Fatty acid synthesis and degradation PDF

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First year metabolic tutorial essay on fatty acid synthesis and degradation with reference to human diseases...

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Compare the pathways of fatty acid synthesis and degradation, indicating how reducing equivalents are transferred to the oxidising agents during degradation. How is reciprocal regulation of the two pathways achieved? Under what conditions are the products of degradation channelled into ketone body synthesis?

Fatty acids have four major physiological roles in the body: 1. They are used to make up biological membranes. (They are amphipathic molecules) 2. They can be covalently bonded to proteins during modification, which targets the protein to specific membrane locations. 3. They are high in energy, so act as a fuel. (Stored in adipose tissues as triacylglycerols) 4. Derivatives of fatty acids, such as hormones and intracellular messenger are used for cell communication.

FATTY ACID SYNTHESIS Fatty acids are synthesised in the cytoplasm of the cell from acetyl-CoA, by fatty acid synthases. A sequence of condensation, reduction, dehydration and then reduction again repeats to create the long fatty acid chains. The glycolytic pathway provides the acetyl-CoA, as well as the glycerol molecules needed to produce triglycerides. (Glycerol is derived from dihydroxyacetone phosphate (DHAP) in glycolysis) Glycolysis occurs in the cytoplasm, with the main aim of generating pyruvate from glucose. The pyruvate made in this pathway is then transported into the mitochondria, where pyruvate dehydrogenase catalyses the conversion of pyruvate to acetyl-CoA. This irreversible step is significant, in that it means mammals are able to synthesise fats from carbohydrates, but not carbohydrates from fats! Acetyl-CoA cannot move across the inner mitochondrial membrane, so instead it combines with oxaloacetate in the first stage of the TCA cycle to produce citrate. Citrate is pumped out of the mitochondria by the tricarboxylate transport system. Once in the cytosol, citrate is broken back down into oxaloacetate and the acetyl-CoA needed for fatty acid synthesis by an enzyme called ATP-citrate lyase. This reaction requires energy from ATP, so it is important that the oxaloacetate is recycled back to the mitochondria to allow the TCA cycle to continue. In the cytosol, malate dehydrogenase reduces oxaloacetate to malate using NADH. Malic enzyme and NADP+ then oxidise and decarboxylate malate to pyruvate, which the inner mitochondrial membrane is permeable to. Pyruvate diffuses back into the mitochondria with the other pyruvate molecules produced in glycolysis, and is reused in the TCA cycle. (See Figure.1) The NADH and NADPH made will be used as reducing agents later in fatty acid synthesis.

Figure.1

In the first step of fatty acid synthesis, acetyl-CoA is carboxylated to its ‘charged’ form – malonyl-CoA. This reaction is energetically unfavourable, so must be ATP driven. The biotin enzyme acetyl-CoA carboxylase catalyses the reaction. This irreversible, committed step, is the rate limiting reaction in the pathway, so the activity of this enzyme if highly regulated. (I will discuss this later with regards to the reciprocal regulation of the synthetic and degradative pathways) The biotin prosthetic group of acetyl-CoA carboxylase is covalently attached to the ϵ amino group of a lysine residue by its carboxyl group. A carboxybiotin intermediate is formed using energy from the hydrolysis of a molecule of ATP. The intermediate then has an activated CO2 group which is transferred to acetyl CoA to form malonyl CoA. Biotin-enzyme + ATP + HCO3- ⇌ CO2-biotin-enzyme + ADP + Pi CO2-biotin-enzyme + acetyl-CoA → malonyl-CoA + biotin-enzyme

The intermediates in fatty acid synthesis are linked to the sulfhydryl terminus of a phosphopantetheine group, attached to a serine residue of an acyl carrier protein. (ACP) ACP, also known as ‘macro CoA’, is a single polypeptide made up of 77 residues that acts as a giant prosthetic group.

The enzyme fatty acid synthase catalyses the elongation cycle in fatty acid synthesis from acetyl CoA, malonyl CoA and NADPH. The synthesis of long-chain fatty acids starts with the formation of acetyl ACP and malonyl ACP, whereby Acetyl CoA and Malonyl CoA are attached to sulfhydryl groups on ACP. (Catalysed by acetyl transacylase and malonyl transacylase) Acetyl CoA + ACP ⇌ Acetyl ACP + CoA Malonyl CoA + ACP ⇌ Malonyl ACP + CoA Malonyl transacylase is a highly specific enzyme, but acetyl transacylase can also transfer acyl groups other than the acetyl unit, although this occurs much more slowly - For example, in order to produce fatty acids with an odd number of carbon atoms, synthesis must start with propionyl ACP, formed from propionyl CoA by acetyl transacylase. Acetyl ACP and malonyl ACP undergo a condensation reaction to produce acetoacetyl ACP. (C4-β-ketoacyl ACP) This reaction is catalysed by the acyl-malonyl ACP condensing enzyme. Acetyl ACP (2C) + malonyl ACP (3C) → acetoacetyl ACP (4C) + ACP + CO2 During this reaction, a four-carbon unit is made from a 2C and 3C unit, and carbon dioxide is released. It is not possible to combine two acetyl ACP molecules because it is too energetically unfavourable, however using malonyl ACP makes the equilibrium favourable because it can be decarboxylated to give a substantial decrease in free energy. Since ATP is used to carboxylate acetyl CoA to malonyl CoA, ATP effectively drives the condensation reaction despite the fact that it doesn’t participate directly.

In the next step of fatty acid synthesis, acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP. This molecule is then dehydrated to form crotonyl ACP, which is a trans-Δ2-enoyl ACP. The final step in the cycle reduces crotonyl ACP to butyryl ACP. NADPH is again the reducant. Enoyl ACP reductase catalyses this step. Acetoacetyl ACP + NADPH + H+ ⇌ D-3-hydroxybutyryl ACP + NADP+ D-3-hydroxybutyryl ACP ⇌ crotonyl ACP + H2O Crotonyl ACP + NADPH + H+ → butyryl ACP+ NADP+

In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to make C6-β-ketoacyl ACP. (Chain is lengthened by two carbons each time) Reduction, dehydration, and then a second reduction convert C6-β-ketoacyl ACP into C6-acyl ACP, ready for the third round of elongation. The cycle continues, adding two carbon each time until C16acyl ACP is made, which is then hydrolysed to give the fatty acid palmitate and ACP. (Fatty acid length controlled by thioesterase – elongation by the fatty acid synthase complex stops on the formation of palmitate (C16))

Figure.2 Intermediates formed in the first round of synthesis

Overall, the equation for the formation of palmitate is: 8Acetyl CoA + 7ATP + 14NADPH + 6H+ → Palmitate + 14NADP+ + 8CoA + 6H2O + 7ADP + 7Pi

In eukaryotes, fatty acids longer than the 16 carbon atom palmitate can be formed by elongation reactions catalysed by enzymes on the cytosolic face of the endoplasmic reticulum membrane. In these reactions two-carbon units are added to the carboxyl ends of fatty acyl substrates whether they are saturated or unsaturated. This condensation is driven by the decarboxylation of malonyl CoA, as described previously. The endoplasmic reticulum has membrane bound enzymes to generate unsaturated fatty acids, by introducing double bonds into long acyl CoA chains. Such desaturation reactions require NADH and O2, and are carried out in a flavoprotein complex with a cytochrome and a non-heme iron protein. As a result, a variety of different fatty acids can be produced by combinations of elongation and desaturation reactions.

FATTY ACID DEGRADATION Degradation of fatty acids (Also known as lipolysis) converts an aliphatic compound into a set of activated acetyl units. This acetyl CoA can then be taken into the TCA cycle, and metabolised to release energy. In order for the body to use fatty acids as fuel, they must first be processed. To start, the lipids are mobilised – triglycerides must be degraded to their constituent fatty acids and glycerol. The glycerol formed by lipolysis is absorbed by the liver and phosphorylated to dihydroxyacetone phosphate. An isomerisation reaction changes this to D-glyceraldehyde-3phosphate (an intermediate in glycolytic and gluconeogenic pathways), so that it can be used in the respiratory chain to release energy, or converted back to glucose for storage. The fatty acids are released from the adipose tissue for transport to the energy demanding tissues. Since fatty acids are not soluble in the blood plasma, they must be transported bound to serum albumin. Unsaturated fatty acids must undergo an additional step before degradation, since their double bonds may prevent them from becoming substrates for the βoxidation pathway. Cis bonds must be converted into trans bonds. An isomerase handles oddnumbered double bonds, and even-numbered double bonds are dealt with by the reductase and isomerase together. Fatty acids with an odd number of carbon atoms give propionyl CoA and acetyl CoA after their final round of degradation, compared to the more common two molecules of acetyl CoA from even numbered fatty acids. Propionyl CoA is an activated three-carbon unit, and is able to enter the TCA cycle after conversion to succinyl CoA. Before they are oxidised, fatty acids are linked to coenzyme A. ATP drives the formation of a thioester linkage between the fatty acid carboxyl group and the sulfhydryl group of CoA. This occurs outside the mitochondria, and is catalysed by acyl CoA synthetase. RCOO- + CoA + ATP + H2O → RCO-CoA + AMP + Pi + 2H+ The activated long chain fatty acids are then transported across the inner mitochondrial membrane by conjugation with carnitine. The acyl group of CoA’s sulfur atom is transferred to the hydroxyl group of carnitine to form acyl carnitine. (Catalysed by carnitine acyltransferase I, which is membrane bound) A transferase moves acyl carnitine across the inner mitochondrial membrane, so that the acyl group can be returned to CoA on the matrix side of the membrane. Carnitine acyltransferase II catalyses this reverse reaction of what occurred in the cytosol. This is another important regulation control point which I will discuss later. There are four steps in each round of β-oxidation – oxidation, hydration, a second oxidation and then cleavage (Thiolysis). Flavin adenine dinucleotide (FAD) conducts the oxidation, NAD+ the second oxidation, and the thiolysis is carried out by CoA. The structures of NAD+ ad FAD are shown in Figure 3. The aromatic ring of NAD+ is able to accept a proton and two electrons to form NADH. In doing so it acts as an oxidising agent. FAD can pick up two electrons and two protons to give FADH2.

Figure.3

In the first stage of the degradation pathway, the activated fatty acid (Linked to CoA) undergoes oxidation. The acyl CoA is oxidised by an acyl CoA dehydrogenase to give an enoyl CoA with a trans double bond. (Double bond between the α and β carbons – C-2 and C3) The enzyme bound FAD is reduced to FADH2. Acyl CoA + E-FAD → trans-Δ2-enoyl CoA + E-FADH2 FAD is used as the electron acceptor here instead of NAD+ because the ΔG value of this reaction is too small to drive the reduction of NAD+. The FADH2 prosthetic group of reduced acyl CoA dehydrogenase transfers its electrons to the electron-transferring flavoprotein (ETF). The ETF will then donate electrons to the iron-sulfur protein, ubiquinone reductase. Ubiquinone is reduced to ubiquinol, which can in turn deliver its high-potential electrons to the second proton pumping site of the respiratory chain. This mean that 1.5 molecules of ATP are produced for every FADH2 formed in this dehydrogenation step. (See Figure 4)

Figure.4

The hydration of the new trans-Δ2 double bond follows this, and is stereospecific – the L isomer of 3-hydroxyacyl CoA is produced. This is catalysed by enoyl CoA hydratase. Trans-Δ2-Enoyl CoA + H2O ⇌ L-3-hydroxyacyl CoA After that, a second oxidation reaction, this time involving NAD+, converts the hydroxyl group at C-3 into a keto group. This is catalysed by L-3-hydroxyacyl CoA dehydrogenase. (Specific for the L isomer of the substrate) L-3-hydroxyacyl CoA + NAD+ ⇌ 3-ketoacyl CoA + NADH + H+

The final step is thiolytic cleavage the 3-ketoacyl CoA by the thiol group of a second molecule of CoA, which gives acetyl CoA and an acyl CoA two carbons shorter in length. (The previous reactions served to oxidise a methylene group at C-3 to a keto group) This is catalysed by β-ketothiolase. 3-Ketoacyl CoA + HS-CoA ⇌ acetyl CoA + acyl CoA (n carbons)

(n-2 carbons)

The acetyl CoA produced will proceed to the TCA cycle, and the acyl CoA will undergo further cycles of oxidation until they are completely degraded.

So, the equation for one cycle of the degradation of palmitate is: C16-acyl CoA + FAD + NAD+ + H2O + CoA → C14-acyl CoA + FADH2 + NADH + acetyl CoA + H+

The complete oxidation of palmitate will require 7 reaction cycles. In the final cycle, two molecules of acetyl CoA will be left from the thiolysis of C4-ketoacyl CoA. For the complete oxidation of palmitate the stoichiometry is as follows: Palmitoyl CoA + 7FAD + 7NAD+ + 7H2O + 7CoA → 8 acetyl CoA + 7FADH2 + 7NADH + + 7H+

Since approximately 2.5 ATP are made from oxidation of NADH in the respiratory chain, 1.5 ATP are made from each FADH2, the oxidation of each acetyl CoA by the TCA cycle gives 10 molecules of ATP, and the equivalent of 2 ATP are consumed to activate palmitate, the complete oxidation of palmitate yields 106 ATP molecules.

COMPARISON BETWEEN THE SYNTHETIC AND DEGRADATIVE PATHWAYS The fatty acid synthetic and degradative pathways cannot be considered the reverse of each other, since there are several important differences between the reactions they involve. Firstly, in synthesis the reductant is NADPH, whereas NAD+ and FAD are the oxidants in fatty acid degradation. - This demonstrates the fact that NADPH is consumed in biosynthetic reactions, and NADH is generated in energy-yielding reactions. Secondly, the intermediates of synthesis are bound to the sulfhydryl groups of an acyl carrier protein (ACP), but in degradation the intermediates are covalently attached to the sulfhydryl groups of coenzyme A. The location of the two pathways is also different: synthesis occurs in the cytosol, whereas degradation primarily occurs in the mitochondrial matrix. Fatty acids can also be oxidised in the peroxisomes, where a flavoprotein dehydrogenase transfers electrons to O2 to yield H2O2 instead of generating FADH2, as occurs in mitochondrial β oxidation. The enzyme catalase then converts the hydrogen peroxide that was made into water and oxygen. Different isoforms of the mitochondrial enzymes carry out the subsequent identical degradation steps. In higher organisms, the enzymes of fatty acid synthesis are joined in fatty acid synthase - a single polypeptide chain. However, the enzymes involved in degradation do not appear to be associated.

Finally, in the synthetic pathway, D-3-hydroxybutyryl ACP is made instead of the L isomer seen in degradation.

Figure 22.2 on pg 602

RECIPROCAL REGULATION OF THE TWO PATHWAYS The first highly regulated, committed step in fatty acid synthesis involves acetyl-CoA carboxylase. This step is irreversible and the kinetically slowest step in the pathway, so it can be used to control the rate of synthesis. Allosteric factors (Local regulation) can be used to control the activity of this enzyme, for example increased concentrations of citrate will stimulate faster catalysis. (Increased citrate concentrations means there will be more acetyl CoA in the cytosol for the enzyme to decarboxylate) Conversely, the presence long chain fatty acids inhibits the action of the decarboxylase. This is useful for the cell, since if there are too few long fatty acids in the cell then more should be made, and if there are too many, the synthesis pathway should be slowed and degradation should take precedence to oxidise them for energy. Hormonal factors are also involved – high levels of insulin will cause increased activity of acetyl-CoA carboxylase, so that fatty acid synthesis occurs more rapidly. Insulin is released when the blood sugar level is too high, so the fatty acid synthesis pathway can act to convert glucose molecule into lipids for storage. This helps to prevent hypoglycaemia. On the other hand, glucagon is released when blood sugar levels drop (Hyperglycaemia), and inhibits acetyl-CoA carboxylase. The degradative pathway can then become dominant to metabolise fats and release energy in times of starvation. The global regulation of acetyl-CoA carboxylase is carried out by reversible phosphorylation. Phosphorylating the enzyme inhibits it, whereas dephosphorylation activates it. An AMPdependent protein kinase (AMPK) modifies a serine residue to convert the carboxylase into its inactive form. Protein phosphatase 2A removes the phosphoryl group of the inhibited enzyme to activate it. Therefore the rate of fatty acid synthesis depends on the proportion of carboxylase in the active dephosphorylated state – controlled by the relative rates of the opposing AMPK and protein phosphatase 2A. (See Figure.5)

Figure.5

AMPK is activated by AMP and inhibited by ATP, so acts to gauge the fuel availability in the cell. As a result, the carboxylase is activated when the ATP levels are high in the cell, allowing energy to be stored in fatty acids for later use. Phosphatase 2A is inhibited when protein kinase A phosphorylates it. Epinephrine and glucagon activate protein kinase A, so these hormones switch off fatty acid synthesis by keeping the carboxylase in its inactive phosphorylated state. Insulin dephosphorylates phosphatase 2A, so it can dephosphorylate acetyl CoA carboxylase, and activate the fatty acid synthesis pathway.

Acetyl CoA will accumulate when oxidative phosphorylation is slowed down, or when glycolysis speeds up. Fatty acid synthesis is faster when more acetyl CoA is available, so factors increasing the rate of glycolysis or decreasing the activity of the electron transport chain will cause more fatty acids to be made. Conversely, if little glucose is available, less glycolysis makes less acetyl CoA, so more fatty acids will be degraded to help make up for the energy demands of the cell. The lipase enzymes in adipose tissue are activated when cells are treated with the hormones epinephrine, norepinephrine, glucagon, and adrenocorticotropic hormone. These chemicals bind to receptors 7TM on the cell to activate adenyl cyclase to produce cyclic AMP. Increased concentrations of cAMP in the cell stimulates protein kinase A to phosphorylate, and thereby activate, lipases in the cytoplasm. However, insulin has an inhibitory effect of lipolysis. Malonyl CoA inhibits carnitine acyltransferase I, thereby preventing the fatty acyl CoA molecules from moving across the inner mitochondrial membrane. If the fatty acyl CoAs cannot get into the mitochondrial matrix, then the degradative enzymes cannot break up the fatty acids. This is an effective way to slow degradation when energy is plentiful in the cell. Finally, NADH inhibits 3-hydroxyacyl CoA dehydrogenase, and acetyl CoA inhibits thiolase in the oxidation pathway, so when the cell has plenty of energy fatty acids will by synthesised as energy stores instead of broken down to release more energy.

KETONE BODY SYNTHESIS The brain cannot metabolise fatty acids for energy, since they are unable to cross the bloodbrain barrier. During starvation, prolonged intense exercise or in diabetes, the brain adapts to use acetoacetate, a derivative of acetyl CoA. Ketogenesis occurs when blood glucose levels are low, and glycogen has been used up in the liver. (Lack of carbohydrates available in the body) Ketone bodies are water soluble molecules produced by the liver, (A transportable form of acetyl units from fatty acid degradation) where D-3-hydroxybutyrate is oxidised to produce acetoacetate and NADH. (Figure.6)

Th ne

Figure.6

other organs. Acetoacetate is activated by the transfer of CoA from succinyl CoA, catalysed by this specific transferase. Thiolase then cleaves acetoacetyl CoA to yield two acetyl CoA molecules which...