Release of the energy of fatty acids is accomplished primarily by chopping them into two-carbon units of Acetyl-CoA, which enters the TCA cycle. An overview of fatty acid oxidation is shown in Figure 18.13. Evidence for the two carbon unit metabolites of fatty acids was suggested in a famous experiment by Franz Knoop, in which dogs were fed derivatized fatty acids linked to benzenes at their end (See Figure 18.12). Dogs fed derivatized fatty acids with even numbers of carbon chains excreted phenylacetic acid (benzene plus two carbon acid), whereas dogs fed derivatized fatty acids with odd number of carbon chains excreted benzoic acid (one carbon acid on benzene).
Fatty acids must be activated for degradation by conjugation with coenzyme A (CoA) in a reaction catalyzed by acyl-CoA ligases (thiokinases). The enzymes are associated with the endoplasmic reticulum and outer mitochondrial membrane and require ATP. Note that ATP is cleaved to AMP plus PPi. Cleavage of PPi to 2 Pi by inorganic pyrophosphatase helps to drive the acylation reaction to completion. (See Figure 18.14).
Fatty acid activation occurs in the cytosol, but they are oxidized inside the mitochondrion. Fatty acyl-CoA must be transported across the impermeable inner mitochondrial membrane. First the acyl groups is transesterified to carnitine (see Figure 18.15) in a reaction catalyzed by Carnitine acyl transferase I (located on external surface of inner mitochondrial membrane) or II (inner surface of inner mitochondrial membrane. The protein responsible for transferring acyl carnitine transfers acyl carnitine into the mitochondrion as it transfers free carnitine out. Once inside, the acyl carnitine is transferred to mitochondrial CoA and free carnitine is transported back out in another reaction of the above shuttle.
Inside the mitochondrion, acyl-CoAs are acted upon in four reactions. (See Figure 18.16)
1. Acyl-CoA dehydrogenase (mitochondria have three such enzymes, specific for short, long, and medium acyl groups) removes two hydrogens between carbons 2 and 3 (numbered from the CoA attachment carbon #1), forming a trans enoyl-CoA and FADH2.
2. Water is added across the double bond by enoyl-CoA hydratase, forming 3-L-hydroxyacyl-CoA (note that the product is stereospecific).
3. 3-L-hydroxyacylCoA dehydrogenase removes hydrogens, forming 3-ketoacyl CoA, and generating NADH.
4. The terminal acetyl-CoA group is cleaved in a thiolysis reaction with CoA catalyzed by Beta-ketothiolase (thiolase), forming a new acyl-CoA two carbons shorter than the previous one.
FADH2 produced by reaction of the flavoenzyme acyl-CoA dehydrogenase (reaction 1 above) passes its electrons to the ETS after a series of electron transfer reactions. First, electron transfer flavoprotein (ETFP) transfers FADH2's electron pair to ETFP:ubiquinone oxidoreductase which then passes the electron pair to CoQ of the ETS. (See Figure 18.17)
Medium Chain Acyl-CoA Dehydrogenase has been implicated as a deficiency in up to 10% of infants who die from Sudden Infant Death Syndrome (SIDS).
Fatty acid oxidation yields one NADH and one FADH2 per acetyl CoA formed from Acyl-CoA. Oxidation of acetyl-CoA in the TCA cycle generates more NADHs and FADH2. Complete oxidation of one palmitate molecule (16 carbons) generates 129 ATPs.
Most biological unsaturated fatty acids are in the cis configuration, and most often these begin with unsaturation between carbons 9 and 10. Additional double bonds are never conjugated and occur at three carbon intervals (conjugation involves double bonds at two carbon intervals). Double bonds in fatty acids must be altered during the oxidation process. This is depicted in Figure 18.18 for oxidation of linoleyl-CoA. After three cycles of Beta oxidation, double bonds at carbon position 3-4 interfere with the oxidation process used for saturated fatty acids because that process normally creates a double bond between carbons 2 and 3. As a way around the problem, enoyl-CoA isomerase catalyzes movement of the double bond to the 2-3 position. After that acetyl-CoA is removed, the first oxidation (removal of hydrogens) creates a delta4 double bond - a conjugated double bond. Enoyl-CoA hydratase will not act on this substrate. Another pair of enzymes solve the problem. 2,4 dienoyl-CoA reductase uses reducing equivalants of NADPH to convert the conjugated double bonds to a single Beta-gamma (carbons 3-4) bond. The Beta-gamma bond is flipped to the 2-3 position by Enoyl-CoA isomerase to form trans-2-enoyl-CoA, which is then metabolized as earlier.
Another factor to consider in fatty acid metabolism is odd-chain fatty acids. Most fatty acids have even numbers of carbon, owing to their conversion from Acetyl-CoA. A few organisms, however, synthesize fatty acids with odd numbers of carbons. Oxidation of these fatty acids generates Propionyl-CoA, a three carbon fatty acid by-product (See Figure 18.19). Propionyl-CoA is carboxylated to D-methylmalonyl-CoA by propionyl-CoA carboxylase (biotin co-factor) with ATP and bicarbonate. D-methylmalonyl-CoA is converted to L-methylmalonyl-CoA by methylmalonyl-CoA racemase. L-methylmalonyl-CoA is converted to Succinyl-CoA by methylmalonyl-CoA mutase (uses a coenzyme B12 prosthetic group). Methylmalonyl-CoA mutase is one of only two mammalian enzymes to use a cobalamin group.
Beta oxidation of fatty acids occurs in the peroxisomes (a eukaryotic organelle) in addition to the mitochondria, though in a modified form. A primary function of peroxisomal beta oxidation is to shorten very long chain fatty acids so they can be degraded in the mitochondrion. Further, in plants, fatty acid oxidation occurs exclusively in peroxisomes and glyoxosomes. Peroxisomal oxidation has advantages over mitochondrial oxidation - fatty acids can diffuse across the peroxisomal membrane. After very long chain fatty acids are partly degraded, they are attached to carnitine for transport to mitochondria. Deficiency in the ability to metabolize very long chain fatty acids in peroxisomes is linked to the disease X-Adrenoleukodystrophy, in which myelin is destroyed by the unmetabolized fatty acids..
Alpha Oxidation - The presence of an alkyl group at the beta carbon of a fatty acyl-CoA blocks beta oxidation of it (See Figure 18.20). Phytanic acid (a metabolic breakdown product of chlorophyll's phytyl group) is one such fatty acid. Oxidation of these branched fatty acids is accomplished by alpha oxidation. This process involves hydroxylation of the alpha carbon, removal of the terminal carboxyl group and concomitant conversion of the alpha hydroxyl group to a terminal carboxyl group, and linkage of CoA to the terminal carboxyl group. This branched substrate will function in the beta-oxidation process, ultimately yielding propionyl-CoA, acetyl CoAs and, in the case of phytanic acid, 2-methyl propionyl CoA. Reduced alpha oxidation is associated with Refsum's disease.
Omega Oxidation - involves oxidation of medium and long chain fatty acids beginning from the end opposite the carboxyl group followed by beta-oxidation inwards. This oxidation, which occurs in the endoplasmic reticulum, is a minor pathway of fatty acid oxidation.
The brain has a high need for glucose as a source of energy. Under starvation, the body is unable to supply glucose in sufficient quantities for the brain to use. When this happens, a process called ketogenesis, which occurs in liver mitochondria, activates. In ketogenesis, ketone derivatives of acetyl-CoA groups are made. These include acetoacetate, acetone, and hydroxybutyrate (see Figure 18.21). These reactions proceed as follows:
Acetoacetate is readily broken down (non-enzymatically) to acetone. Individuals suffering from ketosis have acetone on their breath because they make acetoacetate faster than they can metabolize it. Acetoacetate and D-beta-hydroxybutyrate are released by the liver and taken up by peripheral tissue, where they are readily converted back to acetoacetyl-CoA. Notably, liver lacks the enzyme (3-ketoacyl-CoA transferase) necessary to create acetoacetyl-CoA, facilitating release of acetoacetate. Acetoacetyl-CoA is, of course, an intermediate in beta-oxidation.
Fatty Acid Oxidation