Beta-oxidation of Fatty Acid

Fatty acids are one of the major sources of energy for the human body, especially in the postabsorptive and fasted states when glucose supply is limiting. Fatty acids are stored as triglycerides in adipose tissue and are released into the bloodstream as free fatty acids when needed. Fatty acids can then enter various tissues, such as muscle, heart, liver and brain, where they undergo a catabolic process called beta-oxidation (also β-oxidation) to produce acetyl-CoA, which enters the citric acid cycle, and NADH and FADH2, which are co-enzymes used in the electron transport chain .

Beta-oxidation is named as such because the beta carbon of the fatty acid undergoes oxidation to a carbonyl group. Beta-oxidation is primarily facilitated by the mitochondrial trifunctional protein, an enzyme complex associated with the inner mitochondrial membrane, although very long chain fatty acids are oxidized in peroxisomes. Beta-oxidation consists of four sequential steps: dehydrogenation, hydration, oxidation and thiolytic cleavage. These steps are repeated until all the carbons of a fatty acid are converted to acetyl-CoA or propionyl-CoA.

Beta-oxidation is regulated by the mechanisms that control oxidative phosphorylation (i.e., by the demand for ATP). Activators such as epinephrine stimulate beta-oxidation by activating a hormone-sensitive lipase that releases fatty acids from adipose tissue. Inhibitors such as insulin inhibit beta-oxidation by dephosphorylating the lipase and preventing the release of fatty acids from adipose tissue. Moreover, malonyl-CoA, an intermediate in fatty acid synthesis, inhibits the transport of fatty acyl-CoA into mitochondria by inhibiting carnitine palmitoyltransferase I (CPT I), thus preventing a futile cycle of synthesis and degradation.

Beta-oxidation is an important metabolic pathway for generating energy from fatty acids. It also provides substrates for other pathways, such as ketogenesis in the liver and gluconeogenesis from propionyl-CoA. Defects in beta-oxidation can cause various metabolic disorders, such as fatty acid oxidation disorders (FAODs), which are characterized by hypoglycemia, hypoketosis, cardiomyopathy and muscle weakness. Therefore, understanding the biochemistry and physiology of beta-oxidation is essential for maintaining energy homeostasis and health in the human body.

Beta-oxidation is the process by which fatty acids are broken down into smaller units that can be used for energy production. The location of beta-oxidation depends on the type of cell and the length of the fatty acid chain.

• In eukaryotic cells, beta-oxidation takes place in the mitochondria, the organelles that are responsible for cellular respiration and ATP synthesis . However, before entering the mitochondria, fatty acids have to be activated and transported across the mitochondrial membrane by different mechanisms depending on their chain length.

  • Short-chain fatty acids (less than 6 carbons) can diffuse freely through the mitochondrial membrane and bind to coenzyme A (CoA) inside the matrix.
  • Medium-chain fatty acids (6 to 12 carbons) can also cross the mitochondrial membrane, but they require a specific transporter protein called carnitine-independent transporter (CIT) or monocarboxylate transporter (MCT) to do so.
  • Long-chain fatty acids (more than 12 carbons) cannot cross the mitochondrial membrane by themselves. They have to be converted to fatty acyl-CoA in the cytosol by an enzyme called acyl-CoA synthetase, and then transferred to carnitine by another enzyme called carnitine palmitoyltransferase I (CPT I) on the outer mitochondrial membrane. The resulting fatty acyl-carnitine can then be transported across the membrane by a protein called carnitine-acylcarnitine translocase (CAT), and reconverted to fatty acyl-CoA by carnitine palmitoyltransferase II (CPT II) on the inner mitochondrial membrane. This process is known as the carnitine shuttle.
  • Very long-chain fatty acids (more than 20 carbons) are too large to be processed by the mitochondrial enzymes. They have to be oxidized in another organelle called peroxisome, where they are shortened to medium-chain fatty acids that can then enter the mitochondria via the CIT or MCT transporters. Beta-oxidation in peroxisomes differs from that in mitochondria in that it produces hydrogen peroxide (H2O2) instead of FADH2 and NADH, which are then degraded by catalase.

• In prokaryotic cells, beta-oxidation takes place in the cytosol, where all the enzymes and cofactors required for the process are present. Prokaryotes do not have mitochondria or peroxisomes, so they do not need any transport mechanisms for fatty acids. However, they have different types of acyl-CoA synthetases and acyl-CoA dehydrogenases that can handle a variety of fatty acid chain lengths and degrees of unsaturation.

In summary, beta-oxidation is a universal pathway for fatty acid degradation that occurs in different cellular compartments depending on the type and size of the fatty acid molecule. The main location of beta-oxidation is the mitochondria in eukaryotes and the cytosol in prokaryotes.

Beta-oxidation is the process by which fatty acids are broken down into smaller units of acetyl-CoA, which can then enter the citric acid cycle and generate energy. The substrates and products of beta-oxidation depend on the type and length of the fatty acid chain, as well as the location of the process.

The main substrate for beta-oxidation is a fatty acyl-CoA molecule, which is formed by the activation of a free fatty acid with coenzyme A (CoA) in the cytosol. This reaction requires ATP and produces AMP and pyrophosphate (PPi). The fatty acyl-CoA can have different chain lengths and degrees of saturation (presence or absence of double bonds).

The main products of beta-oxidation are acetyl-CoA, NADH, and FADH2. Acetyl-CoA is a two-carbon unit that can enter the citric acid cycle and produce more energy. NADH and FADH2 are electron carriers that can donate their electrons to the electron transport chain and generate ATP through oxidative phosphorylation.

For each cycle of beta-oxidation, one acetyl-CoA, one NADH, and one FADH2 are produced, and the fatty acyl-CoA chain is shortened by two carbons. The cycle repeats until the entire fatty acyl-CoA chain is converted to acetyl-CoA units. The number of cycles and the total energy yield depend on the initial chain length of the fatty acyl-CoA.

For example, a 16-carbon saturated fatty acid (palmitate) undergoes seven cycles of beta-oxidation and produces eight molecules of acetyl-CoA, seven molecules of NADH, and seven molecules of FADH2. The net ATP yield from palmitate oxidation is 106 ATP (108 ATP from products minus 2 ATP from activation).

Some fatty acids have odd numbers of carbons or double bonds in their chains. These fatty acids require additional enzymes and cofactors to undergo beta-oxidation. For example, odd-chain fatty acids produce propionyl-CoA as their final product, which can be converted to succinyl-CoA and enter the citric acid cycle. Unsaturated fatty acids require an isomerase and a reductase to deal with the cis-double bonds in their chains.

Beta-oxidation can occur in different cellular compartments depending on the chain length of the fatty acyl-CoA. In eukaryotes, most beta-oxidation takes place in the mitochondria, where the fatty acyl-CoA must be transported across the inner mitochondrial membrane by a carnitine shuttle system. However, very long-chain fatty acids (>20 carbons) are oxidized in peroxisomes, where they produce hydrogen peroxide instead of FADH2. In prokaryotes, beta-oxidation occurs in the cytosol without the need for carnitine transport.

In summary, beta-oxidation is a catabolic pathway that converts fatty acids into acetyl-CoA, NADH, and FADH2, which can be used to generate ATP. The substrates and products vary depending on the type and length of the fatty acid chain and the location of the process. Beta-oxidation is an important source of energy for many organisms, especially during fasting or prolonged exercise.

Beta-oxidation is the process by which fatty acids are broken down into acetyl-CoA, which can enter the citric acid cycle and generate ATP. Beta-oxidation occurs in the mitochondria of eukaryotic cells and in the cytosol of prokaryotic cells.

The pathway of beta-oxidation consists of four main steps that are repeated until the fatty acid chain is completely converted to acetyl-CoA. These steps are:

1. Dehydrogenation: The first step is catalyzed by an enzyme called acyl-CoA dehydrogenase, which removes two hydrogen atoms from the alpha and beta carbons of the fatty acyl-CoA and forms a double bond between them. This produces an enoyl-CoA and a reduced coenzyme FADH2, which can donate electrons to the electron transport chain and generate ATP.
2. Hydration: The second step is catalyzed by an enzyme called enoyl-CoA hydratase, which adds a water molecule across the double bond and forms a beta-hydroxyacyl-CoA. This step is similar to the hydration of fumarate to malate in the citric acid cycle.
3. Oxidation: The third step is catalyzed by an enzyme called beta-hydroxyacyl-CoA dehydrogenase, which oxidizes the beta-hydroxy group to a keto group and produces a beta-ketoacyl-CoA and a reduced coenzyme NADH, which can also donate electrons to the electron transport chain and generate ATP. This step is similar to the oxidation of malate to oxaloacetate in the citric acid cycle.
4. Thiolysis: The fourth step is catalyzed by an enzyme called thiolase, which cleaves the bond between the alpha and beta carbons of the beta-ketoacyl-CoA and releases a two-carbon unit as acetyl-CoA and a shortened fatty acyl-CoA that is two carbons shorter than the original one. The acetyl-CoA can enter the citric acid cycle and generate more ATP, while the shortened fatty acyl-CoA can undergo another round of beta-oxidation until it is completely converted to acetyl-CoA.

The following code block shows a schematic representation of one cycle of beta-oxidation:

 FAD + NAD+ + H2O + CoA
| | | |
| | | |
V V V V
R-CO-CH2-CH2-CO-S-CoA --> R-CO-CH=CH-CO-S-CoA --> R-CO-CH(OH)-CH2-CO-S-CoA --> R-CO-CO-CH2-CO-S-CoA --> R-CO-CO-S-CoA + CH3-CO-S-CoA
| | | | |
| | | | |
+------------------------->+------------------------->+------------------------->+ V
FADH2 NADH + H+ Acetyl-CoA

Before fatty acids can undergo beta-oxidation, they must first be activated by a reaction that attaches them to coenzyme A (CoA). This reaction is catalyzed by an enzyme called fatty acyl-CoA synthetase or acyl-CoA synthase . The reaction occurs in two steps:

1. The fatty acid reacts with ATP to form a fatty acyl-adenylate and pyrophosphate (PPi).
2. The fatty acyl-adenylate reacts with CoA to form a fatty acyl-CoA and AMP.

The overall reaction is:

$$\text{Fatty acid} + \text{CoA} + \text{ATP} \rightarrow \text{Fatty acyl-CoA} + \text{AMP} + \text{PPi}$$

The hydrolysis of PPi by pyrophosphatase drives the reaction forward by lowering the free energy of the products. The activation of fatty acids requires the equivalent of two molecules of ATP for each fatty acid molecule.

The activation of fatty acids occurs in the cytosol of the cell for long-chain fatty acids, and in the mitochondria for short-chain fatty acids. The activated fatty acyl-CoAs are then transported into the mitochondrial matrix for beta-oxidation, as described in point 6.

Before fatty acyl-CoA can undergo beta-oxidation in the mitochondrial matrix, it must be transported across the inner mitochondrial membrane, which is impermeable to fatty acyl-CoA. To overcome this barrier, a special transport system involving carnitine, a quaternary amine derived from lysine and methionine, is used .

The transport of fatty acyl-CoA into mitochondria involves three steps:

• First, fatty acyl-CoA reacts with carnitine in the outer mitochondrial membrane, forming fatty acylcarnitine and releasing coenzyme A. The enzyme that catalyzes this reaction is carnitine acyltransferase I (CAT I), also known as carnitine palmitoyltransferase I (CPT I) .
• Second, fatty acylcarnitine is transported across the inner mitochondrial membrane by a translocase enzyme that exchanges it for free carnitine . This antiport mechanism ensures that the concentration of carnitine remains constant on both sides of the membrane.
• Third, fatty acylcarnitine is converted back to fatty acyl-CoA and carnitine in the mitochondrial matrix by another enzyme, carnitine acyltransferase II (CAT II), also known as carnitine palmitoyltransferase II (CPT II) . The regenerated fatty acyl-CoA can then enter the beta-oxidation pathway.

The transport of fatty acyl-CoA into mitochondria is regulated by the availability of substrates and products, as well as by hormonal signals. The rate-limiting step is the first one, catalyzed by CAT I, which is inhibited by malonyl-CoA . Malonyl-CoA is an intermediate in fatty acid synthesis that occurs in the cytosol. Therefore, when fatty acid synthesis is active, malonyl-CoA prevents the transport of fatty acids into mitochondria for oxidation, avoiding a futile cycle . Conversely, when fatty acid oxidation is active, malonyl-CoA levels are low and CAT I is not inhibited.

The activity of CAT I is also influenced by hormonal factors. Insulin, which stimulates lipogenesis and inhibits lipolysis, decreases the expression of CAT I in the liver and adipose tissue. Glucagon and epinephrine, which stimulate lipolysis and inhibit lipogenesis, increase the expression of CAT I in these tissues. Thus, hormonal signals coordinate the balance between fatty acid synthesis and oxidation according to the metabolic state of the organism.

The transport system involving carnitine is required only for long-chain fatty acids (more than 12 carbons). Medium-chain fatty acids (6 to 12 carbons) can diffuse across the inner mitochondrial membrane without carnitine . Short-chain fatty acids (less than 6 carbons) are not activated by coenzyme A in the cytosol, but rather in the mitochondria . Therefore, they do not need any transport system to enter the beta-oxidation pathway.

β-Oxidation is the process by which fatty acids are broken down into acetyl-CoA, NADH and FADH2 in the mitochondria. The term β-oxidation refers to the oxidation of the β-carbon of the fatty acyl-CoA molecule. β-Oxidation consists of four sequential steps that are repeated until the entire fatty acid chain is converted to acetyl-CoA. These steps are:

1. Dehydrogenation: The first step is catalyzed by acyl-CoA dehydrogenase, an enzyme that removes two hydrogen atoms from the α and β carbons of the fatty acyl-CoA and transfers them to FAD, forming FADH2 and a trans-Δ2-enoyl-CoA. FADH2 can enter the electron transport chain and generate ATP.
2. Hydration: The second step is catalyzed by enoyl-CoA hydratase, an enzyme that adds a water molecule across the double bond between the α and β carbons, forming a L-3-hydroxyacyl-CoA.
3. Oxidation: The third step is catalyzed by L-3-hydroxyacyl-CoA dehydrogenase, an enzyme that oxidizes the hydroxyl group on the β-carbon to a ketone group, forming a β-ketoacyl-CoA. This reaction also produces NADH and H+, which can enter the electron transport chain and generate ATP.
4. Thiolysis: The fourth step is catalyzed by β-ketothiolase, an enzyme that cleaves the bond between the α and β carbons of the β-ketoacyl-CoA, releasing acetyl-CoA and a fatty acyl-CoA that is two carbons shorter than the original one. Acetyl-CoA can enter the citric acid cycle and generate more ATP.

The shortened fatty acyl-CoA then undergoes another cycle of β-oxidation until it is completely converted to acetyl-CoA. For example, a 16-carbon palmitoyl-CoA undergoes seven cycles of β-oxidation, producing eight molecules of acetyl-CoA, seven molecules of FADH2 and seven molecules of NADH.

The following figure illustrates the β-oxidation of even-chain fatty acids:

The products of beta-oxidation can be used to generate ATP through oxidative phosphorylation. The amount of ATP produced depends on the length and saturation of the fatty acid chain. For even-chain fatty acids, the following steps can be used to calculate the net ATP yield:

1. Subtract two from the number of carbon atoms in the fatty acid chain to get the number of cycles of beta-oxidation. For example, a 16-carbon fatty acid (palmitate) undergoes 7 cycles of beta-oxidation.
2. Multiply the number of cycles by 5 to get the number of ATP produced from the FADH2 and NADH generated in each cycle. Each FADH2 yields about 1.5 ATP and each NADH yields about 2.5 ATP. For example, 7 cycles x 5 ATP = 35 ATP.
3. Add one to the number of cycles to get the number of acetyl-CoA molecules produced from the fatty acid chain. Each acetyl-CoA enters the citric acid cycle and produces about 10 ATP. For example, 7 cycles + 1 = 8 acetyl-CoA x 10 ATP = 80 ATP.
4. Subtract two from the total ATP to account for the activation of the fatty acid by acyl-CoA synthase, which uses two high-energy phosphate bonds equivalent to two ATP. For example, 35 ATP + 80 ATP - 2 ATP = 113 ATP.

Therefore, the net ATP yield from a 16-carbon fatty acid is 113 ATP. This can be generalized as follows:

• Net ATP yield = (n/2 - 1) x 5 + (n/2 + 1) x 10 - 2
• Where n is the number of carbon atoms in the fatty acid chain.

The oxidation of other even-chain fatty acids will yield different amounts of ATP depending on their length. For example, a 14-carbon fatty acid (myristate) will yield 97 ATP, while an 18-carbon fatty acid (stearate) will yield 129 ATP.

Most of the fatty acids in our body have even numbers of carbon atoms, but some have odd numbers. Moreover, some fatty acids have one or more double bonds in their chains, making them unsaturated. These fatty acids require some additional enzymes and steps to be oxidized by beta-oxidation.

Odd-chain fatty acids

Odd-chain fatty acids produce acetyl-CoA and propionyl-CoA when they undergo beta-oxidation. Propionyl-CoA has three carbons and cannot be further oxidized by beta-oxidation. It has to be converted to succinyl-CoA, which can enter the citric acid cycle. The conversion involves three steps:

1. Propionyl-CoA is carboxylated by propionyl-CoA carboxylase, which requires biotin as a cofactor. The product is D-methylmalonyl-CoA.
2. D-methylmalonyl-CoA is isomerized to L-methylmalonyl-CoA by methylmalonyl-CoA epimerase.
3. L-methylmalonyl-CoA is rearranged to succinyl-CoA by methylmalonyl-CoA mutase, which requires vitamin B12 as a cofactor.

The overall reaction is:

Propionyl-CoA + ATP + HCO3- + CoA + vitamin B12 -> Succinyl-CoA + ADP + Pi + vitamin B12

Succinyl-CoA can then be metabolized in the citric acid cycle or used for gluconeogenesis.

Unsaturated fatty acids have one or more double bonds in their chains, which can be either cis or trans configuration. Beta-oxidation can only handle trans double bonds between the alpha and beta carbons, so unsaturated fatty acids need an isomerase and a reductase to modify their double bonds.

If the double bond is between the alpha and beta carbons (as in acyl-CoA), beta-oxidation can proceed normally until the double bond is eliminated.

If the double bond is between the beta and gamma carbons (as in enoyl-CoA), an enzyme called enoyl-CoA isomerase can shift the position of the double bond to make it between the alpha and beta carbons. Then beta-oxidation can continue.

If there are two double bonds between the alpha and gamma carbons (as in dienoyl-CoA), an enzyme called 2,4-dienoyl-CoA reductase can reduce one of them using NADPH as a cofactor. This produces a trans double bond between the beta and gamma carbons, which can then be acted upon by enoyl-CoA isomerase.

The overall effect of these enzymes is to convert unsaturated fatty acids into saturated fatty acids that can be oxidized by beta-oxidation. However, this process consumes one NADPH and produces one less FADH2 per double bond, resulting in a lower ATP yield for unsaturated fatty acids compared to saturated fatty acids of the same length.

Unsaturated fatty acids have one or more double bonds in their carbon chains, which affect the ATP yield from their beta-oxidation. Depending on the position and configuration of the double bonds, unsaturated fatty acids may require additional enzymes and co-factors to be oxidized.

The ATP yield for unsaturated fatty acids can be calculated by subtracting the ATP equivalents lost due to the presence of double bonds from the ATP yield for saturated fatty acids of the same length. The ATP equivalents lost depend on the number and type of double bonds:

• If the double bond is at an odd-numbered carbon position (such as 3, 5, 7, etc.), then one FADH2 is not produced in the first step of beta-oxidation, resulting in a loss of 1.5 ATP equivalents .
• If the double bond is at an even-numbered carbon position (such as 4, 6, 8, etc.), then one NADPH is consumed in the reduction of a 2,4-dienoyl-CoA intermediate by 2,4-dienoyl-CoA reductase, resulting in a loss of 2.5 ATP equivalents.

For example, oleic acid (18:1 cis-Δ9) has one double bond at carbon 9. Therefore, it loses 1.5 ATP equivalents compared to stearic acid (18:0), which has no double bonds. The ATP yield for oleic acid can be calculated as follows:

• Activation: -2 ATP
• Beta-oxidation cycles: (18/2 - 1) x 14 = 7 x 14 = 98 ATP
• Acetyl-CoA oxidation: (18/2) x 10 = 9 x 10 = 90 ATP
• Double bond penalty: -1.5 ATP
• Total: -2 + 98 + 90 - 1.5 = 184.5 ATP

For comparison, the ATP yield for stearic acid is:

• Activation: -2 ATP
• Beta-oxidation cycles: (18/2 - 1) x 14 = 7 x 14 = 98 ATP
• Acetyl-CoA oxidation: (18/2) x 10 = 9 x 10 = 90 ATP
• Total: -2 + 98 + 90 = 186 ATP

Therefore, oleic acid produces 1.5 ATP less than stearic acid due to its double bond.

Another example is linoleic acid (18:2 cis,cis-Δ9,12), which has two double bonds at carbon 9 and carbon 12. Therefore, it loses both one FADH2 and one NADPH compared to stearic acid. The ATP yield for linoleic acid can be calculated as follows:

• Activation: -2 ATP
• Beta-oxidation cycles: (18/2 - 1) x 14 = 7 x 14 = 98 ATP
• Acetyl-CoA oxidation: (18/2) x 10 = 9 x 10 = 90 ATP
• Double bond penalty: -1.5 - 2.5 = -4 ATP
• Total: -2 + 98 + 90 -4 = 182 ATP

Therefore, linoleic acid produces 4 ATP less than stearic acid due to its two double bonds.

In general, the more double bonds a fatty acid has, the lower its ATP yield will be compared to a saturated fatty acid of the same length. However, unsaturated fatty acids are still an important source of energy for cells and tissues, especially in situations where glucose availability is limited or impaired.

The overall reaction of beta oxidation is the process by which a fatty acid molecule is broken down into smaller units of acetyl-CoA, NADH, and FADH2. Each cycle of beta oxidation removes two carbon atoms from the fatty acid chain and produces one molecule of acetyl-CoA, one molecule of NADH, and one molecule of FADH2. The acetyl-CoA can enter the citric acid cycle and the electron transport chain to generate more ATP, while the NADH and FADH2 can donate electrons to the electron transport chain and contribute to the proton gradient that drives ATP synthesis.

The general formula for the overall reaction of beta oxidation is:

$$C_n\text{-acyl-CoA} + FAD + NAD^+ + H2O + CoA \rightarrow C{n-2}\text{-acyl-CoA} + FADH_2 + NADH + H^+ + \text{acetyl-CoA}$$

where $n$ is the number of carbon atoms in the fatty acid chain.

For example, if we take palmitoyl-CoA (16:0) as a model substrate, it will undergo seven cycles of beta oxidation and produce eight molecules of acetyl-CoA, seven molecules of NADH, and seven molecules of FADH2. The overall reaction is:

$$\text{palmitoyl-CoA} + 7FAD + 7NAD^+ + 7H_2O + 7CoA \rightarrow 8\text{acetyl-CoA} + 7FADH_2 + 7NADH + 7H^+$$

The net energy yield from one molecule of palmitoyl-CoA is about 106 ATP, after subtracting the two ATP equivalents used for activation. This is because each FADH2 can generate about 1.5 ATP, each NADH can generate about 2.5 ATP, and each acetyl-CoA can generate about 10 ATP through the citric acid cycle and the electron transport chain.

The overall reaction of beta oxidation can vary depending on the type and length of the fatty acid. For odd-chain fatty acids, the final cycle will produce one molecule of propionyl-CoA instead of acetyl-CoA, which can be converted to succinyl-CoA and enter the citric acid cycle. For unsaturated fatty acids, additional enzymes are required to deal with the double bonds in the fatty acid chain, which may reduce the energy yield by using NADPH or skipping FADH2 production.

Beta oxidation is an important pathway for generating energy from fatty acids, especially during fasting or prolonged exercise when glucose levels are low. It also provides acetyl-CoA for other metabolic processes such as ketogenesis and cholesterol synthesis.

Beta-oxidation involves a series of enzymatic reactions that break down fatty acids into acetyl-CoA, NADH and FADH2. The main enzymes involved in this process are:

• Acyl-CoA dehydrogenase: This enzyme catalyzes the first step of beta-oxidation, which is the oxidation of acyl-CoA by FAD to form a trans double bond between the alpha and beta carbons. This produces enoyl-CoA and FADH2. There are different isoforms of this enzyme that act on different chain lengths of fatty acids.
• Enoyl-CoA hydratase: This enzyme catalyzes the second step of beta-oxidation, which is the hydration of the trans double bond to form a beta-hydroxyacyl-CoA. This is a reversible reaction that can also convert beta-hydroxyacyl-CoA to enoyl-CoA.
• 3-Hydroxyacyl-CoA dehydrogenase: This enzyme catalyzes the third step of beta-oxidation, which is the oxidation of beta-hydroxyacyl-CoA by NAD+ to form a beta-ketoacyl-CoA. This produces NADH and H+. This enzyme is specific for the L-isomer of beta-hydroxyacyl-CoA.
• Acyl-CoA acyltransferase: This enzyme catalyzes the fourth and final step of beta-oxidation, which is the cleavage of beta-ketoacyl-CoA by CoA to release acetyl-CoA and a shortened acyl-CoA. This enzyme is also known as thiolase or ketoacyl-CoA thiolase.

These four enzymes form a cycle that repeats until the fatty acid chain is completely converted to acetyl-CoA units. However, some fatty acids may require additional enzymes to undergo beta-oxidation, such as:

• Enoyl-CoA isomerase: This enzyme converts cis double bonds at odd-numbered positions to trans double bonds at even-numbered positions, allowing them to be hydrated by enoyl-CoA hydratase. This enzyme is required for the oxidation of unsaturated fatty acids with cis double bonds.
• 2,4-Dienoyl-CoA reductase: This enzyme reduces conjugated double bonds at even-numbered positions to form a single trans double bond at an odd-numbered position, allowing it to be isomerized by enoyl-CoA isomerase. This enzyme requires NADPH as a cofactor and is required for the oxidation of unsaturated fatty acids with conjugated double bonds.
• Propionyl-CoA carboxylase: This enzyme converts propionyl-CoA to methylmalonyl-CoA by adding a carboxyl group. This enzyme requires biotin as a cofactor and is required for the oxidation of odd-chain fatty acids.
• Methylmalonyl-CoA mutase: This enzyme converts methylmalonyl-CoA to succinyl-CoA by rearranging the carbon skeleton. This enzyme requires vitamin B12 as a cofactor and is required for the oxidation of odd-chain fatty acids.

These enzymes allow the oxidation of different types of fatty acids with varying chain lengths and degrees of saturation. The products of beta-oxidation can then enter the citric acid cycle and the electron transport chain to generate ATP.

Beta-oxidation of fatty acids is regulated by the mechanisms that control the availability of fatty acids and their entry into the mitochondria, as well as by the demand for ATP and the levels of coenzymes and intermediates.

• The availability of fatty acids depends on the balance between lipolysis and lipogenesis in adipose tissue. Lipolysis is stimulated by hormones such as epinephrine, glucagon and cortisol, which activate a cAMP-dependent protein kinase that phosphorylates and activates hormone-sensitive lipase (HSL). HSL hydrolyzes triglycerides and releases free fatty acids and glycerol into the blood. Lipogenesis is stimulated by insulin, which inhibits HSL and activates acetyl-CoA carboxylase (ACC), the rate-limiting enzyme for fatty acid synthesis. ACC converts acetyl-CoA to malonyl-CoA, which is a substrate for fatty acid synthase and an inhibitor of carnitine palmitoyltransferase I (CPT I), the enzyme that transfers fatty acyl-CoA to carnitine for transport into mitochondria. Therefore, when fatty acids are being synthesized in the cytosol, malonyl-CoA inhibits their transport into mitochondria and prevents a futile cycle of synthesis and degradation.

• The entry of fatty acids into the mitochondria depends on the activity of CPT I, which is regulated by malonyl-CoA as mentioned above, and by the availability of carnitine. Carnitine is synthesized from lysine and methionine in the liver and kidneys, and can also be obtained from dietary sources such as meat and dairy products. Carnitine deficiency can impair fatty acid oxidation and cause symptoms such as muscle weakness, cardiomyopathy and hypoglycemia.

• The demand for ATP determines the rate of oxidative phosphorylation, which in turn affects the levels of NAD+ and FAD, the coenzymes required for beta-oxidation. When ATP demand is high, NAD+ and FAD are regenerated by donating electrons to the electron transport chain, which creates a favorable gradient for beta-oxidation. When ATP demand is low, NAD+ and FAD are reduced and accumulate, which inhibits beta-oxidation.

• The levels of intermediates and products of beta-oxidation can also affect its rate by feedback inhibition or activation. For example, high levels of acetyl-CoA can inhibit beta-ketothiolase, the enzyme that cleaves acetoacetyl-CoA to two acetyl-CoA molecules. High levels of NADH can inhibit 3-hydroxyacyl-CoA dehydrogenase, the enzyme that oxidizes 3-hydroxyacyl-CoA to 3-ketoacyl-CoA. Conversely, low levels of acetyl-CoA can activate pyruvate dehydrogenase kinase (PDK), which phosphorylates and inhibits pyruvate dehydrogenase (PDH), the enzyme that converts pyruvate to acetyl-CoA. This reduces the competition between pyruvate and fatty acids for entry into the citric acid cycle.

Beta-oxidation of fatty acids is a significant source of metabolic energy during fasting, starvation, exercise and other conditions that require high energy demand or low carbohydrate intake. Fatty acids are stored as triglycerides in adipose tissue, which can provide more than 10 times the energy yield per gram compared to glycogen. Fatty acids can also cross the blood-brain barrier and be used by the brain as an alternative fuel source when glucose is scarce.

Beta-oxidation of fatty acids also produces important intermediates and products that have various biological functions. For example:

• Acetyl-CoA is not only a substrate for the citric acid cycle, but also a precursor for ketone body synthesis in the liver. Ketone bodies (acetoacetate, beta-hydroxybutyrate and acetone) can be transported to other tissues such as the brain, heart and skeletal muscle, where they can be converted back to acetyl-CoA and enter the citric acid cycle.

• Propionyl-CoA is produced from odd-chain fatty acids or branched-chain amino acids. Propionyl-CoA can be converted to succinyl-CoA by a series of reactions that require biotin and vitamin B12 as cofactors. Succinyl-CoA can enter the citric acid cycle or be used for gluconeogenesis in the liver or kidneys.

• NADH and FADH2 are electron carriers that donate electrons to the electron transport chain and generate ATP by oxidative phosphorylation.

Therefore, beta-oxidation of fatty acids is an essential metabolic pathway that provides energy, substrates and cofactors for various cellular processes.

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