Biochemistry, Fatty Acid Oxidation

Fundamentals

Mitochondrial beta-oxidation can be used to supply acetyl coenzyme A (CoA) to 2 separate pathways, depending on which tissue oxidation occurs. In skeletal and cardiac muscle, mitochondrial fatty acid oxidation leads to substrate production for the tricarboxylic acid cycle in the form of acetyl CoA and provides adenosine triphosphate (ATP) for the myocytes. However, in hepatocytes, fatty acid oxidation provides acetyl CoA for ketone body synthesis during prolonged fasting conditions, in which glycogen stores have been depleted. Mitochondrial beta-oxidation yields 4 ATP equivalents per round of oxidation in the form of one flavin adenine dinucleotide, FAD(2H) molecule and 1 nicotinamide adenine dinucleotide (reduced, NADH) molecule, as well as one acetyl CoA molecule.

Beta oxidation requires specific enzymes to carry out the metabolism of fatty acids. For saturated fatty acids, such as palmitate (16:0) and stearate (18:0), a base set of enzymes catalyze the reactions to yield the above molecules. Additional enzymes may be required if the fatty acids to be oxidized are not saturated and, thus, contain double bonds, such as oleate (18:1). Differences in the molecular structure of fatty acids that require additional enzymes for metabolism, whether they are destined for beta-oxidation or not, will be discussed in the section headed “Molecular.”

Peroxisomal beta-oxidation is specialized in that it metabolizes very long-chain fatty acids, which are composed of 24 to 26 carbon units. Processing of these molecules proceeds similarly to mitochondrial beta-oxidation; however, some enzymatic steps differ and are discussed below. Alpha oxidation of fatty acids occurs in the peroxisome as well; this metabolic pathway exists to degrade by-products of chlorophyll, a component of green vegetables in the diet. Phytanic acid is the primary molecule that requires the enzymes dedicated to alpha-oxidation. It derives from chlorophyll within ingested plant matter.

As previously mentioned, omega-oxidation, the third and final fatty acid oxidation pathway, occurs in the endoplasmic reticulum. This pathway exists to process large, water-insoluble fatty acids that would otherwise be toxic to the cell in higher concentrations. Further details and mechanisms of these accessory pathways/enzymes to beta-oxidation are discussed below under the heading “Molecular.”

Cellular Level

Important concepts about the regulation of mitochondrial beta-oxidation, cellular handling, and transport of fatty acids will be discussed here. As the other forms of fatty acid oxidation are substrate-dependent and are not regulated by feedback or substrate concentrations, they will not receive the same level of discussion as mitochondrial beta-oxidation. Fatty acid beta-oxidation is regulated by the cell’s energy requirements. These molecules become more available in times of increased energy demand of prolonged fasting due to stimulation of hormone-sensitive lipase in adipose tissue epinephrine and glucagon. Serum-free fatty acids increase under the influence of these molecules and enter target cells. Other factors that regulate beta-oxidation include adequate oxygen supply for the continued electron acceptance from carrier substrates produced, namelyf lavin adenine dinucleotide (FAD[H2]) and nicotinamide adenine dinucleotide (reduced, NADH), to maintain a pool of electron acceptors available. Expression of enzymes involved in fatty acid oxidation becomes upregulated through fatty acids behaving as ligands that bind to peroxisome proliferator-activated receptors; these transcription factors form homo-/heterodimers and translocate to the nucleus, where they alter gene expression involved in the production of proteins required for beta-oxidation and mitochondrial biogenesis.[3] 

First, free fatty acids must be taken up in cells by a transporter-mediated mechanism involving the membrane fatty acid-binding protein.[1] Then, fatty acids within target cells must undergo multiple steps to arrive at previously mentioned cellular locations for oxidation to occur. The steps and enzymes involved in this process are highly site and substrate-specific. The major steps involve activation and transportation into cellular compartments.

Activation of fatty acids requires the formation of a thioester bond with CoA, an adenosine triphosphate (ATP)-dependent process carried out by acyl-CoA synthetases.[4] The site and substrate specificity are demonstrated by the fact that long-chain acyl-CoA synthetase is located in the outer mitochondrial membranes, peroxisomal membranes, and endoplasmic reticulum membrane and demonstrates activity towards fatty acids of 12 to 20 carbons in length. However, in the case of very-long-chain fatty acids (VLCFAs), a specific synthetase is required for their activation and is only located in the membrane of peroxisomes, as this is the only location with the enzymatic profile suitable for their oxidation. Similarly, medium-chain acyl-CoA synthetases are only present in the mitochondrial matrix. The production of these smaller, diffusible compounds will be discussed in the section headed “Molecular.”

Transportation of long-chain fatty acids into the mitochondrial matrix requires three enzymes in addition to acyl-CoA synthetase. The transport of fatty acyl-CoA across the outer mitochondrial membrane occurs by carnitine:palmitoyltransferase I (CPT I); this enzyme simultaneously converts fatty acylcarnitine. This step is heavily regulated by the energy status of the cell; malonyl-CoA levels rise during the synthesis of fatty acids and function to inhibit mitochondrial beta-oxidation at this point in the pathway. The enzymatic transportation and conversion completed by CPT I is the rate-limiting step of fatty acid oxidation in the mitochondria. Fatty acyl-carnitine molecules are then transported into the mitochondrial matrix in exchange for carnitine by carnitine:acylcarnitine translocase through an antiport mechanism. The pool of carnitine available to this transporter depends on the functioning of carnitine:palmitoyltransferase II (CPT II), which serves to convert acylcarnitine to fatty acyl-CoA, trapping the molecules within the mitochondrial matrix.

In contrast to this involved, regulated transport mechanism, VLCFAs are not dependent on carnitine for transport into peroxisomes; the transport of branched-chain fatty acids destined for alpha-oxidation is similar to this process, and as previously mentioned, is substrate-dependent. VLCFAs and phytanic acid are transported into peroxisomes by the ABCD1-3 transporters by an ATP-dependent process; deficiencies of these transporters have been demonstrated to have severe implications and are discussed below in “Clinical Significance.”[3] 

Molecular Level

In a similar fashion to previous sections, the process and enzymatic steps of the beta-oxidation spiral will primarily undergo discussion with alternative oxidation pathways mentioned later as they pertain to and produce metabolic products destined for mitochondrial beta-oxidation. Variations of fatty acid molecular structure and additional required enzymes will also be discussed. Mitochondrial beta-oxidation of fatty acids requires four steps, all of which occur in the mitochondrial matrix, to produce three energy storage molecules per round of oxidation, including one NADH, one FAD(H2), and one acetyl CoA molecule.[5]

Step 1. The first enzyme required is called acyl CoA dehydrogenase, and like other enzymes involved in the handling of fatty acids, it is specific to chain length. Members of this enzyme family include long-chain, medium-chain, and short-chain acyl CoA dehydrogenases (LCAD), (MCAD), and (SCAD), respectively. These enzymes catalyze the formation of a trans double bond between the alpha and beta carbons on acyl CoA molecules by removing two electrons to produce one molecule of FAD(H2), which eventually accounts for 1.5 ATP molecules produced in the electron transport chain (ETC).

Step 2. Next, the enzyme enoyl CoA hydratase performs a hydration step of the double bond between the alpha and beta carbons; this results in the addition of a hydroxyl (OH-) group to the beta carbon and a proton (H+) to the alpha carbon. There is no energy production associated with this step.

Step 3. Following hydration, the next step is carried out by beta-hydroxyl acyl CoA dehydrogenase; as the name implies, electrons and two protons are removed from the hydroxyl group, and the attached beta carbon to oxidize the beta carbon and produce a molecule of NADH. Each molecule of NADH will result in the production of 2.5 ATP molecules from the ETC.[6]

Step 4. The final step in Beta oxidation involves cleavage of the bond between the alpha and beta carbon by CoA. This step is catalyzed by beta-keto thiolase and is a thiolytic reaction. The reaction produces one molecule of acetyl CoA and a fatty acyl CoA that is two carbons shorter. The process may repeat until the even chain fatty acid has completely converted into acetyl CoA.

Steps 1 through 4 refer to the beta-oxidation of a saturated fatty acid with an even-numbered carbon skeleton. Unsaturated fatty acids, such as oleate (18:1) and linoleate (18:2), contain cis double bonds that must be isomerized to the trans configuration (enoyl CoA isomerase) or reduced at the expense of an NADPH molecule (2,4-dienoyl CoA reductase).

Odd-chain fatty acids undergo beta-oxidation in the same manner as even-chain fatty acids; however, once a five-carbon chain remains, the final spiral of beta-oxidation will yield one molecule of acetyl-CoA and one molecule of propionyl CoA. This 3-carbon molecule can be enzymatically converted to succinyl CoA, forming a bridge between the tricarboxylic cycle and fatty acid oxidation.

VLCFA beta-oxidation in peroxisomes occurs by a process similar to mitochondrial beta-oxidation; however, some key differences exist, including the fact that different genes encode fatty acid oxidation enzymes in peroxisomes, which is significant in certain inborn errors of metabolism.[7] The enzyme responsible for the production of a double bond between the alpha and beta carbon in the first step of the peroxisomal pathway is an oxidase and donates electrons to molecular oxygen to produce hydrogen peroxide, rather than storing electrons in FAD(H2) as reducing the potential for the electron transport chain. The remaining three steps are similar to the mitochondrial steps. Another notable difference involves the extent to which beta-oxidation occurs; it may occur to completion, ending in the production of acetyl-CoA molecules that are able to enter the cytosol or be transported to the mitochondria bound to carnitine.[8] Carnitine may also transfer short to medium-chain fatty acids to the mitochondrial matrix for the completion of oxidation.[8]

Branched-chain fatty acids also require additional enzymatic modification to enter the alpha-oxidation pathway within peroxisomes. Phytanic acid, 3,7,11,14-tetramethylhexadecanoic acid, requires additional peroxisomal enzymes to undergo beta-oxidation. Phytanic acid initially activates phytanyl CoA; then, phytanyl CoA hydroxylase (alpha-hydroxylase), encoded by the PHYH gene, introduces a hydroxyl group to the alpha carbon.[7] The alpha carbon-hydroxyl bond then undergoes two successive rounds of oxidation to pristanic acid. Pristanic acid undergoes beta-oxidation, which produces acetyl CoA and propionyl CoA in alternative rounds. As with peroxisomal beta-oxidation of VLCFAs, this process generally ends when the carbon chain length reaches 6 to 8 carbons, at which point the molecule is shuttled to the mitochondria by carnitine for complete oxidation to carbon dioxide and water.[7]

Omega-oxidation of fatty acids in the endoplasmic reticulum primarily functions to hydroxylate and oxidize fatty acids to dicarboxylic acids to increase water solubility for excretion in the urine. This enzymatic conversion relies on the cytochrome P450 superfamily to catalyze this reaction between xenobiotic compounds and molecular oxygen.[9] Deficiencies in some enzymes of fatty acid oxidation may result in accumulation. Thus, up-regulation of omega-oxidation, increased serum, and or urine medium-chain dicarboxylic acids can be diagnostic of certain deficiencies and will be discussed under the “Clinical Significance” section.

Clinical Significance

Listed below are a few select diseases that either directly involve defective fatty acid metabolism through intrinsic enzyme deficiencies or indirectly prevent the proper functioning of fatty acid metabolism through extrinsic enzyme deficiencies. Many, but not all, deficiencies of enzymes involved in fatty acid oxidation result in abnormal neurological development and or function early in life; a brief list of signs and symptoms appears under the selected diseases mentioned. 

Medium-Chain Acyl Dehydrogenase Deficiency

Medium-chain acyl dehydrogenase (MCAD) is the most common inherited defect of fatty acid oxidation in humans; as one would expect, medium-chain 6-8 carbon molecules accumulate in this disease. Clinical manifestations of MCAD deficiency primarily present during fasting conditions and include lethargy, weakness, diaphoresis, and hypoketotic hypoglycemia, most commonly in children younger than 5.[10] Serum measurements of octanoyl carnitine are usually elevated in these patients and can aid in the diagnosis. These abundant molecules then undergo oxidation by the cytochrome P450 system involved in omega-oxidation, resulting in dicarboxylic acidemia and dicarboxylic aciduria. This clinical syndrome must be differentiated from Reye’s Syndrome, as salicylates compete with medium-chain fatty acids for binding sites on MCAD.

Zellweger Syndrome

Zellweger syndrome results from autosomal recessive mutations in the PEX genes; these deoxyribonucleic acid sequences code for peroxin proteins, which are involved in the assembly of peroxisomes. Almost 70% of all peroxisomal biogenesis disorders result from a PEX1 gene mutation. Many different fatty acid compounds can accumulate without the oxidative machinery of peroxisomes, including VLCFAs and phytanic acid.[11] Manifestations of this disease generally include the brain, kidneys, and skeleton.[3]

X-Linked Adrenoleukodystrophy 

X-linked adrenoleukodystrophy is a genetic deficiency of the ABCD transporters in the membrane of peroxisomes, as mentioned previously, resulting in the pathological accumulation of VLCFAs within cells and is most clinically significant when the ABCD1 transporter is absent. The disease presents with neurodegenerative and adrenal abnormalities.[3]

Refsum Disease

Refsum disease results from a genetic deficiency of the enzyme phytanyl CoA 2-hydroxylase, which, as previously mentioned, is involved in the alpha-oxidation of phytanic acid, a breakdown product of chlorophyll.[7] Notable clinical manifestations of Refsum disease include cardiac malfunction and defective functioning of the olfactory and auditory nerves due to the accumulation of phytanic acid.[3]