Fatty Acid Biosynthesis

Biological Roles of Fatty Acids

Fatty acid biosynthesis is a fundamental cellular process essential for the creation of biomolecules with three primary roles:

• Energy Storage: Fatty acids are stored as triacylglycerols (fats). These serve as the body’s primary long-term energy reserve. Unlike carbohydrates, fats are highly reduced, allowing them to store more energy per gram.

• Structural Components: Fatty acids are critical components of phospholipids, which form the structural basis (the lipid bilayer) of all biological membranes. This is necessary for cellular compartmentalization.

• Signalling Molecules: Certain lipids act as signaling molecules. For example, derivatives of phosphatidylinositol are involved in complex intracellular signaling cascades.

Metabolic Comparison: Anabolism vs. Catabolism

Fatty acid metabolism consists of two opposing yet coordinated processes: oxidation (catabolism) and synthesis (anabolism).

• Oxidation (Catabolism):

  • Function: Breaks down fatty acids to release energy and reducing equivalents.

  • Localisation: Occurs in the mitochondria (β-oxidation).

  • Coenzymes: Uses $NAD^+$ and produces $NADH$ and $FADH_2$.

  • Units: Involves the removal of 2-carbon units.

• Synthesis (Anabolism):

  • Function: Builds fatty acids; requires energy input and reducing equivalents.

  • Localisation: Occurs in the cytosol.

  • Coenzymes: Uses $NADP^+/NADPH$ and consumes $ATP$.

  • Units: Involves the addition of 2-carbon units and requires $CO_2$ (in the form of $HCO_3^-$).

Thermodynamics and Spatial Separation:

• Breaking chemical bonds releases energy, while forming new bonds requires energy input.

• The spatial separation (mitochondria for breakdown vs. cytosol for synthesis) prevents "futile cycling," a situation where synthesis and degradation would occur simultaneously and waste energy.

Stoichiometry and General Properties of Fatty Acid Synthesis

• Primary Product: In mammalian systems, the main product of fatty acid synthesis is palmitate (palmitic acid or hexadecanoic acid).

• Chemical Formula of Palmitate: $C_{16}H_{32}O_2$.

• Structure: A 16-carbon saturated fatty acid ($C16:0$).

• Carbon Increments: Most naturally occurring fatty acids have an even number of carbons (e.g., 14, 16, 18, 20) because they are built by adding 2-carbon units sequentially.

• Metabolic Flux: Fat metabolism is highly dynamic. In an average human, approximately $200 g$ of fatty acids are synthesized and degraded daily. This turnover accounts for about $8\%$ of basal oxygen consumption.

• Regulation: The process is controlled by energy balance, nutritional state, and hormonal regulation (e.g., the balance between insulin and glucagon).

Experimental Determination of Sub-cellular Localisation

The localization of fatty acid synthesis to the cytosol was determined using cell fractionation and centrifugation:

1. Homogenization: Tissue is broken open at $+4^◦C$.

2. Filtration: Filtrate is passed through cheesecloth.

3. Differential Centrifugation:

   • $600 g$ for $5 min$: Pellets nuclei, cell walls, and intact cells.

   • $2,000 g$ for $5–20 min$: Pellets chloroplasts.

   • $10,000 g$ for $20 min$: Pellets mitochondria and mitochondrial fragments.

   • $100,000 g$ for $2 hours$: Pellets free ribosomes and microsomes (membranous debris).

4. Findings: Fatty acid synthesis activity was found in the soluble fraction (the supernatant after $100,000 g$ centrifugation), which contains soluble cytosolic proteins.

5. Further Fractionation: When the soluble fraction was further separated using ammonium sulphate precipitation, no single fraction could support biosynthesis alone. At least two fractions had to be combined, one of which must contain biotin.

Stage 1: The Carboxylation of Acetyl-CoA

The first stage of synthesis is the formation of malonyl-CoA, which serves as the activated 2-carbon donor for chain elongation.

• Enzyme: Acetyl-CoA Carboxylase (ACC).

• Properties: Irreversible, rate-limiting, and highly regulated.

• Cofactor: Biotin.

• Reaction Equation:     $CH_3C–S-CoA + HCO_3^- + ATP \rightarrow CO_2^-–CH_2C–S-CoA + ADP + P_i + H_2O$

• Activation Mechanism (Two-step process):

  1. Biotin Carboxylation: Biotin is carboxylated using energy from $ATP$.         $\text{Biotin} + HCO_3^- + ATP \rightarrow \text{Biotin-}CO_2^- + ADP + P_i$

  2. Carboxyl Transfer: The activated $CO_2$ group is transferred to acetyl-CoA via the transcarboxylase domain.         $\text{Biotin-}CO_2^- + \text{acetyl-CoA} \rightarrow \text{Biotin} + \text{malonyl-CoA}$

Stage 2: The Fatty Acid Synthase (FAS) Multi-Enzyme Complex

Following the formation of malonyl-CoA, the Fatty Acid Synthase (FAS) complex carries out chain elongation.

• Location: Cytosolic fraction.

• Enzyme Structure: A large multi-functional enzyme complex involving seven separate reactions, each with a specific active site.

• Key Carrier: Acyl Carrier Protein (ACP). It uses a 4’-phosphopantetheine arm to carry intermediates between active sites.

• Intermediate Handling: Intermediates are never released; they remain enzyme-bound until the process is complete.

Detailed Enzymatic Steps of the Elongation Cycle

The chain grows by 2-carbon units supplied by malonyl-CoA in a four-step cycle:

1. Priming and Loading:

   • Acetyl-CoA is attached to ACP by acetyl-CoA transacylase to form acetyl-ACP.

   • The acetyl group is then transferred to the $\beta$-ketoacyl ACP synthase domain, freeing the ACP.

   • Malonyl transacylase transfers a malonyl group from malonyl-CoA to the free ACP, forming malonyl-ACP.

2. Condensation:

   • Catalyzed by $\beta$-ketoacyl-ACP synthase.

   • The acetyl group (on the synthase) and malonyl group (on ACP) are joined, and a $CO_2$ molecule is released.

   • Result: $\beta$-ketoacyl-ACP (specifically acetoacetyl-ACP in the first cycle).

3. First Reduction:

   • Catalyzed by $\beta$-ketoacyl-ACP reductase.

   • Uses $NADPH + H^+$ to reduce the keto group to a hydroxyl group.

   • Result: $\beta$-hydroxyacyl-ACP.

4. Dehydration:

   • Catalyzed by $\beta$-hydroxyacyl-ACP dehydrase.

   • Removes a water molecule ($H_2O$) to form a double bond.

   • Result: $\alpha,\beta\text{-trans-enoyl-ACP}$.

5. Second Reduction:

   • Catalyzed by enoyl-ACP reductase.

   • Uses $NADPH + H^+$ to reduce the double bond to a single bond.

   • Result: A saturated acyl-ACP (e.g., butyryl-ACP after the first cycle).

Cycle Repetition: The resulting acyl group is transferred back to the $\beta$-ketoacyl synthase domain. This frees the ACP to bind a new malonyl group, and the cycle repeats until a 16-carbon chain is formed.

Termination and Final Product Release

• Termination: Once the fatty acid chain reaches 16 carbons (palmitoyl-ACP), the process stops.

• Release Enzyme: Palmitoyl thioesterase.

• Result: Palmitate and a free $ACP-SH$.

• Tissue Variations: While palmitate is the major product in the mammalian liver, other tissues or species may use secondary thioesterases to produce different fatty acid lengths.

Total Stoichiometry for Palmitate Synthesis

To synthesize one molecule of palmitate ($C16$), the following is required:

• Units: 1 Acetyl-CoA (to prime/methyl terminus) + 7 Malonyl-CoA (each providing 2 carbons).

• Substrates: Since each malonyl-CoA requires one acetyl-CoA and one $ATP$, the total input from acetyl-CoA is 8 units.

The Overall Chemical Equation: $8\text{ acetyl-CoA} + 7\text{ ATP} + 14\text{ NADPH} + 14 H^+ \rightarrow \text{palmitate} + 8\text{ CoA} + 6 H_2O + 7\text{ ADP} + 7 P_i + 14\text{ NADP}^+$

Note on Reducing Power: The required $NADPH$ is typically supplied by the Pentose Phosphate Pathway or the Malic Enzyme.