Page1 بسم رلاهللا On Thursday, we discussed the synthesis of fatty acids and its regulation. We also went on to talk about the synthesis of Triacylglycerol (TAG). Last time, we started talking about the synthesis of fatty acids, so I will shortly recap before explaining the topics discussed in this lecture. The Requirements of fatty acid synthesis include the following: 1) Carbon Source: Acetyl CoA 2) Reducing Power: NADPH 3) Energy Input: ATP The process of fatty acid synthesis starts off with the Carboxylation of Acetyl CoA to form Malonyl CoA by a biotin containing enzyme called Acetyl CoA Carboxylase. Notice that the pink CO 2 was the one added during carboxylation of Acetyl CoA to Malonyl CoA. All remaining steps of fatty acid synthesis are catalyzed by a multifunctional enzyme complex called Fatty Acid Synthase. This enzyme is a dimer composed of two identical chains, each of which has seven catalytic activities. - One of the activities of this enzyme is the Condensing Enzyme portion that has a reactive SH group. - One Domain is linked to Phosphopantetheine also with a reactive SH group. This domain carries Acyl intermediates such as Acetyl and Malonyl groups during catalysis, it is thus known as the Acyl Carrier Protein (ACP). The Acyl Carrier Protein carries an acetyl group and then transfers to the condensing enzyme of Fatty Acid Synthase. Following the carboxylation of another acetyl group to Malonyl CoA by Acetyl CoA Carboxylase as discussed earlier, this Malonyl CoA is transferred to the Acyl carrier protein of Fatty Acid Synthase, which already has an acetyl group linked to its condensing enzyme portion.
Page2 Now that both the Acetyl CoA and Malonyl CoA are attached to different portions of the same Fatty Acid Synthase Enzyme complex, condensation takes place by combining the acetyl group with only 2 of the 3 carbons of Malonyl CoA, hence producing Ketoacyl ACP. In other words, the CO 2 that was added to make Malonyl CoA from the carboxylation of Acetyl CoA, is now released. The Ketoacyl CoA, which is a 4 Carbon unit attached to the Acyl Carrier Protein in Fatty Acid Synthase, undergoes 3 reactions after condensation, finally resulting in a butyric acid; a 4 carbon fatty Acyl group. 1) Reduction by NADPH. 2) Dehydration by removal of water. 3) Another reduction by NADPH.
Page3 This butyric acid is now transferred to the condensing enzyme of Fatty Acid Synthase. Consequently, the Acyl Carrier Protein is now empty and so it takes on a new Malonyl CoA group and the whole process of condensation takes place again; this time resulting in a 6 Carbon fatty Acyl group with a ketone group at carbon Beta (Carbon # 3). This cycle of condensation, reduction, dehydration and lastly reduction is carried out repeatedly until the fatty Acyl Palmitate (16 Carbon fatty acid) is formed. Why does fatty acid Synthase put an end to the synthesis cycle at 16 Carbons? This is because this enzyme has a specificity to produce a 16 Carbon fatty acid using a cycle of the same steps. The production of a fatty acid, longer than 16 Carbons by Fatty Acid Synthase, is NOT possible. Following the production of Palmitate, hydrolysis of the high energy bond between the fatty acid and ACP, takes place in order to release it.
Page4 Net reaction of Palmitate synthesis *How many cycles of condensation? 7 cycles Why? Since 16/2=8 1 because the first cycle results in the formation of a 4 Carbon fatty Acyl group, after that 2 Carbons are added with every cycle. *How many Malonyl CoA are used throughout the synthesis of Palmitate? 7 Malonyl CoA groups, since in the first cycle, condensation of acetyl CoA and Malonyl CoA takes place, followed by the addition of 2 Carbons of the Malonyl group with every cycle. *How many Acetyl CoA were used directly in the synthesis of Palmitate? Only 1, as acetyl CoA is used in the first cycle only and it forms the methyl omega carbon. Acetyl CoA carboxylated to Malonyl CoA are not directly utilized in the synthesis. *How many NADPH were used? 14, since 2 NADPH are used in every cycle and we have 7 cycles. Note by the doctor: All these numbers can be deduced by studying the reactions above, in the exam he might give you an example of the synthesis of a 12 Carbon fatty acid for example and ask you about the net information such as the ones provided above, so it is important to know how to calculate the number of cycles and number of substrates used. What is the source of Acetyl CoA for fatty synthesis? *It is Pyruvate Dehydrogenase, from the metabolism of carbohydrates or degradation of amino acids that are converted to pyruvate. When there is excess carbohydrate or protein intake, the acetyl CoA is made available for fatty acid synthesis. *The Doctor emphasized the point that the source of Acetyl CoA for fatty acid synthesis is NOT Beta Oxidation of fatty acids, since synthesis and degradation cannot take place at the same time in the same location. Otherwise, it would be a waste of energy. Synthesis and degradation can take place in different places at the same time like glycolysis in muscle tissue and Gluconeogenesis in the liver, but this is a special case. Thus, the synthesis and oxidation of fatty acids should be regulated.
Page5 Production of Cytosolic Acetyl CoA for Fatty Acid synthesis: Fatty Acid synthesis takes place in the cytosol, while Acetyl CoA production, from Pyruvate Dehydrogenase, takes place in the mitochondria. Furthermore, the Inner Mitochondrial membrane is impermeable to Acetyl CoA like it is to Acyl CoA, as demonstrated when we took beta oxidation of fatty acids. So the question here is, how does Acetyl CoA reach the cytosol for fatty acid synthesis? First, Acetyl CoA condenses with Oxaloacetate to form Citrate, which is also the first step in the Citric Acid Cycle. If the cell is in a high energy state, Citrate is not used in the Krebs Cycle and thus leaves the mitochondria by means of a Citrate Carrier. The action of this Citrate carrier depends on the concentration of Citrate in the mitochondria, if high enough; it transports Citrate to the Cytosol where it is cleaved back to Acetyl CoA and Oxaloacetate. *This is not the reverse of the condensation reaction (Acetyl CoA + OAA -> Citrate) that took place in the mitochondria, although it might appear so chemically. This is because the reformation or condensation to form Citrate in the CYTOSOL would require ATP. In other words, the cleavage reaction of Citrate in the cytosol would require energy to reverse it to the formation of citrate, like that in the mitochondria. Thus the irreversible exergonic cleavage of Citrate in the cytosol to give Acetyl CoA, is not the opposite of the first reaction of the Krebs Cycle.
Page6 To validate this point even further, we have been accustomed to the fact that reverse reactions usually utilize the same enzyme, this cleavage reaction in the cytosol uses a different enzyme from the one used in the production of Citrate in the mitochondria. The Names of the enzymes are: - Citrate Synthase in the mitochondria. - ATP Citrate Lyase; the enzyme responsible for the synthesis of Cytosolic Acetyl CoA needed for fatty acid synthesis. Another explanation for the abovementioned reaction, would be that the process of linking Acetyl group to Coenzyme A while cleaving the acetate by lyase enzyme requires ATP (for making the high energy bond), but cleaving the citrate to acetic acid and OAA doesn t require any ATP, so energy is required,because the reaction is actually not reversible and you need to make it reversible. Extra Notes: - Lyases differ from other enzymes in that they require only one substrate for the reaction in one direction, but two substrates for the reverse reaction. - ATP Citrate Lyase is activated by Insulin. After Acetyl CoA was made available in the cytosol for fatty acid synthesis, Oxaloacetate should return to the Mitochondria, however it does not enter as such, as it is reduced to Malate. This is the opposite of the last reaction of the Krebs cycle. Theoretically speaking, Malate can enter the mitochondria, however sometimes according to the Doctor; it can t enter unless it undergoes oxidative decarboxylation to pyruvate. COO is removed as CO 2 and NADP is reduced to NADPH. This is similar to oxidative decarboxylation in the citric acid cycle. The enzyme for this reaction is Malate Dehydrogenase or more commonly known as Malic Enzyme. If Malate can enter the mitochondria as such, why is it converted by Malic enzyme to pyruvate by the process of oxidative decarboxylation? This is because, during oxidative decarboxylation, NADP is reduced to NADPH, which is needed during fatty acid synthesis. 8 out of the 14 NADPH required during fatty acid synthesis of Palmitate are produced in this way when 8 Acetyl CoA leave the mitochondria. After this, in the mitochondria, pyruvate is carboxylated to Oxaloacetate (requiring an ATP molecule ), thus closing the cycle.
Page7 The figure on the right shows the reaction explained on the previous page. The Doctor made a comment about a mistake in one of the old editions of Lippincott. The mistake was that during the reduction of OAA to Malate, NADH is oxidized to NAD + and not the opposite. Should you own one of the earlier editions of Lippincott, correct this mistake. To summarize the net of what happens is: 1) The exit of Acetyl CoA from the mitochondria to the cytosol. 2) The production of NADPH by preventing Malate from returning to the mitochondria before decarboxylating it to pyruvate. 3) 2 ATP consumption per Acetyl CoA, which is energetically costly, but this occurs during high energetic state of the cell. *Pyruvate Carboxylase catalyzes the carboxylation of pyruvate to OAA. The Doctor mentioned that this is an Anaplerotic reaction; which are fill up reactions that form intermediates of metabolic pathways such as the TCA cycle. Now that we ve finished both the synthesis and degradation of fatty acids, let s take a look at the regulation of these two processes. Regulation of Fatty Acid Metabolism The 2 processes should be separated, as no synthesis should take place during degradation and vice versa. Otherwise, energy would be lost since the degradation does not produce ATP while the synthesis requires ATP for Fatty Acid Synthase and transporting Acetyl CoA. Thus, these two processes should be strictly coordinated and regulated. There are mechanisms for the regulation of oxidation and others for the regulation of synthesis. Let s start with the regulation of synthesis.
Page8 Regulation of Synthesis of Fatty Acids The limiting or committed step in fatty acid synthesis is carboxylation of Acetyl CoA catalyzed by Acetyl CoA Carboxylase. There are 3 methods for regulating this step and enzyme: 1) Allosteric Regulation (seconds) 2) By phosphorylation mechanism (minutes) 3) Amount of enzymes (long term) 1) Allosteric Regulation of the enzyme Acetyl CoA Carboxylase: This takes place within a very short period of time (seconds). *Active Form: Polymer converts Acetyl CoA to Malonyl CoA Inactive Form: Dimer *Activator: Citrate Inhibitor: Long Chain Fatty Acyl CoA The significance of stimulation of this enzyme by Citrate is that Citrate is made available to the pathway of fatty acid synthesis when the cell is in high energy state and here is abundance of building blocks. Thus, when the concentration level of Citrate is high during fed state, Acetyl CoA Carboxylase is activated into its polymer form and fatty acid synthesis is stimulation. On the other hand, Fatty Acyl CoA or Palmitoyl CoA to be more specific is a long chain fatty acid or Fatty Acyl CoA group (LCFA). This works as an inhibitor by feedback inhibition of the very early committed step.
Page9 2) Covalent Modification and Regulation by Phosphorylation of the enzyme Acetyl CoA Carboxylase: This requires more time than the Allosteric regulation (minutes). camp dependant protein Kinase converts the active form to inactive form by phosphorylation. Active Form: Dephosphorylated Inactive Form: Phosphorylated This is stimulated by Glucagon and Epinephrine, when there is low blood glucose, the levels of these hormones rise and thus, fatty acid synthesis is stopped. Remember the rule of thumb: phosphorylation corresponds to conserving Glucose, so fatty acid synthesis is stopped. How does the enzyme return to its active form (Dephosphorylated and polymerized)? The enzyme Phosphatase dephosphorylates it, stimulated by Insulin when blood glucose level is high, so fatty acid synthesis resumes. An Insulinoma is a tumor of the insulin producing cells. Upon formation, it causes the person to gain weight very quickly, as insulin is continuously produced by the tumor cells, so Acetyl CoA Decarboxylase is always activated and performing fatty acid synthesis, thus keeping blood glucose level low and creating the hungry sensation. This leads to obesity at a very high rate.
Page10 3) Regulation of Fatty Acid Synthesis not only Acetyl CoA Dehydrogenase, according to the amount of enzymes: The last method of regulation of fatty acid synthesis involves the amount of enzymes present in the body. If the fed state persists, in other words whenever the person feels a little hungry he eats, the number of enzymes involved in fatty acid synthesis increase to accommodate for the high calorific intake. Enzymes including Fatty Acid Synthase, Malic Enzyme, Glucose-6-Phosphate Dehydrogenase, among others increase in number in the body. The number of enzymes, that produce NADPH needed for fatty acid synthesis, also increase. Regulation of Oxidation of Fatty Acids There are also 3 mechanisms regulating the oxidation of fatty acids, they mostly involve the substrate presence or absence. 1) Supply of Fatty Acids 2) Availability of NAD + 3) Entry into the mitochondria 1) If the fatty acid level increases, then their activation into Fatty Acyl CoA increases, followed by increased transport of Fatty Acyl group by Carnitine shuttle into the mitochondria, then Fatty Acyl CoA is oxidized by the 3 steps we studied earlier. When Glucagon stimulates Lipase, fatty acids are mobilized from adipose tissue. When there is a high concentration of fatty acids in the blood, the rate of oxidation increases. (Proportional) 2) The availability of NAD + Oxidation of fatty acids needs NAD +. NAD + is either present oxidized or reduced in cells. If all the NAD + is reduced totally to NADH, then NAD + is not available for fatty acid oxidation. This is an indirect way of regulating fatty acid oxidation. NADH is rapidly reoxidized by the electron transport chain (ETC) in order to make NAD + available. Fatty acid available corresponds to higher oxidation. NAD + available, also a substrate whose availability participates in the regulation of fatty acid oxidation. 3) Malonyl CoA acts as an inhibitor for the entrance of Fatty Acyl to the mitochondria by inhibiting its attachment to Carnitine shuttle. Malonyl CoA is an intermediate during synthesis of fatty acids, thus it inhibits fatty acid oxidation by blocking transfer of fatty acyl to mitochondria. Even though fatty Acyl is available, it does not enter the mitochondria and does not undergo oxidation. This is direct regulation between synthesis and oxidation, as one of the intermediates of synthesis is inhibiting the oxidation.
Page11 *The Doctor then read an article about the discovery of the Acetyl CoA Decarboxylase enzyme to reduce fatty acid synthesis, and its importance in discovering drugs for managing obesity, which is now a major problem in developed and even developing countries. He also mentioned that most drugs inhibit enzymes and do not stimulate them. We will now move to the topic of fatty acid modification which includes both elongation and desaturation (introducing double bonds). Elongation of Fatty Acids Fatty acid synthesis involves reaching a fatty acid of up to 16 Carbons, so the question is how do we obtain fatty acids which are longer than 16 Carbons? - Elongation does not take place in the cytoplasm, it taken place in the endoplasmic reticulum; it involves a similar sequence of reactions (method and cofactors) but with different enzymes. This is because the specificity of fatty acid Synthase enzyme stops at 16 Carbons. The Endoplasmic Reticulum is a membranous structure so the products of fatty acid synthesis enter it directly for elongation. - Elongation can take place in the mitochondria. The reactions involved are similar to those of Beta oxidation but are opposite. However the last reaction that reduces FAD to FADH2 instead oxidizes NADPH to NADP, because synthesis requires NADPH and because FADH 2 is fixed and does not leave the enzyme. The enzyme involved in this step is also different as demonstrated in the figure. If elongation takes place in the mitochondria, fatty acids with less than 16 Carbons are used. Those are obtained from the food we eat ex. 6 Carbon fatty acid. In order to store these short fatty acids in the body, they must be elongated in the mitochondria. They do not go to fatty acid Synthase. This involves similar sequence of steps but with modification.
Page12 Desaturation of Fatty Acids This involves the introduction of double bonds to fatty acids in the endoplasmic reticulum. Let s start with the synthesis of monounsaturated fatty acids. Taking Oleic Acid (18 Carbons) and Palmitoleic Acid (16 Carbons) as examples because they both have a single double bond at carbon number 9. Rule of thumb: No double bonds can be introduced beyond Carbon number 9 in human cells. We can t synthesize fatty acids with double bonds at carbon number 12 and 15 for example. Oleic and Palmitoleic Acids can be synthesized because the double bond is at Carbon number 9. Fatty Acids, with double bonds on carbons higher than 9, must be ingested from dietary sources. Thus the synthesis of monounsaturated fatty acids is possible in humans, because they can be at Carbon 9. How are monounsaturated fatty acids produced? Example: The conversion of Stearoyl CoA (18:0) to Oleoyl CoA (18:1), or Palmitoyl (16:0) CoA converted Palmitoleoyl CoA (16:1)
Page13 This is done by Hydroxylation or introducing a hydroxyl group followed by dehydration (removal of H 2O) to produce a double bond. This is done by enzyme Δ9 Desaturase, Cytochrome b5 which requires NADPH and O 2. We are oxidizing the fatty acid by adding hydroxyl group, and NADPH is required despite its being a reductant. This is because oxygen (O 2) is used as a source for the oxygen atom in hydroxyl group, thus NADPH reduces the other oxygen to prevent free radical formation. Cytochrome b5 transfers 2 electrons to the other oxygen atom in order to reduce it, while the other oxygen atom is introduced into fatty acid. Formation and Modification of Polyunsaturated Fatty Acids (PUFA) We can t synthesize PUFA s but we can modify them by elongation and desaturation. This is done by addition of double bonds at Carbon 4, 5, or 6 but not after Carbon number 9. - Linoleic Acid (18:2) has 2 double bonds at Carbon 9 and 12. If we want to add a double bond, the double bond is introduced at Carbon number 6 not 7 or 5 because the difference between double bonds must be 3 carbons. Desaturase enzyme adds double bonds at 6,9,12. - If elongated, it becomes a 20 carbon fatty acid, each double bond pushed by 2 C s. Thus it is now (20:3 Δ 8, 11, 14). The omega classification is still the same ω6. - Another desaturation at carbon 5 takes place now, not at 6 since 8-3=5. The fatty acid becomes (20:4 Δ 5, 8, 11, 14) which is Arachidonic acid. Note: The Omega classification does not change upon modification, because modification does not take place from the omega side. Thus, fatty acids are divided into omega classification families such as the ω 3 family, the ω 6 family, and the ω 9 family.
Page14 The last topic for this lecture is the biosynthesis of triacylglycerol: Triacylglycerol (TAG) is composed of a glycerol esterified to 3 fatty acids. Triacylglycerol and phosphoacylglycerol are very similar in structure, but the latter has phosphoric acid esterified to the third carbon of glycerol instead of a third fatty acid. Owing to the huge similarity in their structures, their pathways of synthesis should logically share many common steps. Phosphatidic acid is a common intermediate for the synthesis of both, despite the fact that TAG has no phosphate group. Biosynthesis of Triacylglycerol requires: - Adding Fatty Acids in their active form: Acyl CoA - Glycerol phosphate The active form of Acyl CoA is used because Fatty acids can t be added as such, they must be activated so that breaking the high energy thioester bond between Acyl and CoA releases energy needed to bind DAG and Acyl.
Page15 - Hydrolysis of TAG produces Diacylglycerol (DAG), ΔG= ve exergonic. - DAG + FA ΔG=+ve endergonic Fatty acid as Acyl CoA has a high energy bond which when broken releases a lot of energy needed to transfer the fatty acid to Diacylglycerol. This is similar to the synthesis of acetylcholine. DAG + Acyl CoA gives TAG which has ΔG= -ve, that s why the active form is needed. Now let s look at this in a step wise manner; 1) Acyl CoA is transferred to Glycerol 3-phosphate, whereby the fatty acid is transferred to carbon number 1, hence producing a derivative of Phosphatidic acid called Lysophosphatidic acid. *Lysophosphatidic Acid differs from Phosphatidic Acid in that it does not have a fatty acid at carbon number 2.
Page16 2) Next would be the transfer of another Acyl group to carbon number 2 of lysophosphatidic Acid, thus producing Phosphatidic acid; a common intermediate between TAG and phosphoacylglycerol. This Phosphatidic Acid has 2 fatty acids and a phosphoric acid as mentioned earlier. 3) The next step in TAG synthesis would be the removal of phosphate or phosphoric acid by an enzyme called Phosphatase (not phosphorylase). In this manner, Diacylglycerol is produced. 4) Then addition of Acyl group to DAG gives Triacylglycerol. Note: The enzyme used in all these steps except the removal of phosphate group is Acyl Transferase. Step number 3 uses enzyme Phosphatidate Phosphatase. How is glycerol 3-phosphate produced/obtained? By the phosphorylation of glycerol using an enzyme called Glycerol Kinase. Note: This does not take place in adipose tissue; there is another method by which glycerol 3-phosphate is obtained. *Dihydroxyacetone (DHAP); an intermediate of glycolysis, is used to make glycerol phosphate by using NADH. We took this in the mobilization of TAG. Enzyme used: glycerol 3-phosphate Dehydrogenase. This concludes this very long lecture, good luck everyone! Done By: Zeina Hani Kalaji