Lipid Metabolism Biochemistry 202 PDF

BIOENERGETICS AND INTEGRATED METABOLISM BIOC 202 LIPID METABOLISM Discipline of Biochemistry (Westville Campus) 1 OXIDATION OF GLYCEROL About 95% of the biologically available energy of triacylglycerols resides in their three long ch...

BIOENERGETICS AND INTEGRATED METABOLISM BIOC 202 LIPID METABOLISM Discipline of Biochemistry (Westville Campus) 1 OXIDATION OF GLYCEROL About 95% of the biologically available energy of triacylglycerols resides in their three long chain fatty acids. Only 5% of the total energy is contributed by the glycerol moiety (Figure5). 2 2. USE OF FATTY ACIDS FOR ENERGY PRODUCTION: β -OXIDATION OF FATTY ACIDS: The fatty acids that are taken up by the cells can be used for energy production by the.13- oxidative pathway. This system is found in mitochondria of eukaryotes and in prokaryotes. In seeds the β -oxidation system is found exclusively in glyoxysomes and peroxisomes. 2.1. ACTIVATION OF FATTY ACIDS Fatty acids must first be activated in a reaction with ATP and c enzyme A before they will react with the β-oxidative enzymes. Fatty acyl-CoA synthetases, exist specific for short chain, (2-4C), medium chain (4-10C) or long chain fatty acids, (10-20C). The synthetase specific for long-chain fatty acids is an outer mitochondrial membrane bound enzyme. The short and medium chain synthetase enzymes are found primarily in the mitochondrial matrix. Note that only one molecule of ATP is required to activate a fatty acid regardless of the number of carbon atoms in its hydrocarbon chain. 3 ROLE OF CARNITINE lN FATTY ACID OXIDATION: CARNITINE CARRIES LONG CHAIN ACYL CoA ACROSS THE MITOCHONDRIAL MEMBRANE Since the long chain fatty acyl CoA's are formed outside the mitochondrion while the oxidizing machinery is inside the inner membrane, which is impermeable to Co-A, the cell has a major logistical problem. An efficient shuttle system overcomes this problem by using Carnitine as the carrier of acyl groups across the membrane. This process functions primarily in the mitochondrial transport of fatty acyl-CoA1s with chain lengths of C12-C20. By contrast, shorter fatty acids can cross the inner mitochondrial membrane directly and become activated to their Co-A derivatives in the matrix compartment: that is, their oxidation is carnitine independent. 4 2.2. α, β HYDROGENATION OF ACYL -CoA The first reaction is catalysed by a fatty acyl-CoA dehydrogenase which dehydrogenates between the α(2) and β(3) carbons i.e. removal of H atoms to give a trans △2 enoyl-CoA (Trans αβ unsaturated Acyl-CoA). Three acyl-CoA dehydrogenases are found in the matrix of mitochondria and are specific for short, medium, or long chain fatty acyl-CoA’s, respectively. Clinical Correlations - Deficiencies of the various medium-chain acyl-CoA dehydrogenases (MCAD). This usually manifests itself within first 2 years of life after a fasting/staivatlon period of 12 hours or more. Typical symptoms include vomiting, lethargy and frequently coma. Many cases previously diagnosed loosely as cot death or sudden death infants syndrome, were in fact due to MCAD deficiency. 2.3. HYDRATION OF α,β-UNSATURATED ACYL-CoA’s 5 2.4. OXIDATION OF β-HYDROXYACYL-CoA 2.5. THIOLYSIS OR THIOLYTlC CLEAVAGE The broadly specific enzyme thiolase carries out a thiolytic cleavage of the β- Ketoacyl CoA. The carbon-carbon single bond that joins methylene (-CH2-) groups in fatty acids is relatively stable. The β-oxidation sequence represents a solution to the problem of breaking these bonds. Reactions 2-4 of β-oxidation have the effect of creating a much less stable C-C bond. Now the αC or C2 is bonded to 2 carbonyl carbons (C=0). The Ketone function on C3 or β-C makes it a good point for nucleophilic attack by SH of coenzyme A. The acidity of C2 or α-C makes the terminal CH2-CO-S-CoA a good leaving group facilitating breakage of the α-β bond. The enzyme protein has a reactive SH group on a cysteinyl residue that is involved in the following series of reactions: This process (Reactions 2-5) is repeated over and over until the entire fatty acid molecule is degraded to acetyl-CoA which then enters the Krebs cycle directly to be metabolised. 6 Figure 7: β-oxidation of fatty acids. 7 Summary - Energy Yield from fatty acid β-oxidation Mitochondrial oxidation of fatty acids takes place in three stages (Figure 8). In the first stage - β-OXIDATION, the fatty acids undergo oxidative removal of successive 2- carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acy1 chain. In the second stage of fatty acid oxidation, the acetyl residues of ACETYL-CoA are oxidised to CO2 via the citric acid cycle which also takes place in the mitochondrial matrix. The first two stages of fatty acid oxidation produce the reduced electron carriers NADH + FADH2 which in the third stage donate electrons to the MITOCHONDRIAL RESPIRATORY CHAIN through which the electrons are carried oxygen. Coupled to this flow of electrons is the phosphorylation of ADP to ATP. Figure 8: Stages of fatty acid oxidation. 8 We can now write a balanced equation for the overall degradation of Palmitoyl-CoA (16 carbon FA) to 8 mol. of acetyl-CoA. Palmitoyl-CoA + 7CoA-SH + 7FAD + 7 NAD++ 7H2O 8 acetyl-CoA + 7FADH2 + 7NADH + H+ Each of the reduced flavoproteins (FAD) can yield 2 ATP and each NADH can yield 3 ATP when processed through the electron transport chain. So, the reduced nucleotides yield 7 x 5 = 35 ATP per Palmitoyl - CoA. The oxidation of each acetyl CoA through the TCA cycle yields 12 ATP so the 8 X 2C fragments from a palmitate molecule produce 96 ATP. However, 1 ATP equivalent was used to activate pa1mitate to palmitoyl CoA. Therefore, each palmitic acid entering the cell can yield 130 ATP mol (35 + 96) - 1 by complete oxidation to CO2 and H20. From this you can calculate the ATP yield per carbon oxidised to CO2 as 130/16 or about 8.1. NOTE: The same value for glucose is 6.0 (36 ATP's formed per 6 carbons oxidised). This quantitatively supports the statement that the energy yield from fat oxidation is higher than that from carbohydrate. Oxidation of odd-numbered fatty acids Fatty acids with an odd number of carbon atoms are found in significant amounts in the lipids of many plants and some marine organisms. Long chain odd-numbered fatty acids are oxidised by the same pathway as the even-carbon acids, beginning at the carboxyl end of the chain. However, the substrate for the last pass or cycle through the oxidation sequence is a fatty acyl- CoA in which the fatty acid has 5 carbon atoms and NOT 4 carbon Butyryl-CoA (figure 9). When this 5-carbon fatty acyl-CoA is oxidised, the substrate cleaved by the thiolase enzyme is an acetoacetyl-CoA homologue with 5C. The cleavage products are acetyl-CoA (2C) and propionyl-CoA(3C). The acetyl-CoA is, of course, fully oxidized to CO2 + H20 via the citric acid cycle. But Propionyl-CoA must be further catabolised before its carbon atoms can enter the citric acid cycle for complete oxidation to CO2 + H20. Propionyl-CoA takes a rather unusual enzymatic pathway involving 3 enzymes. Clinical correlation: Inability to metabolise propionyl-CoA due to a defective mutase activity or lack of vitamin Bl2 derived cofactor, causes methylmalonyl-CoA concentrations to increase, this is then converted in the body to methylmalonic acid causing a severe acidosis (lowering of blood pH) and damage to central nervous system. This rare condition, called methylmalonic acidaemia is usually fatal in early life. In cases where synthesis of the cofactor from vitamin B12 is deficient, the disease can be successfully treated by administering large doses of vitamin Bl2. 9 Figure 9: Pathway for oxidation of odd-numbered fatty acid carbon chains. 10 3. OXIDATION OF UNSATURATED FATIY ACIDS REQUIRES ADDITIONAL ENZYMES Many fatty acids in natural lipids are unsaturated, that is, they contain one or more double bonds. The many unsaturated fatty acids in the diet are readily available for the production of energy by the human body. However, in several respects, the structures encountered in these dietary acids may differ from those required by the specificity of the enzymes in the β-oxidation pathway. Since the double bonds in these unsaturated fatty acids are in the cis configuration, and in addition the double bonds are not always in the correct position, they cannot be simply acted on by enoyl-CoA hydratase which acts only on trans compounds. Two additional enzymes, enoyl-CoA isomerase and 2,4 dienoyl-CoA reductase must come into play for these fatty acids to be oxidised. 11 LIPID METABOLISM BIOCHEM 202 Ms Seipati Mokhosi (PhD Candidate) Room #: F3 – 04 - 004 mailto: [email protected] LIPIDS – what you will know::: 1. Classification and functions  LIPIDS – what you will know::: Classification and overview Digestion, absorption and mobilisation Metabolism: B-oxidation, biosynthesis, regulation and fate Lipid Storage Diseases LIPIDS  LIPIDS – classification, biochemical function What are we actually referring to? Some common names: fat, fatty acids, lipids. Are all fats bad? What about cholesterol?  LIPID METABOLISM – biosynthesis, breakdown and regulation What is the fate of lipids in our bodies? Does it all just end up in the tummy or the bum?  LIPIDS  Lipids are organic molecules which are soluble in organic non-polar solvents and are sparingly soluble in aqueous solutions  Commonly associated with protection from loss of moisture; such in birds’ feathers, coating leaves, and in humans – camphor cream, oil moisturisers, Vaseline etc.; the hump on Camel’s back is largely a deposition of fat, which provides energy and water during long-term starvation  In good or bad diet, as source of energy (and grease ) – such as in margarine, Upper Café chips / Green Bean samoosas /Amagwinya, Canola/Sunflower / Olive / Coconut/ Avocado / Fish / Black Castor Oil etc.  LIPIDS  Why we can’t go Herbex-Hlasela Amafutha on all of our body fat?  Is it even possible to attack or eliminate all of our body fat?  It is an essential biomolecule as with carbohydrates and proteins – and found in all living organisms including prokaryotes and eukaryotes CLASSES OF LIPIDS  What lipids? Nomenclature used in lipid metabolism LIPIDS – CLASSIFICATION 5 general groupings – unlike with other biomolecules, lipids are classified on basis of their physical properties i.e. solubility in organic solvents  Fatty acids and their derivatives: saturated, unsaturated and eicosanoids,  Esters of fatty acids and glycerol: triglycerides, phosphoglycerides  Lipids without glycerols: sphingolipids  Sterol derivatives: cholesterol, steroid hormones and vitamin D  Terpene and isoprenoid derivatives  LIPID FUNCTIONS Wide variety of roles in biological systems – relates to the chemical and physical properties:  Fatty acids and their derivatives (especially triacylglycerols) are high energy storage molecules; 6X more energy/weight of glycogen – hydrophobic, anhydrous nature  Layers of lipids form good insulators, and limited metabolic activity of adipose tissue – reduced heat exchange  Membranes are generally composed of fatty acid derivatives – hydrophobic barrier  Lipids can be used as signaling molecules, such as in prostaglandins and steroids; and as enzyme cofactors.  Precursors of several hormones and some fat-soluble vitamins: A, D, E, K  WHAT? WHERE? WHEN? DEFINITIONS. THE VARIOUS UNDER WHAT LOCALITIES, ORGANS, CONDITIONS? COMPONENTS ETC. LIPID DIGESTION WHY? HOW? SOME OF THE PROCESSES AND REASONS. THE ACTUAL PROCESS INCLUDING STRUCTURES  LIPID DIGESTION AND ABSORPTION o 90% of normal diet lipids are TAGs and some digestion starts in stomach by some lipases and is oxidised into CO2 and H2O o They are water insoluble and digestion occurs at lipid/water interface – not very efficient in stomach due to aggregation o Facilitation through bile salts which include: ACTION OF BILE SALTS o A bile salt possesses both hydrophobic and hydrophilic surface, thus allowing for oil- water interface o Emulsification through detergent action of TAGs – results in forming smaller units termed micelles o This allows for water-soluble pancreatic lipases to digest, and also facilitates absorption into intestinal mucosa ACTION OF BILE SALTS ABSORPTION o Stabilisation of micelles is crucial before uptake and absorption - due to presence of bile salts. o Thus mixed micelles would comprise monoacylglycerols, lysophosphoglycerols, long chain FAs PLUS bile salts o Absorption is achieved by dissociation of micelles, where the salts remain in the lumen while the digested lipids are taken up by simple transfer from micellar environment into aqueous one  Lipases hydrolyse the ester linkages of TAG at positions 1 and 3 – to yield 2 monoacyl glycerol and (2X) fatty acids ACTION OF LIPASES CLINICAL IMPLICATION Pale, bulky, non- smelly stool “Floaties”, difficult to flush Oil droplets etc.  Steatorrhoea Biliary duct blockage / liver diseases??? Low bile production/secretion – what happens? Severe reduced absorption (malabsorption) of dietary fats and fat- soluble vitamins (vitamins A, D, E, and K) from the digestive tract into the bloodstream PROBLEM: Sufficient levels of fats, cholesterol, and vitamins are necessary for normal growth, development, and maintenance of the body's cells and tissues, particularly nerve cells  o TAGs are hydrolyzed in periphery by lipoprotein lipase. 1) glycerols are transported back transport to liver and kidney, 2) FA uptake bound to serum albumin for ß-oxidation in TRANSPORTATION peripheral tissues o Transportation is a dynamic process, dependant on varying status of the body: to tissue in use, from storage in adipose tissue (if not absorbed) to maintain metabolism  TRANSPORTION o Once small enough, re-absorption into the epithelial cells via re-esterification of fatty acids back into TAGs o These combine with lipoproteins released by the intestines to produce chylomicrons (in ER and Golgi apparatus) ~75nm diameter, which act as serum transport particles for TAGs o Released into lymph or blood stream and are either taken up by the liver or delivered to adipose tissue o TAGs made by liver are packaged into VLDLs and released into blood LIPOPROTEINS o Chylomicrons – least dense form; VLDL – very low density lipoproteins; LDL – low density lipoproteins; HDL – high density lipoproteins (aka cholesterol scavengers; associated with reduced risk of heart diseases o Apolipoproteins refer to proteins in lipoproteins. Lipoprotein lipase – enzyme located in capillary walls; removes fatty acids from chylomicrons and VLDLs o CLINICAL IMPLICATION: what is abetalipoproteinemia??? GREAT DIGESTION! DIGESTION OF LIPIDS  BIOENERGETICS AND INTEGRATED METABOLISM LIPID METABOLISM Discipline of Biochemistry (Westville Campus) 1 Lipids play roles both in energy metabolism and in aspects of biological structure and function. The great bulk of lipid in most organisms is in the form of triacylglycerols (triglycerides). A mammal may contain 5-25% of its body weight as lipid and 90% of this in the form of triacylglycerols. Most of this fat, is stored in adipose tissue. Triacylglycerols are derived from 2 primary sources: a) the diet-digestion, absorption, and transport of fats to adipose tissue b) mobilization of fat stored in adipocytes. 1) DIGESTION OF DIETARY FATS An adult man ingests about 60-100g of fat per day. Triacylglycerides constitute more than 90% of the dietary fat. The rest is made up of phospholipids, cholesterol, cholesterol esters, and free fatty acids. · Lipids are soluble in organic solvents. Conversely, they are sparingly or not at all soluble in aqueous solutions. This poor water solubility presents problems for digestion because the substrates are not easily accessib1e to the digestive enzymes in the aqueous phase. In addition, even if ingested lipids are hydrolysed into simple constituents, the products tend to aggregate to larger complexes that make poor contact with the cell surface and therefore are not easily absorbed. These problems are overcome by i) increases in the interracial area between the aqueous and lipid phase and ii) ‘solubilization’ of the hydrolysis products with detergents. Essential to the normal digestion and intestinal absorption of lipids are bile salts, detergent substances secreted from the gallbladder. These are salts of bile acids such as cholic and chenodeoxycholic acid which are synthesized in hepatocytes (liver cells) from cholesterol. These bile acids are composed of 24 carbon atoms containing 2 or 3 hydroxyl groups. They have a side chain that ends in a carboxyl group that is ionized at pH 7.0. The carboxyl group of the primary bile acids is often conjugated via an amide bond to either glycine (NH2-CH- COOH) or taurine (NH2-CH2-CH2-S0 3H) to form glycocholic or taurocholic acid respectively and constitute the forms that are secreted into bile. 2 A bile salt molecule contains a hydrophobic surface and a hydrophilic surface. This characteristic allows bile salts to dissolve at an oil-water interface, with the hydrophobic surface in contact with a polar phase and the hydrophilic surface in contact with the aqueous phase. This detergent action emulsifies triacylglycerol to form particles approximately l µm in diameter yielding micelles, which allow digestive attack by water-soluble enzymes e.g. pancreatic lipase and facilitates the absorption of lipid through the intestinal mucosa. Figure 1: Action of bile salts in emulsifying fats in the intestine. The digestion of triacylglycerol occurs mainly in the duodenum of the small intestine into which flow both bile and the secretion of the pancreas. Pancreatic lipase hydrolyses ester links in the 1 and 3 positions of the triacylglycerol to yield the 2 monoacylglycerol and fatty acids: 3 The products of triacylglycerol digestion, mainly monoacylglycerol and long chain fatty acids must form a stable interaction with water before uptake and absorption into the epithelium of the intestine can occur. This stabilization is achieved by the action of the bile salts present in bile salts micelles. These bile salt micelles incorporate monoacylglycerols, lysophosphoglycerols and long chain fatty acids to form 'mixed' micelles. The mixed micelle formation conveys, the non-polar lipid molecules through the aqueous contents of the intestinal lumen, to the epithelial cell surface. Here the micelle dissociates to produce locally high concentrations of monoacylglycerols, lysophosphoglycerols and fatty acids which are absorbed while the bile salts remain in the lumen. The digested lipids are taken up into the absorptive cells by simple transfer from the favourable micellar environment into an apparently unfavourable aqueous one. Clinical implication The importance of bile salts is emphasized by the diminished fat absorption and the steatorrhoea which results from an abnormally low concentration of bile salts in the lumen of the small intestine. There are a number of causes of this low luminal bile salt concentration, the most obvious is biliary obstruction in which the bile duct is blocked, but it can also occur when the liver is diseased, thus causing decreased bile production. Consequently, patients with reduced bile salt concentration are maintained on a low-fat diet. A number of other non-specifiable lipids of importance for example, vitamins A, D, E and Kare also absorbed from mixed bile salt micelles. Consequently, patients suffering from inadequate concentrations of bile salts in the intestine are prone to deficiencies of these vitamins. In such patients, these vitamins can be supplemented for by being administered by injection. Figure 2: Diagrammatic representation of triacylglycerol digestion and absorption (not to scale). 4 After absorption into the intestinal epithelial cells fatty acids are re-esterified to form triacylglycerols. Subsequently, in the small intestine, triacylglycerol, phospholipids, cholesterol and a specific protein called apolipoprotein combine to form spherical chylomicrons with a diameter > 75nm. Chylomicrons contain approximately 85% triacylglycerol, 8% phospholipid, 2% cholesterol, 3% cholesterol ester and 2% protein. They arise solely in the intestine and contain triacylglycerol of dietary origin only. The chylomicrons are released into the blood stream and are subsequently taken up by the liver and by adipose tissue. The association of lipids with proteins not only solubilizes lipids but also aids in their transport into cells. Triacylglycerols are transported to tissues either in chylomicrons or in VLDL. At the cell surface the triacylglycerols are cleaved by lipoprotein lipase to give glycerol and free fatty acids. After absorption into the cell, the glycerol and fatty acids derived from lipoprotein lipase action can be either catabolized to generate energy or, in adipose cells, used to resynthesize triacylglycerols Figure 3: Diagrammatic representation of chylomicron synthesis in an intestinal absorptive cell. Not to scale 5  2) MOBILISATION OF FAT STORED IN ADIPOCYTES The mobilisation of fatty acids from triglyceride deposits in adipose tissue is deeply influenced by hormones. The consumption of meals rich in starch ensures a high concentration of glucose in the blood and hence a high concentration of the hormone insulin. If the concentration of insulin is greater than the hormones, glucagon, and adrenalin, then glycolysis, glycogenesis and the synthesis of fatty acids and other lipids are stimulated. At the same time, the oxidation of fatty acids (β-oxidation) and gluconeogenesis are inhibited. Subsequently under these conditions the mobilization of fats would be inhibited, rather the excessive glucose would be used 1st as energy source. By contrast, under stress conditions, the concentration of blood glucose and [insulin] drops dramatically, whilst the concentration of glucagon and adrenalin become highly elevated. This type of hormonal scenario occurs in starvation, diabetes, trauma, and toxic conditions. The net result would be that glycolysis and glycogenesis would be inhibited due to lack of substrate glucose. Similarly, lipid (TAG) and fatty acid biosynthesis would be inhibited. Since stress conditions would involve an active new source of energy, fatty acid catabolism and gluconeogenesis would now be promoted. Consequently, when blood glucose is low and insulin is low while glucagon and adrenalin concentrations are high, the mobilisation of fats from adipose tissue is promoted. (Figure 4) Figure 4: Mobilization of free fatty acids from adipose tissue by cyclic AMP- mediated cascade system. 6  LIPIDS – what you will know::: Classification Metabolism: B-oxidation, and overview biosynthesis, regulation and fate Digestion, absorption and mobilisation Lipid Storage Diseases OVERVIEW: FA β-OXIDATION FUNDAMENTAL QUESTIONS TO ASK: o What? What is beta-oxidation? The actual process? o Where? The various locations involved, which inform the process? o When / Why? When does the body kick into this process? o How? The actual reactions? you need to know the structures; as well as regulation FA β-OXIDATION (FA BREAKDOWN PATHWAY) o The actual breakdown of free fatty acids takes place in the mitochondria o 1) Activation of fatty acids o 2) Transport (for long chain fatty acids) o 3) Fatty acid ß-oxidation reactions ACTIVATION OF FATTY ACIDS  ACTIVATION OF FATTY ACIDS This system located in the mitochondria in eukaryotes and prokaryotes, while in seeds it is found in glyoxysomes and peroxisomes Before undergoing the β-oxidation reactions, FAs require activation This is achieved by conjugation to coenzyme A (CoA) Acyl-CoA synthetase catalyzes the formation of a thioester bond (high-energy) between a fatty acid and CoA-SH ACTIVATION OF FATTY ACIDS o The reaction is reversible – an ATP molecule is used to supply the energy for the process and AMP (not ADP) is the product o The enzyme for long-chain fatty acid (> 10carbon) is an outer mitochondrial membrane-bound enzyme o For short-to medium-chain FAs, the enzyme is found in the mitochondria matrix TRANSPORT (FOR LONG CHAIN FATTY ACIDS) o In a multi-step process, entry is through a separate small molecule, carnitine o The cytosolic enzyme carnitine acyltransferase I reversibly exchanges the thioester bond to CoA-SH in the acyl-CoA for an ester bond to carnitine o Carnitine acyltransferase I acts as a major control point for fatty acid breakdown – due to location implication. This enzyme is inhibited by malonyl-CoA which is substrate for FA biosynthesis  TRANSPORT o Acyl-carnitine - ligand for a specific transporter, the carnitine/acyl-carnitine antiport o Once inside the mitochondrion, carnitine acyl-transferase II reforms the Acyl-CoA o This is a reversible reaction, no energy is added or lost – carnitine merely acts as a means of transport TRANSPORT FATTY ACID β-OXIDATION REACTIONS o Why? The chemistry involves the β-carbon of the acyl-CoA substrate o Through a series of 4 reactions – resulting in release of the 2- carbon acetyl-CoA, and an acyl-CoA molecule two carbons shorter o This shorter acyl-CoA then re-enters pathway – and so on and so on, always releasing 2-carbon acetyl CoA and a 2 carbons shorter acyl-CoA molecule FA β-OXIDATION REACTIONS o OVERALL REACTION: o Hydrolysis of the bond between the α and β carbons in order to release a 2-carbon unit o A 3-enzyme pathway must: 1) first activate the β- carbon, 2) then cleavage of the bond between the methylene α-carbon and the ketone on the oxidized β- carbon DEHYDROGENATION o Facilitated by enzyme acyl-CoA dehydrogenase o Oxidation of the α-β bond single bond to a trans double bond via FAD+ reduction to give rise to FADH2 (equating to 2 ATPs) o Acyl-CoA dehydrogenase is not located in the mitochondrial inner membrane, but instead uses a short chain of soluble electron carriers to donate electrons  HYDRATION o Facilicated by enzyme enoyl CoA hydratase o Hydration reaction where a water molecule is added across the double bond formed by acyl-CoA dehydrogenase o Results in formation of a hydroxyl group on the β- carbon of the acyl chain DEHYDROGENATION o Facilitated by enzyme β-hydroxyl CoA dehydrogenase o Oxidation of the β-hydroxyl to a ketone using NAD+ as a cofactor, resulting in NADH (equivalent to 3 ATPs) o Formation of β-ketoacyl-CoA, containing a ketone on the carbon β to the thioester carbon  THIOLYSIS o Facilitated by enzyme thiolase a.k.a. Acyl- CoA:acetyltransferase o Cleavage of the β-ketoacyl-CoA thus releasing an acyl-CoA 2 carbons shorter and acetyl-CoA - leads to formation a thioester bond between the β-ketone carbon and an additional coenzyme A. o Hydrolysis of the bond between the α and β carbons of the original acyl-CoA. CLINICAL IMPLICATION o Deficiencies of various medium-chain fatty acyl CoA dehydrogenase (MCAD) o In the first 2 years of a child – following starvation of 12 hours or more o Symptoms include: lethargy, vomiting and frequently a coma. Misdiagnosis of cot deaths (SIDS) has occurred – later found to be due to MCAD-related. β-OXIDATION PATHWAY REACTIONS oLet’s compare bioenergetics of glucose and FA (Stearate: 18-carbon) SATURATED VS UNSATURATED VS ODD-NUMBERD  ODD-NUMBERED FAS o Found in some marine animals, many herbivores, microorganisms, and in plants (LOOK UP SOME NAMES/EXAMPLES???) o The final β-oxidation spiral results in the production of the 3-carbon propionyl-CoA (pg 15) o Methylmalonyl-CoA mutase is one of two known vitamin B12-dependent enzymes in humans. Most vitamin B12- dependent enzymes catalyze carbon-transfer reactions ODD-NUMBERED FAS  CLINICAL IMPLICATION o In the case of methylmalonyl-CoA mutase, the carbonyl of the thioester is moved from the branched α-carbon to the methyl carbon o Accumulation of methylmalonyl-CoA due to this, or mutase defect results in its metabolism into methylmalonic acid can also result in methylmalonic acidaemia: severe acidosis and damage to CNS o Vitamin B12 (cobalamin) is used only in animals and some microorganisms – so it can be problematic for strict vegetarians (may develop pernicious anaemia)  UNSATURATED FAS o Oxidation of unsaturated carbons requires additional reactions (in case of odd-numbered double bonds o The double bond needs to be moved to the 2-position, a reaction catalyzed by enoyl-CoA isomerase  UNSATURATED FAS o In case of even-numbered double bonds, normal β-oxidation will eventually result in the presence of a Δ2-Δ4 conjugated intermediate o BUT the enoyl-CoA hydratase cannot use the conjugated compound as a substrate OTHER FAS o Short-chain FAs of < 10 carbons enter the mitochondrial matrix without carnitine shuttle o Long-chain FAs of > 22 carbons cannot enter mitochondria and are metabolised in the peroxisome – peroxisomal β- oxidation is similar to mitochondrial with few exceptions o These do not require activation nor carnitine shuttle for entry into this organelle