetoacetate spontaneously (non-enzymatically) or via acetoacetate decarboxylase undergoes decarboxylation to acetone, which is volatile and eliminated via the lungs.

     Acetoacetate → Acetone + CO₂

3.3 Utilization of Ketone Bodies in Extrahepatic Tissues

Ketone bodies diffuse from liver mitochondria into the blood and are transported to extrahepatic tissues (brain, heart, skeletal muscle, renal cortex).

Extrahepatic Activation Steps:

1. β-Hydroxybutyrate is re-oxidized to acetoacetate by β-hydroxybutyrate dehydrogenase, producing 1 NADH.
2. Acetoacetate is activated to acetoacetyl-CoA by thiophorase (β-ketoacyl-CoA transferase) using succinyl-CoA as the CoA donor.

   Acetoacetate + Succinyl-CoA → Acetoacetyl-CoA + Succinate

3. Acetoacetyl-CoA is cleaved by thiolase into 2 Acetyl-CoA, which enter the TCA cycle for oxidation and ATP production.

3.4 Clinical Significance and Diabetic Ketoacidosis