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Once the fatty acid is inside the mitochondrial matrix, beta-oxidation occurs by cleaving two carbons every cycle to form acetyl-CoA. The process consists of 4 steps.

  1. A long-chain fatty acid is dehydrogenated to create a trans double bond between C2 and C3. This is catalyzed by acyl CoA dehydrogenase to produce trans-delta 2-enoyl CoA. It uses FAD as an electron acceptor and it is reduced to FADH2.
  2. Trans-delta2-enoyl CoA is hydrated at the double bond to produce L-3-hydroxyacyl CoA by enoyl-CoA hydratase.
  3. L-3-hydroxyacyl CoA is dehydrogenated again to create 3-ketoacyl CoA by 3-hydroxyacyl CoA dehydrogenase. This enzyme uses NAD as an electron acceptor.
  4. Thiolysis occurs between C2 and C3 (alpha and beta carbons) of 3-ketoacyl CoA. Thiolase enzyme catalyzes the reaction when a new molecule of coenzyme A breaks the bond by nucleophilic attack on C3. This releases the first two carbon units, as acetyl CoA, and a fatty acyl CoA minus two carbons. The process continues until all of the carbons in the fatty acid are turned into acetyl CoA.

β-Oxidation of unsaturated fatty acids poses a problem since the location of a cis bond can prevent the formation of a trans-Δ2 bond. These situations are handled by an additional two enzymes, Enoyl CoA isomerase or 2,4 Dienoyl CoA reductase.Whatever the conformation of the hydrocarbon chain, β-oxidation occurs normally until the acyl CoA (because of the presence of a double bond) is not an appropriate substrate for acyl CoA dehydrogenase, or enoyl CoA hydratase:

  • If the acyl CoA contains a cis-Δ3 bond, then cis-Δ3-Enoyl CoA isomerase will convert the bond to a trans-Δ2 bond, which is a regular substrate.
  • If the acyl CoA contains a cis-Δ4 double bond, then its dehydrogenation yields a 2,4-dienoyl intermediate, which is not a substrate for enoyl CoA hydratase. However, the enzyme 2,4 Dienoyl CoA reductase reduces the intermediate, using NADPH, into trans-Δ3-enoyl CoA. As in the above case, this compound is converted into a suitable intermediate by 3,2-Enoyl CoA isomerase.

So, odd-numbered double bonds are handled by the isomerase. Even-numbered double bonds by the reductase (which creates an odd-numbered double bond).

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