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 (exception - elongation of palmitate in the mitochondrion - see below). Figure 18.23 shows the relationship between fatty acid biosynthesis and 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). It will be noted in Figure 18.26 that there are structural similarities between the Coenzyme A portion of CoA and the Phosphopantetheine moiety of ACP. The reaction has a different stereochemisry from Beta-oxidation and the form of the unit added is actually a three carbon unit (malonyl-CoA) which is decarboxylated to incorporated a net 2 carbon unit. A depiction of the cyclic route of synthesis and catabolism of acetyl-FA in fatty acid metabolism is shown in Figure 18.22. As we shall see, fatty acid biosynthesis can be broken in to three separate pathways shown below:
Acetyl-CoA is produced in two ways in the mitochondria -
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 (Figure 18.31). 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 and NADP+. The resulting pyruvate is permeable to the inner mitochondrial membrane and diffuses in. Inside the mitochondrion, pyruvate can be converted to oxaloacetate by pyruvate carboxylase (along with bicarbonate ion, and ATP), completing the cycle. An alternative path is to transport malate across the inner membrane and convert it to oxaloacetate.
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 Reaction #1, Figure 18.24). 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 (See Figure 18.25). The polymer appears to be the active form of the enzyme. Monomeric units are inactive. Citrate shifts the polymer - monomer equilibrium towards polymer formation. 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 hormones. Glucagon, epinephrine, and norepinephrine trigger a cAMP dependent phosphorylation (remember the cascade system) of the enzyme that shifts the equilibrium towards monomer formation. Insulin, conversely, stimulates desphosphorylation, favoring polymerization. The enzymes responsible for phosphorylating Acetyl-CoA carboxylase are cAMP-dependent protein kinase and AMP-dependent protein kinase (AMPK). E. coli's Acetyl-CoA carboxylase is regulated by guanine nucleotides, which are a function of those cells' growth requirements.
This multifunctional enzyme catalyzes the seven different reactions whereby two carbon units from malonyl-CoA are linked together, ultimately to form palmitoyl-CoA. In some systems, the activities are present on separate enzyme units. In other cells, a single polypeptide chain has multiple activities that can be isolated after protease treatment. The enzyme complex can exist as both a monomer and dimer. The dimeric form is the fully functional form of the enzyme. The overall synthesis of palmitate from acetyl-CoA requires 14 NADPHs, and 7ATPs.
Steps of fatty acid synthesis starting with Acetyl-CoA and Malonyl-CoA are shown in Figure 18.24 and Figure 18.27. The reactions are as follows:
The multiple enzymatic activities integrated into Fatty Acid Synthase complex are probably related to the growing fatty acid being "swung" into the appropriate catalytic region of the synthase, as depicted in Figure 18.29.
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 to produce many of the other fatty acids. Elongases are present in mitochondria and the endoplasmic reticulum. Elongation using elongase in the mitochondrion involves a mechanism that is essentially the reverse of Beta-oxidation except substitution of NADPH for FADH2 in the last reaction (see Figures from page 653).
Terminal desaturases produce unsaturated fatty acids. One such enzyme is fatty acyl-CoA desaturase. The reaction catalyzed by this enzyme on a stearoyl-CoA is shown in Figure 18.32. 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. 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.
The synthesis of arachidonic acid from linoleic acid is depicted in Figure 18.33. Note that arachidonic acid contains 4 double bonds. Arachidonic acid is a precursor of a group of compounds called eicosanoids to be discussed later in the course.
Like all metabolic pathways, cells must have appropriate controls on fatty acid metabolism to be able to meet energy needs. Precursors for energy generation - triacylglycerols in chylomicrons and VLDL, fatty acid/albumin complexes, ketone bodies, amino acids, lactate, and glucose - are all carried in the blood as needed for various tissues. One mechanism of regulation involves hormone release.
Both of latter systems control glucose-related metabolism as well. Students should recognize that the regulatory mechanisms of controlling enzymatic reactions we have discussed to date are short-term regulation. They act in minutes (or less). Fatty acid synthesis is controlled partly by short term regulation (mechanisms include substrate availability, allosterism, covalent modification of enzymes) and partly by long term regulatory mechanisms. Long term regulation involves controlling the quantity of enzyme by controlling the rate with which a protein is synthesized and/or degraded. A simple scheme depicting fatty acid biosynthesis regulation is shown in Figure 18.34. One of the reasons fats do not supply emergency energy is that control of their metabolism is largely by long term regulatory mechanisms whereas control of sugar metabolism is more prominent under short term regulatory mechanisms.
Insulin stimulates increased synthesis of acetyl-CoA carboxylase and fatty acid synthase (two critical enzymes for synthesizing fatty acids). Starvation, conversely decreases synthesis of these enzymes. Fatty acid oxidation is regulated by fatty acid concentration in the blood. This is controlled by the amount of hydrolysis of triacylglycerols in adipose tissue by hormone-sensitive triacylglycerol lipase (HSTL). This enzyme is phosphorylated in the hormonally-controlled cAMP-dependent phosphorylation cascade, which activates the lipase, stimulating release of fatty acids. This cascade is turned on by the cell's binding of glucagon or epinephrine It should also be noted that the cAMP-dependent phosphorylation system also causes inactivation of acetyl-CoA carboxylase, an important control enzyme in fatty acid biosynthesis. Insulin opposes the effects produced by glucagon and epinephrine, stimulating glycogen formation and triacylglycerol synthesis, by favoring dephosphorylation of the enzymes phosphorylated as described above.
Synthesis of fatty acids is only half of the process of making triacylglycerols. In the first part of the process, a fatty acyl-CoA is linked to carbon #1 of dihydroxyacetone phosphate (DHAP) or glycerol-3-phosphate (Gly3P) by either dihydroxyacetone phosphate acyltransferase (for DHAP) or glycerol-3-phosphate acyltransferase (for Gly3P) (Figure on page 656 shows the reaction for Gly3P). The product of the reaction for Gly3P is lysophosphatidic acid (LPA)
The product of the DHAP reaction (acyl-dihydroxyacetone phosphate) can be converted to the product of the Gly3P reaction (lysophosphatidic acid) by NADPH and acyl-dihydroxyacetone phosphate reductase.
Lysophosphatidic Acid (LPA) is acylated (with an acyl-CoA) at carbon #2 by 1-acylglycerol-3-phosphate acyltransferase to produce phosphatidic acid. This intermediate can be converted to other phospholipids (such as phosphatidyl choline) or converted to triacylglycerols (see below). Phosphatidic acid is thus an important branch point between triacylglyerol biosynthesis and glycerophospholipid biosynthesis.
The phosphate of phosphatidic acid is removed by phosphatidic acid phosphatase, forming diacylglycerol (shown as phosphate being split out by water in the Figure on page 656). Diacylglycerol is converted to triacylglycerol (with acyl-CoA, of course) by diacylglycerol acyltransferase.
Fatty Acid Synthesis
Fatty Acid Synthesis (for those like to feel pain when looking at metabolism)
Fatty Acid metabolism (long)