The fats in the body are triacylglycerols, cholesterol, or long chains of fatty acids. The fatty acids, a biomolecule, are converted into acetyl-CoA by the method known as beta (𝛃)oxidation.
The acetyl CoA enters the central energy-yielding pathway in many organisms and tissues. Likewise, the electrons removed during 𝛃-oxidation pass through the respiratory chain. Acetyl CoA can also convert to ketone bodies and act as water-soluble fuels for the brain and other tissues.
Beta (𝛃) oxidation is a repetitive four-step metabolic process by which long-chain fatty acids convert into two-chain carbon compounds (acetyl CoA). The fatty acids are insoluble in water and chemically inert, making these perfect storage fuels.
These properties make their catabolism challenging. So, the complete oxidation of fatty acids to CO2 and H2O has primarily three stages; 𝛃-oxidation, oxidation of acetyl CoA in the citric acid cycle, and transfer of electrons to the mitochondrial respiratory chain.
The cells gain fatty acids from three sources; from the diet, fats stored as lipid droplets, and fats synthesized in one part and exported to another. Some species use all three sources and others may use one or two sources. Vertebrates use all three sources, whereas protists obtain fat from their diet only.
Transportation of Fatty Acids
Even though the fats come from different sources, these should enter the mitochondria of the cells before 𝛽-oxidation. The method of transfer of fats depends on the source. Like small intestine absorbs dietary fats. Through blood, it reaches the liver, where it’s catabolism occurs. The fats absorbed from the intestine can also be stored as fat droplets in the adipose and muscle tissues.
The enzymes for the oxidation of fatty acids reside inside the mitochondrial matrix. The fatty acids with 12 or fewer carbon molecules can easily enter the mitochondria without membrane transporters. The free fatty acids (FFAs) with 14 or more carbon molecules cannot enter the mitochondrial membranes. So these must undergo the three-step enzymatic reactions of the carnitine shuttle.
The family of isozymes present in the outer mitochondrial membrane, the acyl-CoA synthetases, catalyzes the first reaction of the shuttle. Thus, forming a fatty acyl-CoA. The thioester linkage between the thiol group of coenzyme A and the carboxyl group of fatty acid forms the fatty acyl-CoA. Here, the cleavage of ATP to AMP and PPi is the energy source for this reaction. The reaction occurs in two steps. The first step is formation of intermediate product, fatty acyl-adenylate, bounded by enzyme. The second step is the formation of fatty acyl CoA. The step is also called activation of fatty acids.
Fatty acid + ATP ⇋ Fatty acyl–adenylate + CoA ⇋ Fatty acyl CoA + AMP + PPi; in presence of acyl-CoA synthetase. △G° = -34 kJ/mol
Like acetyl CoA, fatty acyl CoA is a high-energy compound because the hydrolysis to FFA and CoA has a significant, negative standard free-energy change, i.e.,△G ≅ 31 kJ/mol. The hydrolysis of two phosphate molecules of ATP makes this reaction favorable. In the outer membrane of the mitochondria (face towards the inner membrane), fatty acyl-CoA oxidizes to produce ATP. The ATP returns to the cytosol facing the side of the outer membrane and reused.
In the shuttle’s second reaction, the carnitine hydroxyl group transiently attaches to the fatty acid that will be entering inside the mitochondria. This reaction forms fatty acyl carnitine under the catalyst carnitine acyltransferase I. The CoA is removed either in the outer membrane or intermembrane space. However, the exact place of CoA removal is not discovered. The fatty acyl-carnitine ester enters the mitochondrial matrix by facilitated diffusion through the acyl-carnitine/carnitine transporter of the inner mitochondrial membrane.
The carnitine shuttle’s final reaction is to transfer the fatty acyl group enzymatically from carnitine to mitochondrial CoA by carnitine acyltransferase II. The enzyme is located on the inner face of the inner mitochondrial membrane for releasing fatty acyl-CoA and free carnitine into the matrix. The free carnitine reenters the intermembrane space through the acyl-carnitine transporter.
Steps of Beta (𝛃) Oxidation
The first step of mitochondrial oxidation of fatty acids is the oxidative removal of successive two-carbon units in the form of acetyl CoA from the carboxyl end of the fatty acyl chain. For example, for 16-carbon palmitic acid undergoes seven passes and each pass releases two carbons as acetyl CoA forming 8 acetyl CoA.
𝛃-oxidation of saturated even numbered fatty acids has four basic steps. For unsaturated even numbered fatty acids there is three additional steps. Finally, four additional steps are required for odd numbered fatty acids.
𝛃-Oxidation of Even Saturated Fatty Acids
The beta-oxidation of even saturated fatty acids has four basic steps. The steps repeat until the entire fatty acid completely oxidizes. For example, myristic acid has 14 carbon, and the four basic steps repeat six times to produce seven acetyls CoA.
- The first step of β-oxidation is the dehydrogenation of fatty acyl-CoA to produce a double bond between ɑ and β carbon atoms (C-2 ad C-3) that yields trans-2-enoyl-CoA. Three isozymes of acyl-CoA dehydrogenase catalyzes this reaction. Each enzyme is specific for a range of fatty-acyl chain lengths; very long chain (12-18), medium chain (4-14), and short-chain acyl-CoA dehydrogenase (4-8). A molecule of FAD reduces to FADH2 by the electrons produced after the addition of the double bond.
- Then, in the second step of the β-oxidation, addition of water to the double bond of the trans-2-enoyl-CoA occurs. It forms the L stereoisomer of β-hydroxy acyl-CoA, catalyzed by enoyl-CoA hydratase.
- After that, the third step is the dehydrogenation of L-hydroxy acyl-CoA to form β-ketoacyl-CoA, with the help of catalyst β-hydroxy acyl-CoA dehydrogenase. NAD is the electron acceptor for this step which reduces to NADH + H+.
- Acyl-CoA acetyltransferase (thiolase) catalyzes the last step of β-oxidation where the carboxyl-terminal of the β-ketoacyl-CoA splits to form acetyl-CoA. This step requires a molecule of free coenzyme A. The other byproduct is the coenzyme A thioester of the fatty acid with two fewer carbon atoms. The reaction is called thiolysis because of the removal of the thiol group of coenzyme A.
For fatty acyl chains of 12 or more carbons, the multienzyme complex, the trifunctional protein (TFP), catalyzes the reactions. The TFP is a hetero-octamer 44 subunit. After the long-chain fatty acids shorten to 12 or fewer carbons, four soluble enzymes in the matrix catalyzes the further oxidations.
𝛃-Oxidation of Even Unsaturated Fatty Acids
Most of the fatty acids in phospholipids and triacylglycerols of animals are usually unsaturated, i.e., with one or more double bonds. Enoyl-CoA hydratase cannot act upon the double bonds. β-oxidation of common unsaturated fatty acids requires two added enzymes; isomerase and reductase.
Isomerase changes the position of the double bond to the right position, i.e., from cis to trans in monosaturated fatty acids. Reductase and isomerase work together to change the position of two cis double bonds in polyunsaturated fatty acids.
To understand the concept of 𝛃-oxidation of even monounsaturated fatty acids, let us take an example of oleate, an 18-carbon monounsaturated fatty acid with a cis double bond between C-9 and C-10. Its oxidation includes the following steps:
- The oleate converts to oleoyl-CoA occurs like typical saturated fatty acids and enters the mitochondrial matrix through the carnitine shuttle. Oleoyl-CoA undergoes three cycles of normal 𝛃-oxidation forming three molecules of acetyl-CoA and the CoA ester of a 12-carbon unsaturated fatty acid, cis-3-dodecenoyl CoA.
- The auxiliary enzyme 3,2-enoyl-CoA isomerase changes the cis-3-dodecenoyl-COA to the trans-2-enoyl-CoA which is the substrate for the enzyme enoyl CoA hydratase and converts it into the L-𝛃-hydroxyacyl-CoA and releasing a molecule of acetyl CoA.
- Now, the other enzymes oxidizes the intermediate product in four cycles of beta-oxidation to yield five more acetyls CoA. Hence, a total of nine acetyls CoA forms under the beta-oxidation of oleate.
𝛃-Oxidation of Odd Fatty Acids
Although most of the fatty acids utilized by the body are even-number, odd-number fatty acids are also reasonably common in many plants and marine organisms. The activation of odd fatty acids for oxidation is as same as even-number fatty acids, i.e., beginning at the carboxyl end of the chain.
The substrate for the final pass of ꞵ-oxidation is a 5-fatty acyl-CoA, which oxidizes to form acetyl-CoA and propionyl-CoA. The acetyl-CoA enters the citric cycle, whereas propionyl-CoA enters a different pathway.
In the first step, propionyl-CoA carboxylated to form a D-stereoisomer of methyl malonyl-COA in the presence of propionyl-CoA carboxylase, ATP, and HCO3–. Cleaving of ATP to ADP and Pi provides energy for this reaction. The methyl malonyl-CoA epimerase transforms D-methyl malonyl-CoA into L-stereoisomer (L-methyl malonyl-CoA). methyl malonyl-CoA mutase and coenzyme B12 catalyzes the rearrangement of L-methyl malonyl-CoA to form succinyl-CoA. The succinyl-CoA enters the citric cycle.
Energy Yield During Beta Oxidation
The energy-yielding steps in beta-oxidation are:
- The first step is where fatty acyl-CoA converts into tans-2-Enoyl-CoA, and forming an FADH2 molecule. For the “n” number of cycles, “n” FADH2 forms. Each FADH2 produces 1.5 ATP after oxidative phosphorylation.
- The third step is where ꞵ-hydroxy acyl-CoA converts into ꞵ-ketoacyl-CoA, a molecule of NADH, i.e.,producing “n” NADH after the “n” number of the ꞵ-oxidation cycle. From a single NADH, 2.5 ATP produces after oxidative phosphorylation.
- Each cycle of ꞵ-oxidation produces a molecule of acetyl CoA, so the “n” cycle of ꞵ-oxidation produces “n+1” acetyl CoA. Each molecule of acetyl-CoA produces 10 ATP after Kreb’s cycle.
So for example, beta oxidation of palmitoyl-CoA produces 7 FADH2, 7 NADH, and eight acetyl-CoA, which enters the citric acid cycle and oxidative phosphorylation to produce 108 ATP molecules (10.5 from FADH2, 17.5 from NADH, and 80 from acetyl CoA).
Two molecules of ATP are used during activation, so 106 ATP is generated after the oxidation of palmitate.
Regulation of Beta 𝛃 Oxidation
The ꞵ-oxidation of fatty acid is highly regulated as it is a necessary fuel, and oxidation must only begin in dire situations.
The three-step process (carnitine shuttle) that transfers fatty acids from cytosol to mitochondrial matrix is rate limiting for ꞵ-oxidation and a key regulation point. It is because once the fatty acyl CoA group enters the mitochondria, it will be converted into acetyl CoA. The following is the way this shuttle is regulated:
- The first intermediate byproduct of cytosolic biosynthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, increases in concentration whenever there is a good supply of carbohydrates. This byproduct inhibits acyltransferase I inhibiting the ꞵ-oxidation of fatty acids in the liver.
- When NADH/NAD ratio is high, inhibition of ꞵ-hydroxy acyl-CoA dehydrogenase occurs.
- High concentrations of acetyl CoA inhibit thiolase.
- Insulin also inhibits ꞵ-oxidation by dephosphorylating hormone-sensitive lipase, which inhibits the release of fatty acids from adipose tissues.
Activating ꞵ-oxidation occurs due to the epinephrine activating a cAMP-dependent protein kinase. The cAMP-dependent protein kinase phosphorylates and activates hormone-sensitive lipase. Hormone-sensitive lipase releases fatty acids and glycerol from adipose tissue for ꞵ-oxidation.
Importance of 𝛃-Oxidation
Ꞵ-oxidation is an essential metabolic process because it yields energy during exercising (in humans) or during hibernation in hibernating animals like bears. Other importance of ꞵ-oxidation include:
- It helps control blood glucose levels, i.e., people with genetic defects have reported low blood glucose levels (hypoglycemia).
- The non-alcoholic fatty liver syndrome occurs due to impaired beta-oxidation.
- Nelson, D., Lehninger, A., Cox, M., & Nelson, D. (2005). Lecture notebook for Lehninger principles of biochemistry, fourth edition (pp. 631-643). W.H. Freeman.
- Talley JT, Mohiuddin SS. Biochemistry, Fatty Acid Oxidation. [Updated 2023 Jan 16]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK556002/