Fatty Acid Metabolism
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. Fatty acids are obtained either by breakdown of fat (endogenous or from the diet) or by synthesis starting with acetyl-CoA. Breakdown of fats to provide fatty acids is an important energy source for the body. Breakdown of Dietary Fat

Fats are very insoluble compounds in water. Pancreatic Lipase acts to hydrolyze triacylglycerols at positions 1 (last point of hydrolysis) and 3 (first point of hydrolysis). The products of this reaction are Na+ and K+ salts of fatty acids (also known as soaps). Soaps, of course, help emulsify fats. Further, the activity of pancreatic lipase actually increases when it contacts the lipid-water interface (interfacial activation). Binding of pancreatic lipase to the lipid-water interface requires a complex with another factor, pancreatic colipase. In the absence of substrate (lipids), the enzyme complex's active site is buried by a lid-like structure. In the present of lipid, the active site is exposed and in the process a hydrophobic entrance to the active site is exposed.Remember that a micelle is formed when soaps surround a non-polar substance in water. Lipid digestion by pancreatic lipases generates mono and diacylglycerols that are absorbed in the small intestine. Bile acids aid in this process too, forming micelles. A common bile acid is glycocholate. Blocked bile ducts inhibit absorption of fats considerably.

The mono and diacylglycerols in the digestive cells are converted back to triglycerides, and packaged into lipoproteins in the bloodstream called chylomicrons. They travel into the blood stream via the lymph system to target tissues. Upon arrival in adipose tissue and muscle cells, a hormone-sensitive triacylglycerol lipase cleaves fats to free fatty acids and glycerol. Note that this pathway is stimulated by epinephrine and goes through the G-protein mediated receptor system that also regulates glycogen metabolism. Binding of epinephrine to the receptor ultimately activates triacylglycerol lipase by phosphorylation (as in the other system), resulting in breakdown of triacylglycerol ultimately to free fatty acids and glycerol.

Fatty acids are broken down for energy as described below. Glycerol from fat breakdown is transported back to the liver where it is converted into DHAP or G3P. Glycerol is the only component of fats that can be used to make glucose in net amounts in animals.

Fatty Acid Oxidation

Fatty acids must be activated for degradation by conjugation with coenzyme A (CoA) in a reaction catalyzed by acyl-CoA ligases (thiokinases). Note that creation of the thioester bond converts ATP to AMP. This is the equivalent of using two ATPS. The enzymes are associated with the endoplasmic reticulum and outer mitochondrial membrane and require ATP. 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.

Transport Into the Mitochondrion

First the acyl groups is transesterified to carnitine 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.

Oxidation Reactions

Inside the mitochondrion, acyl-CoAs are acted upon in four reactions.

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 (ETF) transfers FADH2's electron pair to ETF:ubiquinone oxidoreductase which then passes the electron pair to CoQ of the ETS.

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. Complete oxidation of one palmitate molecule (16 carbons) generates 106-120 ATPs, depending on how you count ATPs from oxidative phosphorylation. Fatty acids that have odd numbers of carbons or that have unsaturated bonds in them required additional metabolic reactions.

Oxidation of Unsaturated Fatty Acids

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. 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. This fatty acid is now in the normal configuration for beta oxidation and beta oxidation proceeds.

Another possible configuration is found in oxidation of linoleoyl CoA. Three oxidations here create a template with a double bond between carbons 3 and 4. Enoyl-isomerase, as previously, moves the cis bond between carbons 3 and 4 to a trans bond between carbons 2 and 3. A single round of beta oxidation yields a structure with a double bond between carbons 4 and 5. Acyl-CoA dehydrogenase will act on this template, yielding an intermediate with double bonds between carbons 2/3 and 4/5. These are conjugated double bonds. Enoyl-CoA hydratase of beta oxidation 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 cis bond between carbons 3-4 (incorrectly shown as a trans bond in your book). This bond is flipped to the 2-3 position by Enoyl-CoA isomerase (as before) to form trans-2-enoyl-CoA, which is then metabolized as earlier IS a substrate for enoyl-CoA hydratase and beta oxidation proceeds. Thus, using only two different enzymes, cells are able to create substrates from ALL unsaturated fatty acids for oxidation in the normal beta oxidation pathway of saturated fatty acids.

Oxidation of Odd-chain Fatty Acids

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. 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.

Peroxisomal Beta Oxidation

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. Zellweger syndrome results from the absence of functional peroxisomes and is caused by the lack of the ability of the peroxisomes to import enzymes.

Ketone Bodies

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. In addition, whatever oxaloacetate normally present in the citric acid is taken to make glucose, so there is nothing to put the acetate of acetyl-CoA onto. Acetyl-CoA will thus accumulate. 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. 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 Biosynthesis

Fatty acid biosynthesis occurs similarly to Beta-oxidation - acetyl groups are added to a growing chain, but the mechanism of the pathway is distinctly different from being simply the reverse of Beta-oxidation. Fatty acid biosynthesis occurs in the cytosol (not mitochondria). It uses a moiety called Acyl-carrier protein (ACP) instead of CoA and the reducing agent NADPH (not NAD/FAD). There are structural similarities between the Coenzyme A portion of CoA and the Phosphopantetheine moiety of ACP. The initial form of the unit added is actually a three carbon molecule (malonyl-CoA) which is decarboxylated and incorporated as a 2 carbon acetate. As we shall see, fatty acid biosynthesis can be broken in to three separate pathways shown below: 

Steps in the process of fatty acid synthesis starting with malonyl ACP and acetyl ACP are as follows:

Enzymes

You will note to this point that I have not given enzyme names for the reactions of fatty acid biosynthesis in my notes. There is a very good reason for this. After malonyl-CoA is made by acetyl-CoA carboxylase (more on this below), all of the enzymatic activities for the reactions above are found in a single complex. This complex is called Fatty Acid Synthase. I do not require you to know the names of the individual activities of the fatty acid synthase.

The enzyme complex can exist as both a monomer and dimer. The dimeric form is the fully functional form of the enzyme. In E. coli, the complex contains activities on individual enzyme units, whereas in eukaryotes, all of the activities are found on a single polypeptide chain. The overall synthesis of palmitate from acetyl-CoA requires 14 NADPHs, and 7ATPs.

The multiple enzymatic activities integrated into Fatty Acid Synthase complex are probably made possible by the "swinging" action of the ACP.

Movement of Acetyl CoA

Acetyl CoA will accumulate when the ETS/oxidative phosphorylation slows (why? - a good exam question). Under these conditions, acetyl-CoA is transported out of the mitochondrion to the cytosol where it can be used in fatty acid synthesis. This is accomplished using the tricarboxylate transport system in the inner mitochondrial membrane which pumps citrate out. Acetyl-CoA, of course is used in synthesis of citrate when combined with oxaloacetate. Citrate transferred into the cytosol is broken back to oxaloacetate and acetyl-CoA by ATP-citrate lyase (using ATP and CoA). Oxaloacetate can be reduced to malate by malate dehydrogenase and NADH. Malate can be converted to pyruvate by malic enzyme, creating NADPH. Using this pair of reactions, electrons have been transported from NADH to NADP, forming NADPH. The resulting pyruvate can be transported into the mitochodrial matrix. Inside the mitochondrion, pyruvate can be converted to oxaloacetate by pyruvate carboxylase (along with bicarbonate ion, and ATP), completing the cycle.

Acetyl-CoA Carboxylase

The first committed step of fatty acid biosynthesis is catalyzed by Acetyl-CoA carboxylase. The enzyme contains biotin, and adds a CO2 (resulting in a carboxyl group) to the methyl end of acetyl CoA (see the reaction on the bottom of page 617 - no figure available). Note that this reaction is an energy requiring process (1 ATP per malonyl-CoA formed). Acetyl-CoA carboxylase is an interesting enzyme. Studies of the enzyme from birds and mammals indicate that it forms long linear polymers. The polymer appears to be the active form of the enzyme. Monomeric units are inactive. Allosterically, citrate shifts the polymer - monomer equilibrium towards polymer formation and palmitoyl-CoA shifts the equilibrium towards monomer formation. Of the two compounds affecting enzyme form, palmitoyl-CoA probably exerts the greater influence. Another regulation of Acetyl-CoA carboxylase is by hormone-stimulated phosphorylation. Glucagon, epinephrine, and norepinephrine trigger a cAMP dependent phosphorylationof the enzyme that shifts the equilibrium towards monomer formation, inactivating it. Insulin, conversely, stimulates desphosphorylation, favoring polymerization and activation. The enzyme responsible for phosphorylating Acetyl-CoA carboxylase is AMP-dependent protein kinase (AMPK). AMPK acts like a "fuel gauge", being activated when AMP is present (low fuel) and inactivated when ATP is present (high fuel). Thus when the fuel is low, the acetyl-CoA carboxylase gets phosphorylated, turning it off and stopping fatty acid biosynthesis. Conversely, when the fuel is high, the AMPK is inactive and protein phosphatase dephosphorylates acetyl-CoA carboxylase, activating it and turning on fatty acid biosynthesis. The regulation by citrate can at least partially overcome the inhibition of the enzyme when it is phosphorylated.

Elongation of Palmitate

The product of fatty acid synthase action, palmitate, is but of course one of many fatty acids synthesized by cells. Elongases are enzymes that act to lengthen palmitate (and unsaturated fatty acid, as well) to produce many of the other fatty acids. Elongases are present in the endoplasmic reticulum and use malonyl-CoA to add two carbon units in reactions like those in the fatty acid synthase reactions.

Desaturation of Fatty Acids

The transfer of electrons from a fatty acid through electron carriers to NAD, forms NADH. Note the unusual electron transferring pathway in which electrons from NADH are ultimately passed to oxygen, forming water. The energy released in this process drives oxidation of stearoyl-CoA to oleyl-CoA. In the process, a double bond is formed. Terminal desaturases are the enzymes that catalyze these reactions. From the free methyl end, mammals cannot make double bonds closer to the end than the Delta-9 position (Oleic acid is a Delta-9 fatty acid). Thus, linoleic acid (Delta 9,12 double bonds) and linolenic acid (Delta 9,12,15 double bonds) must be provided in the diet of mammals, and are called essential fatty acids.

A polyunsaturated fatty acid commonly found in cells is arachidonic acid. Arachidonic acid is synthesized from linoleic acid and contains four double bonds. Arachidonic acid is a precursor of a group of compounds called eicosanoids. These include the prostaglandins, thromboxanes, and leukotrienes. Arachidonate is not normally free in cells. Arachidonate is released from phospholipids or diacylglycerols by action of lipases (PLA2 or DG lipase). Prostaglandin synthase (a cyclooxygenase) converts arachidonate to prostaglandin H2, which is a precursor of many other prostaglandins and thromboxanes. Prostaglandins are associated with pain and many other bodily responses, including inflammation, control of blood flow, platelet stickiness, uterine contractions, and others. There are two ways to inhibit the production of prostaglandins. One is the use of aspirin, ibuprofen, or similar compounds. These are called COX inhibitors because they inhibit the cyclooxygenase. They are also called NSAIDs (non-steroidal anti-inflammatory drugs) to differentiate them from the other inhibitors used to treat inflammation. These are steroids that are used to inhibit the lipases that release arachidonate. By prohibiting the release of arachidonate, all of the subsequent products are avoided.