Lipid Biosynthesis 2 PDF

Aurora Killi 31.08.21 Biosynthesis of lipids Catabolism and anabolism of fatty acids proceed through different pathways - B-oxidation...

Aurora Killi 31.08.21 Biosynthesis of lipids Catabolism and anabolism of fatty acids proceed through different pathways - B-oxidation - Biosynthesis Catabolism of fatty acids Anabolism of fatty acids Produces acetyl-CoA Requires acetyl-CoA and malonyl-CoA Produces reducing agents (NADH + H , + Requires reducing power from NADPH + H+ FADH2) Takes place in cytosol in animals and Takes place in the mitochondria Imatrix chloroplasts in plants Fatty acid biosynthesis Place of action: Every cell with mitochondria, happens in cytosol. High amount in adipose tissue. Substrate: Acetyl-CoA Product: Acyl-CoA ATP count: 23 ATP to make 16C long chain (including transport of acetyl-CoA) Fatty acid synthesis is related to the cell compartments where there is high NADPH + H+ concentration. This is related to the reducing power that is necessary for the fatty acid synthesis. One of the major producers of NADPH + H+ is the pentose phosphate pathway, however in adipocytes an enzyme named malic is an important contributor. In hepatocytes and mammary gland, the pentose phosphate pathway will be the main metabolic pathway that ensures NADPH + H+ production. Malic enzyme: Acetyl-CoA is transported into the cytosol for fatty acid synthesis In non-photosynthetic eukaryotes acetyl-CoA is made in the mitochondria, but fatty acids are made in the cytosol. So, acetyl-CoA is transported into the cytosol with a cost of 2ATPs for fatty acid synthesis. Therefore, the cost of fatty acid synthesis is 3ATP per 2C unit. Oxaloacetate - Citrate and acetyl-CoA used for formation of citrate, once transported out into the cytosol is reconverted into Acetyl-CoA. Then for the fatty acid biosynthesis, the cycle will involve formation of malate and pyruvate through the malic enzyme. NADPH + H+ created in the process is the one that ensures 50% of the phosphorylated NADH + H+ for the biosynthesis of the fatty acids in adipose tissue. Overview of the fatty acid synthesis: Fatty acids are built in several places, processing one acetate unit at a time. The acetate is coming from activated malonate in the form of malonyl-CoA. Each pass involves reduction of a carbonyl carbon to a methylene carbon. Acetyl-CoA carboxylase reaction – committed step: By the enzyme Acetyl-CoA carboxylase acetyl-CoA is converted into malonyl-CoA. The acetyl-CoA carboxylase reaction is the committed step of the fatty acid biosynthesis. Once malonyl-CoA is formed, we know that it will be used for the fatty acid synthesis. C-2 - c - c c 14 - c c c c c 2 c - - c L - C c - - - - - - - - -  & 1 ATP 102 > Acetyl-CoA + - Aurora Killi Malonyl-cot 31.08.21 How does this conversion happen? In the middle of this enzyme there is a biotin carrier protein and a Biotin vitamin (B7) attached to it through the lysin side chain. This vitamin Biotin performs carboxylation reaction (addition of CO2). The CO2 is added as a bicarbonate ion in its soluble form. Here you can see the extra ATP unit that is needed for each 2 added carbons in the fatty acid chain. There is an addition of bicarbonate ion as a form of the carboxyl group to the biotin vitamin. Once this has happened it is easier to add carboxyl-group to the Acetyl-CoA. The first part of the enzyme Biotin carboxylase now is flipped over to 3ATP O.. the other part of this enzyme’s active site. There can therefore be an addition of the Acetyl-CoA. You can also notice that this addition of carboxyl-group allows Acetyl-CoA to become more reactive. The reason for this is because this CH3-group is stable and EP 1 thus not willing to react with other molecules, however once you have added this Acety' t carboxyl group to it this molecule is more reactive due to the electron distribution around these various atoms. Imalonyl - 7. M. 1AT)> ↓ In this carboxylation, an extra ATP is needed for addition of the bicarbonate ion which means that the cost of fatty acid synthesis is 3ATP per 2C unit (we needed 2ATPs for acetyl-CoA transportation into the - cytosol). 7 T Fatty acid synthase: Once malonyl-CoA is formed synthesis of fatty acids can start. This is catalyzed by an enzyme called fatty acid synthase. This is a large enzyme that has 7 different active sites in vertebrates. Fatty acid synthase always leads to the formation of a single product: palmitate (palmitic acid in other words). This is a saturated fatty acid with 16 carbon atoms. All the active sites in the system are in different domains within a single large polypeptide chain. In plants and bacteria’s, the fatty acid synthase is separate different enzymes, soluble ones also located in the cytosol of the cell. They do tend to produce more kinds of various products, meaning that the fatty acids can both be saturated, unsaturated, and branched. The different enzymatic activities are: 𝛽-ketoacyl-ACP synthase (KS) Malonyl/acetyl-CoA-ACP transferase (MAT) 𝛽-hydroxyacyl-ACP dehydratase (DH) Enoyl-ACP reductase (ER) 𝛽- reductase (KR) Overall goal of the fatty acid synthesis: to attach acetate unit (2C) from malonyl-CoA to a growing chain and then reduce it. The fatty acid synthesis consists of four reactions that repeat These reactions are the themselves from cycle to cycle. Each cycle adds 2C units: reverse reactions of the Condensation of the growing chain with activated acetate beta oxidation. Reduction of carbonyl to hydroxyl 15  Aurora Killi 31.08.21 Dehydration of alcohol to trans-alkene Reduction of alkene to alkane Acyl carrier protein: Acyl carrier protein (ACP) serves as a shuttle in the fatty acid synthesis. ACP is attached to the fatty acid synthase, and it has a long flexible side chain also called arm, that has prosthetic group called 4-phosphopantetheine. Part of it consists of the pantothenic acid (vitamin B5). This flexible arm has the important role of transferring fatty acid from one active site to another in the large fatty acid synthase complex, thus ensuring proceeding reactions without releasing any intermediates in between (substrate channeling). ACP it the one that delivers acetate (in the first step) or malonate (in all the next steps) to FAS. acetyl-CoA Malonyl-con Charging: Before each reaction can occur, the fatty acid synthase needs to be charged. We need to distinguish synthesis de novo which means from scratch or from zero. This is done by the addition of acetyl-CoA and malonyl-CoA 1. Acetyl-CoA addition (de novo): The acyl-carrier protein binds the acetyl-CoA and CoA is released. The acyl-carrier protein shifts the acetyl-group to the ketoacyl-ACP synthase that has a thiol group attached. This first thioester bound between the acetyl group is formed. Now we have added one of the units necessary for the fatty acid synthesis. 2. Malonyl-CoA addition (elongation): This is the unit that is used for the elongation of the fatty acid. Before each fatty acid cycle starts it must be added to the acyl-carrier protein. Malonyl-CoA is also added to the acyl carrier protein by the same steps as the acetyl-CoA. By using the activated malonyl-CoA group and at the same time we have this activated acetate created in the fatty acid breakdown. The cell with these two activated forms of these molecules ensures that both processes are energetic favorable, besides the fact that they are opposite. Step 1: Condensation by 𝜷-ketoacyl-ACP synthase (KS) Once malonyl-CoA has been added to the ACP the elongation of fatty acid can start this is performed by the 𝛽-ketoacyl-ACP synthase (KS). The carboxyl group that was added in the formation of malonyl- CoA is removed which releases a free electron that can react and perform the condensation reaction with the added acetate unit. Acetyl-CoA and the CH3 group is inward, but once we remove the carbon dioxide, we can see that there opens a space where the condensation reaction can occur. The two-carbon atom in the acetate unit is transferred so the fatty acid chain (or from de novo) has been elongated by 2 carbons. The result of this reaction is the β-ketobutyryl-ACP (𝛽-ketoacyl-ACP is a more general name for this molecule). Step 2: Reduction by 𝜷-ketoacyl-ACP reductase In the next step reduction takes place. We want to proceed the reverse step of the 𝛽-oxidation. We want to form a hydroxyl group. We perform this by using the NADPH+H+ as a cofactor that gives 2 hydrogen ions to the ketogroup and we can form the hydroxyl-group (secondary alcohol group). The intermediate created is the D- 𝛽-hydroxybutyryl-ACP (D- 𝛽-hydroxyacyl-ACP). 16  Aurora Killi 31.08.21 Here we can see one difference from the 𝛽 -oxidation since there it was formed the L- 𝛽-hydroxybutyryl-ACP which means that this is the stereoisomer. Step 3: Dehydration by 𝜷-hydroxyacyl-ACP dehydratase: We are removing the water molecule and thus reforming the double bond between the alpha and beta carbon atom. As a result, we gain the intermediate trans-∆^2-Butonoyl-ACP (trans-∆^2-Enoyl-ACP). This is an intermediate after the 1st of the 𝛽-oxidation Step 4: Enoyl-ACP reductase: NADPH + H+ is used and we are now saturating the fatty acid and the carbon corresponding atoms at the alpha and beta position. We are getting the methane group that is typical for the fatty acid. This also tells us that the saturated fatty acid is primely the ones that are synthesized and produced in our body. At least when it comes to the fatty acid synthesis in the cytosol. Finishing touches: To finish up the fatty acid synthesis cycle and start the next one we must relocate the extended chain of the fatty acid to the ketoacyl-ACP synthase domain. ACP would be free and able to bind the next malonyl-CoA. Once this has happened the recharging of Acyl- carrier protein can take place and the next round can occur. Instead of the acetate we already have a four-carbon atom group and the next malonyl-CoA used would add two more carbon atoms. At the end it will look something like this: each time the malonyl- CoA is used and added the acetate group after the decarboxylation to the existing fatty acid sidechain (or if we start synthesis denovo just to the acetate group). Each time we are adding these carbon atoms until the palmitate or palmitic acid is produced. Regulations of fatty acid synthesis: The fatty acid synthesis is tightly regulated by the Acetyl-CoA carboxylase. There are three major types of regulation: (1) Allosteric regulation: Citrate will ensure the feed-forward regulation through allosteric modifications. Citrate availability in the cytosol for the fatty acid synthesis suggest that there is plenty of energy in the cell. Thus, citrate is also the inhibitor for the glycolysis Palmitoyl-CoA as an end-product ensure negative feedback regulation through similar allosteric mechanisms. That is important to remember that this kind of modifications are short term regulation and a quick response to the changes of the available various products in the cell or substrates. (2) Covalent modification: 17  Aurora Killi 31.08.21 Related to the hormonal regulations that occur inside the cell. Epinephrine and glucagon: Once energy is needed for the cell glucagon and epinephrin is released. They reduce the synthesis of fatty acid. This is through the phosphorylation and inactivation of the acetyl-CoA carboxylase. Acetyl- CoA carboxylase is inactivated when it is phosphorylated, once dephosphorylation occurs the enzyme regains its active form. Once it is dephosphorylated it forms filaments. (3) Reciprocal regulation by malonyl-CoA: The formation of malonyl-CoA stops the breakdown of the fatty acids meaning it interrupts the 𝛽-oxidation. More precisely it interrupts the transport of fatty acids into the matrix of mitochondria by the acyl- carnitine/carnitine transferase-1. In case of a lot of available energy the malonyl-CoA will inhibit fatty acid transport into the matrix. It will also allow further breakdown glucose through the glycolysis since we cannot store as much glucose as we can store different fats. Palmitate can be lengthened to longer-chain fatty acids: In the human body we can also form longer fatty acid than the palmitate. This process is slightly different since it uses the elongation systems that are found in the endoplasmic reticulum and the matrix of mitochondria. The use of CoA is prevalent instead of the Acyl carrier protein. The palmitate can be elongated to stearate and longer saturated fatty acids (not common for human metabolism). There is also a possibility to desaturate these saturated fatty acids by the enzyme Acyl-CoA desaturase and> - oleate is in this case unsaturated f - formed. Humans do have the desaturases for the double bond formation at C4, C5, C6 and C9 atoms, but not to the further ones. This means that polyunsaturated fatty acid as the omega-3 and omega-5 families we are not able to form. Omega-3 and omega-6 can be formed in the plants. Linoleate and alpha-linolenate are necessary for mammals, including humans and we need to take up these fatty acids with the food. These polyunsaturated fatty acids ensured the membrane fluidity and especially in plants and bacteria they will reduce the temperature at which the membrane remains its properties. At different temperatures the saturated and unsaturated fatty acids are soluble. This varies a lot based on the bonds one fatty acid have. Synthesis of fat (TAGs) and phospholipids Animal and plants store fat for the fuel and the plants store these TAG in seeds, nuts, and fruits. The typical human being weights around 70 kg in which 15 kg are fat. This is energy would be sufficient for 12 weeks. If we compare it to the glycogen storage in the muscles and liver, it will last 12 hours. Phospholipids are used for the cell membrane formation. Both the phospholipids and the TAGs contain one glycerol backbone and 2-3 fatty acids attached to them. Phospholipids in the cell are primarily used for growth. New organelle and cell formation TAGs (fats) are used for the energy storage The start of synthesis of TAGs and phospholipids: 18  Aurora Killi 31.08.21 Firstly, we have to synthesis the backbone glycerol-3-phsophate. There are two mechanisms that can happen: Most of the glycerol-3-phosphate comes from the dihydroxyacetone phosphate that is created in the glycolysis. This is done by the enzyme glycerol-3- phosphate dehydrogenase Another option is that there is possible to phosphorylate the glycerol and form glycerol-3-phosphate using glycerol kinase. This enzyme is only found in the liver and kidney, and it is a minor pathway in comparison to the dihydroxyacetone phosphate. Synthesis of phosphatidic acid: Once the glycerol-3-phosphate is formed it is a need of addition of 2 fatty acids to it, no matter if we are forming TAGs or phospholipids, this part is the same. To add these fatty acids to the backbone they should be activated first. Acyl-CoA synthase is the enzyme that is used and ATP energy, we will activate the fatty acid which can be added to the glycerol backbone. Acyl transferase is the enzyme that adds this fatty acid. By the addition of Acyl-CoA we create phosphatidic acid also called diacylglycerol-3- phosphate. Phosphatidic acid is the precursor that starts up and at the same time finalized the TAGs and phospholipid synthesis. Phosphatidic modification Phosphatidic acid can be modified to form phospholipids or TAGs. Once phosphatidic acid is formed, then in case if there is extra energy that we want to store, the enzyme phosphatidic acid phosphatase (lipin) ensures dephosphorylation of phosphatidic acid. By removing the 3-phosphate from the phosphatidic acid, 1,2-diacylglycerol is formed. That allows the enzyme acyl transferase to add one more fatty acid to the third carbon on the glycerol backbone and finally form the triacylglycerol. Regulation of TAG synthesis by insulin Regulation of the TAG synthesis is dependent on insulin, as insulin is the hormone that suggest that there is an energy rich state in the body. Normally, insulin will result into breakdown of dietary carbohydrates into glucose, dietary proteins into amino acids we are going to form acetyl-CoA which we can use for the fatty acid synthesis and once we have free fatty acids, we can join them and form TAGs. If there is a lack of insulin due to diabetes, we are not able to use the dietary carbohydrates and proteins to synthesize fatty acids, and thus two will happen: 1. The lack of insulin will result in the opposite process even though substrates for energy such as carbohydrates are available. Still, the body will go into a state of energy breakdown and lipolysis will occur. Lipolysis will release TAG Oxidation a lot of free fatty acid for oxidation. > ↳ - B-Ox 2. Fatty acids will be converted into ketone bodies if citric acid cycle intermediate concentration (oxaloacetate) is low. Oxaloacetate is fueled into the gluconeogenesis because of the body´s ides of lack of energy due to lack of insulin. If the patient is not aware of diabetes, there can be weight loss experience due to the breakdown of the TAG reserves. 19  Aurora Killi 31.08.21 TAG cycle The role of the triacylglycerol cycle is not yet fully understood amongst scientists. The cycle explains the breakdown of triacylglycerols through lipolysis in the adipose tissue which will release glycerol and fatty acids. At the same time, re-esterification of fatty acids into triacylglycerols, or re-esterification in the liver and correspondingly these fats are brought back to the adipose tissue through lipoproteins. Various lipoproteins like VLDL will deliver these triacylglycerides back and by the help of lipoprotein lipase release fatty acids for reformation of triacylglycerols. 75% of free fatty acids released by lipolysis are re-esterified to form TAGs, rather than be used for fuel under all metabolic conditions (even in case of starvation). Therefore, we can think of this cycle as a futile cycle as we do not understand the function of it. One of the ideas that have been brought up is that somehow the availability of TAGs in the blood could be representing the energy that would be needed in case of urgent flight or fight response. Another question would be where the glycerol-3-phosphate come from in the adipose tissue. The glycerol kinase that makes the glycerol 3-phosphate out of glycerol is not present in the adipose tissue. Glyceroneogenesis Glycerol 3-phospohate can be formed through the glyceroneogenesis. The formation of the glycerol 3- phosphate in the adipocytes is performed with the glyceroneogenesis. During lipolysis, which is stimulated by glucagon and epinephrine, the glycolysis itself in the adipocytes will be inhibited. For this reason, the intermediates of the glycolysis cannot be used for the synthesis of glycerol 3-phosphate and the re- esterification in the triacylglycerol cycle. Thus, adipocytes use an alternative way of glycerol 3-phosphate formation which basically is a type of gluconeogenesis or a part of it, because the sequence of reactions that take place up to the dihydroxyacetone phosphate which further on is used for the glycerol 3- phosphate formation is identical to the gluconeogenesis but in the adipocytes. Thus, the substrates for the glyceroneogenesis would be pyruvate, but pyruvate could be derived from alanine, glutamine, or any substance from the citric acid cycle. In this way, adipocytes ensure that there is enough of the glycerol 3- phosohate stored that can perform this re-esterification and 75%of the fatty acid re-esterification in the triacylglycerides. Thiazolidinediones Facilitation of glyceroneogenesis activity is used in treatment of diabetes type 2. More specifically, the drugs thiazolidinediones are used to upregulate the synthesis of phosphoenolpyruvate carboxykinase which is one of the steps of glyceroneogenesis in the adipocytes. Upregulation of glycerol 3-phosphate will bind more of the fatty acids and form more triacylglycerol’s. How does this happen for diabetic patients? It reduces availability of the free fatty acids found in the blood and otherwise are disturbing and interfering with glucose utilization in the muscle tissue which thus promote the insulin resistance that is typical syndrome in case of type 2 diabetes. Common patterns for synthesis of phospholipids: No matter what phospholipid that is synthesized, it follows a few common patterns: 1. Synthesis of the backbone molecule (glycerol or sphingosine) 2. Attachment of fatty acid(s) to the backbone through an ester or amide linkage 20  Aurora Killi 31.08.21 3. Addition of a hydrophilic head group to the backbone through a phosphodiester linkage 4. In some cases: alteration or exchange of the head group to yield the final phospholipid product Phospholipid biosynthesis Place of action: Every cell, primarily on the surface of the smooth ER and inner mitochondrial membrane Every cell has a need to build up the cell membrane and the membrane of various organelles. The transport of phospholipids to other cellular locations is not fully understand. Step 1: Phosphatidic acid The phospholipid biosynthesis begins with phosphatidic acid (diacylglycerol) in most cases. The reaction between alcohol group of the diacylglycerol molecule and the phosphoric acid would not be necessary if phosphatidic acid is already available. A condensation reaction with elimination of water molecule between the phosphorous group and the head group would be necessary to form the phosphodiester bond that is glycerophospholipids. Step 2: attaching phospholipid head group The attachment of the phospholipid head group requires activation by cytidine diphosphate. This can be done in two ways: 1. Diacylglycerol becomes activated with CDP and thus the head group can perform a nucleophilic attack while simultaneously releasing CMP (cytidine monophosphate), forming glycerophospholipid. 2. The head group becomes activated/charged up by the CDP, and thus the 1,2-Diacylglycerol has a free alcohol group. This alcohol group is the one that performs a nucleophilic attack on the head group bounded to the cytidine diphosphate (CDP), releasing CMP. In either of the cases, we get to the same final product, which is the glycerophospholipid. Example: Phospholipid synthesis in E. coli In E. coli the basic phospholipid synthesis can happen in two main pathways: 1. CDP-Diacylglycerol formation into phosphatidylserine by attachment of serine group and further on decarboxylated to phosphatidylethanolamine. 2. CDP-Diacylglycerol is bounded to the head group glycerol 3-phosphate, forming phosphatidylglycerol-3-phosphate. The glycerol 3-phosphate would in this case replace the CMP and form a polar head group. Hydrolysis by the enzyme PG-3-phosphate phosphatase hydrolyses phosphatidylglycerol 3- phosphate, removing the phosphor group to form phosphatidylglycerol. By adding another phospholipid to the phosphatidylglycerol we get the final product called cardiolipin. Further modification to cardiolipin can be activated by replacement of the glycerol head group with another phospholipid 21  Aurora Killi 31.08.21 Phosphatidylserine synthesis in mammals The synthesis of Phosphatidylserine in mammals does not take place. For this reason, we use another bypass reaction compared to in E. coli. In mammal’s phosphatidylserine can be created from: Phosphatidylethanolamine Phosphatidylcholine These processes are catalyzed by specific catalyzes that we will not go into detail about. Summary of phospholipid biosynthesis pathways in eukaryotes Phospholipids are not only important in cell membranes, but they also ensure signal transduction and anti- inflammatory properties, and they have a role in endocytosis, phagocytosis, and micropinocytosis. Catabolic Anabolic Summary of lipid anabolism: Fatty acid synthesis and storage are essential components of body energy homeostasis Fatty acid synthesis takes place in the cytosol; its committed step (and rate limiting) is the reaction catalyzed by acetyl-CoA carboxylase Elongation of the fatty acid chain (up to 16C → palmitic acid) is carried out by the dimeric fatty acid synthesis, which possesses 7 enzyme activities; both acetyl-CoA carboxylase and fatty acid synthase are subject to a complex regulation The citrate facilitates the transfer of two-carbon units from the mitochondria to cytoplasm for use in fatty acid synthesis The reducing power for fatty acid synthesis in form of NADPH + H + is supplied by the pentose phosphate pathway and by the malic enzyme The essential unsaturated fatty acids are linoleic and linolenic acid; linoleic acid is converted to arachidonic acid, which in turn serves as the precursor of prostaglandins The synthesis of triacylglycerides and phospholipids starts after the formation of phosphatidic acid Further synthesis of triacylglycerides involves dephosphorylation and acylation and is associated with energy storage promoted by insulin Glyceroneogenesis is adipocytes is required to ensure the TAG cycle; activation of glyceroneogenesis is used in the treatment of diabetes type II Further synthesis of phospholipids involves the addition of the polar head group and is associated with growth processes in cells/body promoted by insulin 22