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Homeostasis Panel Revision Guide

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Homeostasis Panel Revision Guide

  1. 1. Homeostasis Panel Lecture Notes James Carter April 2012
  2. 2. 1 Contents Histology of the Kidney............................................................................................................................. 2 Cellular Metabolism................................................................................................................................ 14 Central Glycolytic Pathways................................................................................................................ 14 Common Themes: A Glossary of Terms, Reactions, Concepts and Themes....................................... 22 Glycolysis............................................................................................................................................. 26 Link Reaction....................................................................................................................................... 39 Citric Acid Cycle, Kreb’s Cycle or Tricarboxylic Acid Cycle .................................................................. 41 Electron Transport Chain .................................................................................................................... 44 Gluconeogenesis................................................................................................................................. 56 Pentose Phosphate Pathway .............................................................................................................. 60 Lipid Metabolism................................................................................................................................. 62 Cholesterol Synthesis.......................................................................................................................... 76 Glycogen Metabolism – Breakdown and Synthesis............................................................................ 77 Whole Body Metabolism 1.................................................................................................................. 84 Whole Body Metabolism 2.................................................................................................................. 89 Whole Body Metabolism 3.................................................................................................................. 97 Amino Acid Metabolism.......................................................................................................................... 99
  3. 3. 2 Histology of the Kidney Introduction The urinary system consists of 2 kidneys, 2 ureters, a bladder and a urethra with the overall purpose of the system being to maintain homeostasis by regulating blood pressure, blood composition and fluid volume. This is achieved by filtration of blood in glomeruli, reabsorption of nutrients and substances from filtrate and secretion or excretion of waste products. The kidneys produce urine and ensure it is the correct composition to maintain body homeostasis. The bladder is the region in which urine is stored prior to excretion. The two are connected by the pelvicalyceal and ureters that transfer urine from the kidney to the bladder and excretion is via the urethra. Shown below is a very simplistic way of looking at kidney function so that the histology may be applied. Blood comes into the kidney. Water and small molecules are removed from it, but not large substances. As this filtrate moves through the kidney, those things that are needed by the body are reabsorbed at the correct level back into the blood, while others can be secreted into from the blood into the filtrate. Regulatory systems continually sense body requirements and adjust the level of reabsorption and secretion so that homeostasis is maintained. These mechanisms and the hormones involved will be learnt within this panel. The waste products are excreted in urine via bladder.
  4. 4. 3 Histologically, we are looking for the adaptations that enable filtration and allow reabsorption and secretion to occur so that specific substances can be excreted or retained dependent on the body’s requirements Gross Structure of the Kidney Kidneys are bean-shaped organs located high in the posterior abdominal wall underneath the peritoneum. As their major function is filtration of blood they receive 20-25% of cardiac output. The kidney is surrounded by a connective tissue capsule (sheath) that has fibroblasts in the outer layer and myofibroblasts (fibroblasts having contractile properties) in the inner layer. The opening in the sheath through which the renal blood vessels enter and exit is known as the hilum. Grossly, when a kidney is cut open we can distinguish 2 regions:  The cortex, an outer reddish brown part  The medulla, an inner part, lighter in colour The Cortex is formed from the cortical labyrinths and medullary rays with associated connective tissue and an extensive vascular network. The cortical labyrinth is composed of renal corpuscles and cross-sections of proximal and distal convoluted tubules and the macula densa region of distal tubules. The medullary rays are a continuation of medullary tissue extending into the cortex. They are composed of collecting tubules, parts of the proximal tubules, ascending thick limbs of Henle’s loop and blood vessels. The Medulla is composed of 8-18 renal pyramids which are bordered by cortical columns. The renal pyramids consist of collecting tubules and Henle’s loop, accompanied by a special capillary network called the vasa recta. Together these form the countercurrent exchange system that regulates the
  5. 5. 4 concentration of urine. The apex of the renal pyramid is the renal papilla, whose perforated tip is the area cribosa where the large collecting ducts deliver urine to the minor calyx. The base is the boundary of the medulla and the cortex. Caps of cortical tissue extend peripherally around the lateral portion of the pyramid and between them forming the renal columns. Despite containing the same components as the cortical tissue, they are part of the medulla. Kidney Vasculature This is unusual as contains a capillary bed within the arteriole, in addition to the normal capillary bed. Each kidney receives its blood from the renal artery, which then divides to form interlobar branches that span out into the kidney. These go between the pyramids in the cortex and then run between the medulla and cortex where they are known as arcuate arteries. Branching from the arcuate arteries are interlobular arteries that go through the cortex to the capsule. Branching from the interlobular arteries are the afferent arterioles, one to each glomerulus. The glomerular capillaries reunite to form an efferent arteriole (this is unusual as usually capillaries are expected to form a venule). A second capillary bed is formed from the efferent arterioles. From the efferent arterioles to the cortical glomeruli, a peritubular capillary network is formed between the cortical tubules. While the arterioles from the juxtamedullary glomerulus form a capillary network (the vasa recta) in the medulla alongside the tubular system.
  6. 6. 5 The venous flow is a reverse of the arterial. The peritubular cortical capillaries drain into the interlobular veins, into the arcuate veins then to the interlobar veins and so in to the renal vein. Upon injection of vasculature dye, the following features may be observed:
  7. 7. 6 Nephron The nephron is the functional unit of the kidney, with each kidney containing approximately 2 million nephrons (figures vary from 1-4 million dependent on source). The nephron is responsible for the production of urine. Dependent on the location of the renal corpuscle in the kidney, nephrons are termed cortical or juxtamedullary nephrons. General Layout of a Nephron The nephron starts at the renal corpuscle, which is the initial site of filtration. The filtrate passes to the descending thick segment comprising the proximal convoluted tubule and the proximal straight tubule, followed by the thin segment of the loop of Henle and into the ascending thick segment consisting of the ascending straight tubules and the distal convoluted tubule. The distal convoluted tubule connects to the collecting tubule. Description of each Region of a Nephron Renal Corpuscle Renal corpuscles are formed from a glomerulus, Bowman’s space and Bowman’s capsule.
  8. 8. 7 The Glomerulus is the capillary network, whose function is the initial filtration of blood. It is composed of tufts of fenestrated (large pores 60-90nm in diameter) capillaries supplied by the afferent glomerular arteriole and drained by efferent glomerular arteriole. The pores occupy 20% of the area of the endothelium and allow passage of small molecules. Bowman’s capsule is formed from 2 membranes. It is lined with epithelial cells which are supported by a basement membrane and reticular fibres, separated by Bowman’s space. Its function is to produce glomerular filtrate. The outer (parietal) membrane is formed from simple squamous cells, whereas the inner (visceral) layer has specialised epithelial cells, known as podocytes. Unlike normal epithelial cells which sit on a basement membrane the podocytes have numerous long, tentacle-like cytoplasmic extensions, called pedicles which wrap around the glomerulus. These attach to the basement membrane, leaving filtration slits between them to allow fluid to more easily move from the glomerulus to Bowman’s space. Bowman’s space (or urinary space) is an epithelial-lined gap between the coated glomerular capillary network and Bowman’s capsule. This receives the fluid filtered through the capillary wall and visceral layer.
  9. 9. 8 The Filtration Membrane is formed from the fusion of the endothelial basement membrane and the inner epithelial cell basement membrane. This thicker membrane ensures that only substances less than 1kDa in the plasma can enter Bowman’s capsule, such as water, ions, glucose and amino acids. Plasma proteins are retained in the plasma. Regulation of the molecules that can pass through the filtration membrane is probably modified by the fact that it is a charged membrane. Mesangium is a connective tissue found in the extracellular matrix, which surrounds mesangial cells and supports the capillary network. The intraglomerular mesangial cells act as ‘macrophage-like’ cells removing any particulate matter that would otherwise clog the filtration slits. These cells secrete the mesangial matrix and some vasoactive factors. They have contractile properties, which may enable them to modify the diameter of the glomerular capillaries in response to vasoactive substances. Some of these vasoactive substances they secrete and some may come from the lacis cells (extraglomerular mesangial cells), which form part of the juxtaglomerular apparatus. Tubular and Collecting Systems The renal tubule from Bowman’s capsule to the collecting duct can be up to 55mm long in humans. It is lined by a single layer of epithelial cells. The class of these differs as move along the tubule dependent on the function of the particular region. The tubular segments of the nephrons are named according to the course they take (convoluted or straight, ascending or descending), location, (proximal or distal) and wall thickness (thick or thin). The tubules have a similar basic structure but the abundance of particular organelles within the cells varies dependent on the function of the particular region.
  10. 10. 9 Proximal Convoluted Tubule The function of a proximal convoluted tubule is in resorption from glomerular filtrate; they are a major site of resorption. The structure originates from Bowman’s capsule and follows a tortuous route to enter the medullary ray, where it becomes the straight proximal tubule. The cells are simple cuboid epithelium and form an acidophilic cytoplasm, due to numerous mitochondria and modifications to increase surface area for absorption. The apical cell surface has numerous microvilli (forming an apical surface brush border), which have canaliculi at their base to increase up-take of macromolecules into the cell, as do the presence of pinocytic vesicles. Basolateral membranes form interdigitations with adjacent cells and contain many Na+ /K+ -ATPase pumps for active Na+ transport. The proximal convoluted tubule absorbs all the glucose and amino acids and some of water and sodium chloride. Henle’s Loops are formed by a thick descending limb (proximal straight tubule), a thin descending limb, a thin ascending limb and a thick ascending limb. This creates a gradient of hypertonicity that influences the concentration of the urine as it follows through the collecting duct. There are differences in the permeability of the different areas to water, with the descending limb being highly permeable to water and the ascending limb only moderately permeable to water, but readily permeable to salts. These differences are due to the epithelium. The thin limb has a wide lumen (despite narrow diameter) as it is formed of squamous epithelial cells whose nuclei protrude only slightly into the lumen. The length depends on the region in which the nephrons are found; cortical nephrons have a short thin segment while that of the juxtamedullary nephrons is longer. Thin segments have flat squamous epithelium with little cytoplasmic Brush border Basal interdigitations Mitochondria
  11. 11. 10 specialisation. Thick segments have simple cuboid epithelial cells which are similar to either proximal or distal convoluted tubule. Distal Tubules consist of the ascending thick limb of Henle’s loop, the macula densa and the distal convoluted tubule. Their function is acid-base balance and controlling urine concentration. The ascending thick limbs are lined by cuboid epithelial cell which interdigitate with each other to increase the surface area. They contain numerous mitochondria to provide the energy for the ion pumps. The thick ascending limb cells have chloride pumps. The distal convoluted tubule is short with wide lumina. The cells (simple cuboid epithelium) are narrow, with clear pale cytoplasm and few microvilli. There is basal indigitations but these are not as extensive as the thick ascending limb as there is less resorption in this region meaning that the surface area does not need to be as great. There are plenty of mitochondria as the main purpose of the region is active resorption of Na+ . These mitochondria provide the energy for ion pumps. Collecting Tubules The distal convoluted tubule leads into the Collecting Tubule. Its function is to conduct fluid and control the final concentration of urine.
  12. 12. 11 Its cells are of 2 types: the majority are known as clear cells, which are cuboid or flat with few organelles; other are intercalated dark cells which are rich in organelles, especially mitochondria, and contain a microvillar system. Juxtaglomerular apparatus The juxtaglomerular apparatus comprises of the macula densa, juxtaglomerular cells and extraglomerular mesangial cells (lacis cells). It acts as a sensory, regulator monitoring ion levels and regulates blood pressure through secretion of renin. The cells are taller and thinner so that the nuclei appear closely packed together. Fewer mitochondria are present in the cells and the basement membrane on which they rest is relatively thin. The macula densa is a specialised region of densely-packed epithelial cell nuclei along the distal convoluted tubule, adjacent to the afferent arteriole at the vascular pole of the corpuscle. Compared to the other cells of the distal convoluted tubule the cells are taller, thinner and have more prominent nuclei situated towards the luminal surface. There is a very thin basement membrane between the macula densa and the juxtaglomerular cells. There is no sodium pump activity. The cells of the macula densa are thought to be sensitive to the concentration of sodium and chloride ions in the fluid within the distal convoluted tubules. The juxtaglomerular cells are modified smooth muscle cells located in the wall of the afferent arteriole immediately before it enters the capsule. Their cytoplasm is full of secretory granules. A major secretion is renin, which is involved in the regulation of blood pressure and activated by a signal from the macula densa.
  13. 13. 12 The extraglomerular mesangial cells also known as lacis cells continue from the mesangium of the glomerulus between the afferent and efferent arterioles. Their function is not fully understood but they may secrete factors that can cause contraction of the intraglomerular mesangial cells. Collecting ducts Collecting ducts collect urine from tubules. They are lined with epithelium, initially identical to tubules, but gradually become regular, straight-sided columnar cells, and finally cuboid. There are two types of cell: principal, which have few organelles, basolateral infoldings and are able to reabsorb Na+ and secrete K+; and intercalated, which have many organelles and are able to secrete H+ and reabsorb HCO3. These cells can alter their permeability to water in response to antidiuretic hormone (ADH), but otherwise have no specialisations. Renal Interstitium The connective tissue of the parenchyma is called the interstitial tissue, which surrounds the nephrons, ducts and blood and lymphatic vessels. In the cortex this occupies a small volume, whilst in the medulla it is more predominant. In the cortex, fibroblast cells are present for synthesis and secretion of collagens and glycosaminoglycans (GAGs), whilst in the medulla, myofibroblast cells are present, orientated to the long axis of tubular structure; myofibroblasts may compress the tubules they surround. These have more muscle-like properties, such as the ability to contract. Interstitial cells are secretory. Rest of Urinary System The collecting ducts merge to form a large duct (ducts of Bellini). The urine is then delivered to the minor and then to the major calyx. The urine then leaves the kidney via the ureter to be taken to the bladder and then excreted via the urethra. The extra-renal tubes are lined by transitional epithelia (see the Foundation Studies epithelia lecture) as these can change from cuboid to squamous to accommodate the emptying and filling of the bladder. The subepithelial connective tissue (lamina propria and submucosa) is composed of fibroelastic tissue. Under the connective tissue is a muscular layer (muscularis), which is composed of an inner longitudinal and outer circular layer of smooth muscle. In the lower end of the ureter and in the bladder there is a third outermost longitudinal layer of muscle. However the layers of muscle often blend in to each other, making it difficult to distinguish individual layers. The muscularis is surrounded by a fibroelastic adventitia. The histology of the urethra is gender specific, as the male urethra also functions as part of the male reproductive system.
  14. 14. 13 Bladder The bladder stores urine until it is convenient to excrete and is also lined by transitional epithelium which is folded in the bladder’s relaxed state. Transitional Epithelia Transitional epithelium (uroepithelium) only occurs in conducting passages of the urinary system and allows distension. The epithelium is stratified and can vary from 3-6 layers dependent on the distension of the bladder; 6 layers when empty and 3 layers when full. The cells are of the basal layer are compact and cuboidal, while those in the middle are more columnar, with their nuclei at right angles to the basement membrane. The surface cells have a domed appearance. These surface cells have the ability to maintain impermeability of the epithelium to urine even when fully stretched. The impermeability also ensures water cannot move into the bladder if the urine is hypertonic. The dome- shape gives the surface a scalloped appearance. There are large numbers of junctional complexes between the cells to ensure cohesion between them preventing urine leaking out between the cells. These histological features ensure urine can be stored without damaging the cells, as urine can be toxic. Urethra The urethra is the final conducting part of the urinary tract. It is a fibromuscular tube which is gender specific in its size, structure and function. In males it serves as the terminal duct for the urinary and genital system and has three distinct segments which are defined by structure: transitional, then pseudostratified columnar, then squamous. In females, the urethra is a shorter tube which consists initially of transitional epithelium, changing to stratified squamous.
  15. 15. 14 Cellular Metabolism Central Glycolytic Pathways Note that this is an overview lecture and future lectures explain the pathways in more detail/clarity. Releasing Energy from Food ATP Adenosine Triphosphate
  16. 16. 15 Energy is required to form ATP. ATP is then broken down again to release energy to power metabolic processes in the body’s cells. ATP itself has a fairly short half-life and therefore breaks down very easily, meaning that most of the energy in the body is not stored. Cells make and break lots of ATP continuously, but it is not used as long term storage. Glycolysis Glycolysis occurs in the cytoplasm of cells and requires glucose, which is turned into two pyruvate molecules. Glycolysis does not require oxygen, but the following reactions do. Link reaction The citric acid cycle takes place in the mitochondrial matrix and requires O2. The Respiratory chain occurs in the mitochondrial matrix.
  17. 17. 16 NB. Black arrows signify where something is being put into the pathway, and white arrows show where useful products are coming out. Therefore, the top portion of the diagram (above fructose-1,6 bisphosphate) requires energy, and the bottom part liberates energy. Energy is being invested in glycolysis and the molecule is therefore destabilised. This means that once the energy has been invested, it takes very little energy to liberate it. Consequently, in glycolysis where ATP has been invested into glucose to make fructose-1,6-bisphosphate, it is relatively easy to break it down into two three carbon molecules. This releases energy and NADH+H+ . Oxygen is not required and carbon dioxide is not produced. The graph overleaf shows the energy which must be invested (in 5 steps) until it can be easily broken down. The original two ATP are liberated and another two are produced, resulting in a net gain of two. Some NADH+H+ is also produced. 6C 3C 3C
  18. 18. 17 Pyruvate Oxidation: The Link Reaction 3C 2C
  19. 19. 18 Other metabolic pathways link in here. More useful product is formed (NADH + H+ ). This is a non- reversible equation. Note that two pyruvate are used from glucose so all products hereon must be doubled. Citric Acid Cycle Again, white arrows signal the useful products coming out: FADH2 (two formed) and NADH+H+ (6 of each) are also used in the electron transport chain to generate more ATP. 6C 4C 2C 6C 5C 4C 4C 4C
  20. 20. 19 Respiratory Chain From the previous reactions, 10 NADH + H+ and 2 FADH2 enter the respiratory chain. The respiratory chain produces 30/32 ATP and turns 6 O2 molecules into water. Each NADH+H+ produces around 2.5 ATP and each FADH2 produces around 1.5 ATP. Shown above are 4 electron carrier molecules (left) and ATP synthase on the right (4 potatoes and a mushroom [which rotates, the mechanical force provides power to combine ADP + Pi, much like a water wheel). Complexes 1, 3 and 4 pump protons. FADH2 deposits electrons into complex 2 rather than 1, so fewer protons are pumped as complex 1 will not pump. Complexes 1 and 2 both pass electrons to complex 3. Complex 1 will never pass electrons to complex 2. Therefore, protons build up in the intermembranal space, forming a proton motive force. Mitochondria provide a path for facilitated diffusion through ATP synthase (chemiosmosis). Oxygen drives the electron transport: it is combined with H+ and electrons to make water. → If there is no oxygen, there is no terminal electron acceptor and the reaction backs up meaning NADH+H+ and FADH2 build up. The citric acid cycle will then cease due to Chatelier’s principle. Following this, the link reaction will stop also. Mitochondrial Matrix NADH+H + FADH2 Lots of H+
  21. 21. 20 Anaerobic Respiration In anaerobic respiration, lactic acid is formed to remove pyruvate and allow the reaction to continue according to Le Chatelier’s principle. Additionally, a build-up of NADH+H+ would also stop these reactions. To prevent this, NADH+H+ is used up in the formation of lactic acid. Lactate can be turned back into pyruvate once oxygen is available; however, if lactate builds up, the reaction will stop, again due to Le Chatelier’s principle. This means that anaerobic respiration cannot take place forever. Most anaerobic respiration occurs in the muscles whilst running or exercising. Therefore, pyruvate builds up in muscles, which convert it to lactate, which is carried away to the liver in the circulation. When oxygen is present, it is converted back into pyruvate. Therefore anaerobic respiration cannot take place forever, as lactic acid will build up throughout the circulation and shut down these pathways. This is when the tissues or muscles involved shut down and cease to function properly. Fructose-1, 6-biphosphate 6C 3C 3C 3C 3C
  22. 22. 21 Chemical Equations Aerobic Respiration → ΔG = -2880Kj/mole, so the reaction is spontaneous. Anaerobic Respiration → 2 hours running can turnover 60kg of ATP. Maths Recommended energy intakes:  Men = 10MJ (2,500 Kcal); BMR = ~7.5MJ  Women = 8MJ (2,000 Kcal); BMR = ~5.4MJ. Around 40% of energy taken in is turned into ATP, whilst 60% is used in other processes, the largest of is head production (ΔH). Some Facts (Set as SDL by Dr Shore)  Assuming glucose is taken in, you turn over your body weight in ATP every day.  BMR stands for Basal Metabolic Rate, and is synonymous with Basal Energy Expenditure or BEE. BMR measurements are typically taken in a darkened room upon waking after 8 hours of sleep; 12 hours of fasting to ensure that the digestive system is inactive; and with the subject resting in a reclining position.  RMR stands for Resting Metabolic Rate, and is synonymous with Resting Energy Expenditure or REE. RMR measurements are typically taken under less restricted conditions than BMR, and do not require that the subject spend the night sleeping in the test facility prior to testing.  MET stands for Metabolic Equivalent of Task. It is a unit used to compare the working metabolic rate (the amount of oxygen used by the body during physical activity) to the resting metabolic rate. It is a way to compare the amount of exertion required for different activities.  1 calorie = 4.2 joules  Energy contents: o Fat/Lipid (RQ = ~0.7) : 1 gram = 9 calories o Protein (RQ = 0.8-0.9): 1 gram = 4 calories o Carbohydrates (RQ = 1): 1 gram = 4 calories o Alcohol (RQ = 0.667) : 1 gram = 7 calories  RQ value corresponds to a caloric value for each litre of CO2 produced.
  23. 23. 22 Common Themes: A Glossary of Terms, Reactions, Concepts and Themes Theme 1 – Equilibrium and Coupling Pathways Catabolic pathways break things down, and anabolic pathways build things up. Generally, anything with ‘lysis’ in the title is catabolic (for example, glycolysis), and anything with ‘genesis’ in the title is anabolic (gluconeogenesis, for example). Not all pathways are this simple, but in many of them it is easy to figure out what is happening. Amphibolic pathways are uncommon; they break some things down and make other things. An example of one is the tricarboxylic acid cycle (also known as the Kreb’s cycle and Citric Acid Cycle; all three names must be known). Normally catabolic and anabolic reactions are distinctly different and they are usually compartmentalised (performed in different regions of the cell). For example, lipid metabolism (β-oxidation) occurs in a different place to lipid formation (fatty acid synthesis). This is partly because the product of one pathway may be the substrate for another, so placing the two together might cause them to cycle (if there is enough energy). Equilibrium and Coupling Organisms require energy for mechanical work, active transport (or processes driven by ATP), and macromolecule synthesis (building large molecules from precursor molecules). Exergenic and Endogenic Exergenic (energy yielding) pathways are usually catabolic. Endogenic (energy requiring) pathways are usually anabolic. Amphibolic pathways will have elements of both, but the sum total will probably make them either energy yielding or requiring. Standard Free Energy Change Gibbs free energy: ΔG = ΔH – TΔS (NB. constant conditions and 1M concentration) A reaction can only occur if ΔG is negative. At equilibrium, ΔG = 0. If the ΔG of a reaction is positive, an input of free energy is required for it to occur. ΔG can be changed by varying the concentration of the reactants or the products. ΔG gives no indication of reaction rate; it is independent of the path or mechanism of the reaction. Adding up all of the ΔG values for each reaction in a pathway will give an overall ΔG. It does not matter if some reactions in a pathway have a positive ΔG; as long as the overall mechanism has a negative value of ΔG, the reaction will occur. In this way, an energy yielding reaction can be used to drive an unfavourable one: A  B + C ΔG = +30kjmol-1 B D ΔG = -40Kjmol-1 ∴ A C+D ΔG = -10Kjmol-1 (just add up the other two).
  24. 24. 23 Other Nucleoside Phosphates  Guanosine  Uridine  Cytidine They are all roughly equivalent energy carriers, but ATP is the primary one. Theme 2 - Carriers Phosphoryl-Transfer Standard free energies provide a gauge of transfer potential. An example in muscle is the PCr reaction, where creatine phosphate is used as an energy store. Creatine phosphate  creatine + phosphate  ΔG=-43.1Kjmol-1 ATP  ADP + Pi  ΔG=-30.5Kjmol-1 In muscles: creatine phosphate + ADP  ATP + creatine This releases energy due to the difference in ΔG. The produced ATP is then broken down, releasing more energy. Therefore, creatine phosphate is a phosphoryl-transfer molecule. Therefore, any carrier with a more negative ΔG than another molecule can make it. The table below provides a few examples: a molecule can make anything below it on the table.
  25. 25. 24 NAD+  NADH+H+ Learn that the nicotinamide ring is synthesised from niacin and vitamin B3, which is why vitamin B3 deficiency gives you a broad spectrum of symptoms (it interferes with electron transport in all cells). These often present as neurological symptoms, because the cells that run out of energy first are neurons, as they have few mitochondria and relatively little way of storing energy. 2C Carriers A 2C carrier is a molecule which carries 2 carbon molecules, such as coenzyme A. Note that coenzyme A itself is actually quite large. We usually shorten the names of 2C carriers, so coenzyme A is written as CoA, even when shown as part of a structural formula. Themes  ATP is an energy carrier and a phosphoryl carrier.  We also have electron carriers, for fuel oxidation: e-  carrier  O2, and biosynthesis (reduction).  2C carriers.  Many carriers are derived from vitamins. Theme 3: 6 Recurring Reactions 1. Oxidation – Reduction This is the oxidation of carbon compounds in order to provide useful energy. Many enzymes involved in these reactions end in reductase or dehydrogenase. Enzymes are shown on the formula sheet given in the exam and their names can be quite useful in order to work out what is going on. 2. Ligation Ligation is (normally) making a carbon to carbon bond and is also (normally) ATP dependent. Therefore, if ATP can be seen to be going in and a molecule with extra carbons comes out, it is usually a ligase reaction. Again, look out for ligase enzymes. 3. Isomerisation Isomerisation is where atoms or groups within a molecule are rearranged in preparation for a future reaction. If nothing is seen entering or leaving the molecule, an isomerisation reaction is taking place. The enzyme involved may include the word isomerase.
  26. 26. 25 4. Group Transfer This is the transfer of a functional group from one molecule to another. This is used all over the pathways and often requires ATP. It has many uses, especially phosphoryl-transfer (mentioned earlier on page 25). 5. Hydrolysis Normally hydrolysis will display as water going into the reaction and more products coming out from the other side. 6. Lyases This is where an atom or group is removed to leave a double bond, or adding an atom or group to a double bond. Again, the enzyme will probably have the word lyase in it. Theme 4: Pathway Regulation Enzyme concentration affects the speed of pathways. As enzymes concentration increases, so does Vmax (maximum velocity). Transcription, translation and proteolysis (breaking down proteins) all regulate enzyme concentration by balancing levels of synthesis and breakdown as required. Enzyme and substrate must be placed together in order for a reaction to proceed. In this way, a pathway can be regulated: limiting the input of substrates into a cell will stop reactions. Substrates are therefore transferred in and out of the cell (and between compartments) as required. To control the catalytic activity of an enzyme, allosteric control can be used (binding an effector molecule at the protein’s allosteric site [that is, a site other than its active site]) with an inhibitor, or a species may be bound to it, usually a phosphate group (this is known as a reversible covalent modification or phosphorylation in this case). Pathways have evolved to keep just enough ATP within a cell, but not so much that the cell will break ATP down when it does not require it. Energy charge (the ratios of ATP and ADP to ATP, ADP and AMP) and phosphorylation potential (the ratio of ATP to ADP+Pi) are used by cells to work out if they need to make any more ATP – this is a feedback loop. Many enzymes are therefore controlled by the concentrations of ATP and ADP within a cell. Whenever ATP or an ATP derived molecule (such as ADP) is involved in regulation of an enzyme (through the donation of a phosphate group, for example), the enzyme is said to be under “adenylate control”.
  27. 27. 26 Glycolysis Glycolysis means “sweet dissolution” and is the breakdown of glucose. Glycolysis is the primary source of ATP in certain cells. These cells have few or no mitochondria. Examples include erythrocytes (red blood cells), the cornea, the lens and the kidney medulla. The best example is that of red blood cells: they turn over vast amounts of glucose but cannot perform aerobic respiration. Such locations are restricted oxygen environments. Anywhere performing anaerobic respiration will be reliant on glycolysis for its ATP. Examples include skeletal muscle, the neonate (for a limited time initially), some disease states, and tissues with restricted blood flow, such as in sickle cell crisis. Essentially, if there is no oxygen or few mitochondria, the cell will be reliant on glycolysis to produce ATP. Erythrocyte – Red Blood Cells These cells have the highest level of glucose consumption. As they can only perform glycolysis, they are very inefficient and therefore have one of the lowest levels of ATP production. This ratio is because each glucose molecule only produces two ATP, so the erythrocytes have to use a lot of it. What to Learn about the Pathways You should have an understanding of what is going on in each reaction – is it, for example, a group transfer reaction, an isomerisation, a lyase reaction, etc. What is the point of the reaction? Does it make ATP or NADH+H+ ? Is it destabilising a molecule by giving it more energy? How is that step regulated, if it is regulated at all? In glycolysis, there are only three steps that are regulated and only one of those is the rate determining step. Often, the enzyme name tells you what is going on and the point of the reaction, as do the reactants and products. Regulatory Points Some reactions are reversible and some are not. Irreversible reactions are normally the points that are the regulatory points. You will need to learn what sort of regulation is occuring. The most common type of regulation is allosteric regulation. This is very quick (it happens in a matter of milliseconds) and is where a non-competitive inhibitor attaches to an enzyme to change the shape of an active site.
  28. 28. 27 The next type of modification is very common: covalent modification. The most common covalent modification is phosphorylation and it occurs in a matter of seconds. This may up-regulate or down- regulate the enzyme. Occasionally, there may be transcriptional or translational regulation, which can take several minutes or even hours. For example, transcriptional regulation may change the amount of protein produced. Hexokinase The first reaction in glycolysis is catalysed by an enzyme called hexokinase: This is a group transfer reaction (specifically phosphoryl transfer). This can be derived by the –kinase suffix of the hexokinase enzyme. This reaction requires a cofactor (something which is essential for an enzyme to work but is not used up in the reaction [it is not a product or substrate]). This enzyme requires divalent metal ions, usually magnesium. The mechanism is induced fit rather than lock and key – the substrate changes the shape of the enzyme’s active site. This is known as substrate-induced cleft closing. This enzyme occurs in different isoforms. Isoforms of enzymes perform the same reaction but are slightly different in their structure. For example, in the liver and pancreatic β-cells, there is a form of hexokinase known as glucokinase. Hexokinase does several things. Adding a phosphoryl group makes glucose 6-phosphate less stable than glucose. It is therefore easier to break down. Additionally, this change in structure prevents glucose transport out of cells (via glucose transporters) and traps it in the cell. This keeps the concentration gradient in favour of moving glucose into the cell as a low concentration is maintained. This step is a regulatory point and will be expanded on overleaf.
  29. 29. 28 Glucose Transporters You do not need to remember Km numbers, but which ones are high and which ones are low is important. GLUT1 and GLUT3 do almost the same job, and have a low Km. Km is the substrate concentration at which the enzyme can reach half of its maximum velocity. This means that GLUT1 and GLUT3 can move glucose quickly at low concentrations. Therefore they are responsible for the normal basal uptake of glucose into the cells: they are essentially always working. However, GLUT2 and GLUT4 are up to 20 and 5 times higher respectively. GLUT4 is in muscle and fat cells and its levels are elevated after training or exercise. Therefore, muscles gain more GLUT4 when athletes train, to allow the athlete to take glucose into their muscle cells faster. GLUT2 is in the liver and pancreatic β- cells, meaning that following eating, when blood glucose levels are elevated, GLUT2 will start to work much faster as its Km value is approached. This works to help lower blood glucose. GLUT5 is not relevant to this lecture, but is in the table to complete the set. A glucose co-transporter is shown below on the left: Na+ and Glucose
  30. 30. 29 This is secondary active transport, as ATP is required by the sodium potassium pump. The glucose transporters are symporters, and the method of transportation is facilitated diffusion. They transport sodium ions and glucose. Part of the ATP that a red blood cell is producing through glycolysis is being used to pump sodium out and by secondary active transport, glucose in, to carry on powering glycolysis. Regulatory Point of Glucose to Glucose 6-Phosphate The enzyme hexokinase is inhibited by its product, glucose 6-phosphate and is therefore self-limiting. If there is lots of glucose 6-phosphate, it is a sign that no more glucose is required to be converted as there is enough ATP in the cell. This is both a competitive inhibitor (it binds into the active site to slow the enzyme down) and is also allosteric (it binds in a secondary site on the same enzyme and changes the shape of the active site). Although this is a regulatory point, this step is not the rate limiting step. In the liver however, both isoforms of the enzyme are present, and glucokinase is not inhibited by its product. This means that lots of glucose 6-phosphate is produced and this is the sign for pancreatic β- cells to release insulin. This is covered in a future lecture. Phosphoglucose Isomerase On the A3 sheet entitled glycolysis, this is shown as a one-step reaction. However, nothing is lost or gained by the molecule because the reaction is an isomerisation, as shown by the enzyme name, phosphoglucose isomerase. This is the conversation of an aldose to a ketose. Essentially, the ring is broken, a group is changed, and the ring is reformed. This is done so that later on in the pathway when the molecule is split by aldolase, it is evenly split into two molecules containing three carbons each.
  31. 31. 30 Phosphofructokinase Again, the –kinase suffix gives away that this is a phosphoryl transfer (group transfer) reaction. Again this reaction requires a cofactor. This enzyme requires divalent metal ions, which is usually magnesium. Again the mechanism is induced fit (substrate-induced cleft closing). This is a very important regulatory point, is regulated allosterically and plays a central role in metabolic integration. Regulatory Point of Fructose 6-Phosphate to Fructose 1,6-Bisphosphate This is the first irreversible reaction which is exclusive to glycolysis. It is a committed, rate limiting step. The whole pathway cannot go faster than this step. The enzyme phosphofructokinase is slowed down or inhibited by ATP by an allosteric mechanism; if there is lots of ATP in the cell, making more makes no sense as it breaks down too easily. However, this inhibition is in turn removed by AMP. AMP is a sign that there is not enough energy, because when cells are short of energy, they take two molecules of ADP and put them together to make one molecule of ATP, resulting in a left over molecule of AMP. This makes sense if you remember that this pathway is trying to make enough ATP for the cell. In muscles, a low pH caused by lactic acid switches the enzyme (phosphofructokinase) off. This is a self-regulating protective mechanism in skeletal muscle, because if lactic acid builds up, it can damage the proteins and cells in the skeletal muscle. Switching off the enzyme slows down glycolysis, therefore preventing the production of pyruvate and therefore lactic acid. Therefore, this regulatory point protects skeletal muscle from being damaged during anaerobic respiration. In the Liver However, in the liver this step is regulated a bit differently. The liver is a store of glucose (in the form of glycogen), so all of the adenylate control discussed previously is the same. However, pH change has no effect – low pH will not downregulate the enzyme in the liver. This is because the lactate from the muscle has been taken to the liver to be recycled, so pH is always lower. This enzyme is actually linked with the recycling of lactate, covered later on. However, it is downregulated by citrate. If there is a lot of citrate in the Krebs cycle, it means that a lot of glycogen has already been broken down and so no more is required and the enzyme’s regulatory point switches glycolysis off. However, this enzyme is upregulated by fructose 2,6-bisphosphate. Increased glucose leads to more fructose 6-phosphate being produced, which leads to higher fructose 2,6-bisphosphate production. This means that there is more glucose to be broken down. This is a feed-forward mechanism.
  32. 32. 31 In the liver, this reaction is upregulated by glucose, because lots of glucose means that it has to be broken down, so glycolysis is required to go faster. Aldolase This is not a regulatory point for glycolysis. It is a lyase reaction, the clue for which is shown by the double bonds. It generates two 3 carbon molecules from a 6 carbon molecule (this is the reason for the earlier isomerisation reaction). Triose Phosphate Isomerase Again, the word “isomerase” shows that this reaction is an isomerisation reaction. Glyceraldehyde 3- phosphate is the molecule which goes down glycolysis, but there is also a dihydroxyacetone
  33. 33. 32 phosphate. This isomerase reaction turns this into a glyceraldehyde 3-phosphate, meaning that there are now two which can both enter the pathway. The reaction is an intramolecular oxidation-reduction and is performed by a kinetically perfect enzyme. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) The enzyme includes the word dehydrogenase and the reaction produces NADH+H+ . This tells us that this is a redox reaction, specifically oxidation of an aldehyde to an acid. The NADH+H+ goes to the electron transport chain. This is the first step in the energy yielding phase of glycolysis; everything before has been investing energy by using up ATP. At the end of this is 1,3-bisphosphoglycerate, which has a high phosphoryl transfer potential – it can easily donate its phosphate group. Note that GAPDH can use arsenate as a substrate and is irreversibly inhibited by arsenic and arsenite. Phosphoryl-Transfer 1,3-Bisphosphoglycerate has a higher phosphoryl transfer potential than ATP (to ADP). This means it can be used to make ATP. Phosphoglycerate Kinase
  34. 34. 33 Again the –Kinase tells us that this is a phosphoryl-transfer (group transfer). Phosphoryl-transfer where ATP is produced is called substrate level phosphorylation. The phosphoryl group from 1,3- Bisphosphoglycerate is removed and added to ADP, producing ATP. 2-3 Bisphosphoglycerate Additionally, 1,3-bisphosphoglycerate can be isomerised into 2-3 bisphosphoglycerate by 2-3 bisphosphoglycerate mutase (mutase as it moves an entire phosphate group from the first carbon to the second carbon). This molecule is a major intermediate in erythrocytes (red blood cells). It lowers the affinity of haemoglobin for oxygen and therefore encourages red blood cells to release oxygen. The enzyme is regulated by pH; when pH is low (acidic), it makes more 2,3-BPG, meaning that haemoglobin more readily gives up oxygen. This is important in muscles which are anaerobically expiring and are therefore producing lactic acid. The same enzyme also has another activity (active site) as a phosphatase, the opposite of a kinase (it removes phosphate groups). It can turn 2,3-BPG into 3-phosphoglycerate, the next step in glycolysis. This function is switched on by normal or raised pH and so removes 2,3-BPG. Overall, slightly less ATP is produced this way because the kinase step in glycolysis is skipped, but it is an important step for red blood cells. Phosphoglycerate Mutase Mutase enzymes signify a group transfer within a molecule – these are not common. A whole functional group is moved from one place on the molecule to another. This makes the molecule less stable and easier to break down, and provides the substrate for the next reaction.
  35. 35. 34 Enolase This is a dehydration reaction (water can be seen leaving) which produces an ‘enol’ molecule, from which the enzymes name is derived. Phosphenolpyruvate has a much greater phosphoryl transfer potential than 2-phosphoglycerate. Therefore, phosphenolpyruvate can give its phosphoryl group to ADP in the next reaction. Pyruvate Kinase This is the last step in glycolysis. It has a kinase suffix so must be group transfer (phosphoryl-transfer). Again, ATP is being made in substrate level phosphorylation.
  36. 36. 35 Regulatory Point of Pyruvate Kinase It is the third irreversible step in the glycolysis pathway. It is inhibited by ATP, because if there is lots of ATP, glycolysis is not required. It is also inhibited by the amino acid alanine because it is an indication that there are lots of “building blocks” (lots of catabolic activity has already happened – nothing else needs breaking down). Alanine is also synthesised from pyruvate and so it signals that there are abundant precursors for its synthesis. Pyruvate kinase is upregulated by Fructose 1,6- bisphosphate in the same feed forward mechanism using the previous irreversible step – an increased concentration of a previous substrate speeds up the reaction ready for when the molecules arrive at that step. In the Liver Pyruvate kinase is regulated differently in the liver. A different isoenzyme performs the same function but has a slightly different structure. It has the same allosteric control mechanism as pyruvate kinase, but is found in an L form rather than an M form. It is covalently regulated by low blood glucose levels, in an AMP cascade triggered by glucagon. The cascade phosphorylates the kinase in the liver. So when there is low blood glucose and lots of glucagon is released from the alpha cells, the cascade is triggered. A kinase at the end of the cascade phosphorylates pyruvate kinase, which switches off the enzyme. This means that the liver stops breaking down glucose, conserving it for tissues such as brain and muscle. Fermentation There are two fermentation of pyruvate. The first and the only one you need to know is reduction in which lactic acid is formed. You do not need to know ethanol fermentation. This reaction removes pyruvate, so prevents the product of glycolysis from building up. Therefore a concentration gradient is maintained. It also removes NADH+H+ , also preventing it from building up. A build-up of either of these two products would switch off glycolysis. There is no other way of making ATP anaerobically so it is essential that glycolysis continues. This reaction is reversible, so that when oxygen is present again, the lactate can be turned back into pyruvate and then put through aerobic respiration. Therefore the energy bound in pyruvate is not lost because lactate is not excreted as a waste product.
  37. 37. 36 Other Sugars in Glycolysis: Galactose Galactokinase Galactokinase ends in –kinase so therefore is a group transfer enzyme (phosphoryl transfer). This enzyme performs almost the same role as hexokinase in glycolysis. It uses ATP to perform this same role but has a different substrate, which it traps in the cell and prevents it from being transported out again. It destabilises the molecule by passing energy to it. Galactose 1-Phosphate Uridyl Transferase
  38. 38. 37 This is another group transfer reaction. It takes what has been made in the previous step, galactose 1- phosphate, and it combines it with a carrier molecule, UDP (uridine). Uridine is donated by UDP- glucose, a uridine carrier molecule. UDP-galactose is formed and glucose 1-phosphate is released and is used in glycolysis (via a mutase enzyme). UDP-glucose is attached, broken off and recycled, by being formed again, each time a molecule of galactose is broken down. UDP-Galactose 4-Epimerase This pathway is concerned with reforming UDP-Glucose. This reaction does not fit any of the other categories; it is an epimerisation reaction and has inverted a hydroxyl group. The UDP-glucose produced then combines with another galactose 1-phosphate and the process repeats. Other Sugars in Glycolysis: Fructose In most Tissues: In most tissues, the top pathway (hexokinase) is active. Hexokinase performs a group transfer (phosphorylation) on fructose, just like the one in glycolysis, and it requires ATP. However, glucose is a strong competitive inhibitor, meaning that any glucose present will outcompete fructose. This means that only a small amount of fructose is normally metabolised in most tissues. Therefore, this pathway does not take place very often unless blood glucose levels are very low. The fructose 6-phosphate produced in this reaction enters glycolysis.
  39. 39. 38 In the Liver: The bottom pathway (fructokinase) occurs primarily in the liver. Again, this kinase performs a group transfer (phosphorylation) reaction on fructose, using ATP to destabilise the molecule for the next step, and turning it into fructose 1-phophate. Next, this molecule is broken by fructose 1-phosphate aldolase in a lyase reaction similar to the one seen in glycolysis. This enzyme is an isoform of the one seen in glycolysis and uses a different substrate as shown. This produces dihydroxyacetl phosphate which enters glycolysis, and glyceraldehyde which must be altered in order to enter glycolysis. Glyceraldehyde kinase phosphorylates it, turning it into glyceraldehyde 3-phosphate which enters glycolysis. This is the path most fructose takes. All the fructose metabolised in the liver enters glycolysis below PFK, the rate determining step. This means that even if this step is glycolysis is downregulated, the fructose would still be metabolised. Furthermore, fructose is a ready source of ATP and glucose, and this is the theory behind high fructose drinks.
  40. 40. 39 Link Reaction The link reaction is irreversible – this is important and is the reason we must have carbohydrates in our diet. Pyruvate dehydrogenase, the enzyme involved, is a large complex having a mass of 4 million to 10 million daltons. Groups transfer from one subunit to another. Subunits are connected to the core by tethers. An overview of the link reaction is shown below: Cofactors Pyruvate dehydrogenase requires cofactors to perform its job. Some of these cofactors (such as thiamine pyrophosphate [TPP]) are made in thiamine, vitamin B1, which is the reason vitamin B1 is essential. Pyruvate also requires other cofactors such as lipoid acid and so called stoichiometric cofactors coenzyme A and NAD+ , which are not normally referred to as cofactors. These are regenerated during the reaction and so are not used up. Note that in the reactions below, it is not necessary to learn which reactions occur at which point (e.g., E1, E2, E3), but for interest it has been included. Pyruvate Dehydrogenase Component (E1) This carries out a decarboxylation reaction as shown on the pathway. This reaction itself is much more complicated but knowledge of this is not required. Following this, an oxidation occurs. Dihydrolipoyl Transacetylase (E2) This carries out a group transfer of an acetyl group (acetylation) and produces acetyl Coenzyme A, which is the fuel for the Kreb’s cycle. Dihydrolipoyl Dehydrogenase (E3) This is an oxidation reaction which produces NADH + H+ , which is taken to the electron transport chain and makes ATP by oxidative phosphorylation. This must occur before further acetyl CoA can be formed from pyruvate.
  41. 41. 40 Regulatory Point: Link Reaction This reaction is irreversible and a very important regulatory point. The enzyme is under adenylate control and part of a big chain of reactions making either ATP or lipid. Therefore, is there is lots of ATP, this enzyme will be switched off; and if there is too little ATP, it will be switched on. Therefore this enzyme is upregulated by ADP and down regulated by ATP. Additionally, the enzyme is upregulated by pyruvate so that it uses up pyruvate. Phosphorylation (via a kinase) turns the enzyme off via covalent modification. Other control mechanisms are involved too. This reaction produces NADH+H+ . If there is too much of this in the cell, the enzyme will be switched off. Therefore, NADH downregulates the enzyme competitively as it is a product of the enzyme and can block the active site. It is also competitively downregulated by acetyl CoA. Essentially, the enzyme is slowed down by what it is producing and sped up if concentrations of these products are low. In addition, the enzyme is upregulated by calcium ions, Ca2+ . Calcium covalently modifies the enzyme to speed it up. This occurs because calcium is used to initiate muscle contraction (as a second messenger). When muscles are contracting, they will use lots of ATP. Therefore, the signal which contracts a muscle also speeds up this pathway to make more ATP, making it harder for muscle cells to run out of ATP.
  42. 42. 41 Citric Acid Cycle, Kreb’s Cycle or Tricarboxylic Acid Cycle This is the final common pathway for fuel oxidation. All of the processes involved are dealing with carbohydrates, fats and amino acids. Most of these enter as acetyl coenzyme A at the top of the cycle, but some substances enter at other points (covered in later lectures). Citrate Synthase This is the first reaction. The name of the enzyme tells us that citrate is produced. This is a condensation reaction, followed by a hydrolysis. Overall, it is a condensation reaction. Acetyl CoA (2 carbons) has been added to the last product in the cycle, oxaloacetate (4C), to make a 6 carbon molecule, citrate. The energy for this reaction comes from breaking a thioester bond in acetyl CoA. Aconitase The enzyme here does not give away the function. It is an isomerisation (involving a dehydration followed by a hydration, but this is not necessary knowledge). The products and reactants give the reaction away, because citrate is the substrate and the product is isocitrate. Specifically, the reaction rearranges a hydroxyl group. This is a preparatory step for the next enzyme, generating a less stable molecule.
  43. 43. 42 Isocitrate Dehydrogenase Dehydrogenase tells us that this involved a redox reaction. Specifically, the substrate is oxidised and NAD+ is turned into NADH+H+ for the electron transfer chain. After this, a decarboxylation takes place. Regulatory Point of Isocitrate Dehydrogenase This is the first enzyme to generate something useful for the electron transfer chain and is hence a key point to be regulated in the cycle. It makes sense that this enzyme is upregulated by ADP and downregulated by ATP and NADH. The control by ATP and ADP is performed under adenylate control, whereas the downregulation by NADH is via competitive inhibition as NADH is a product of the reaction. This regulation is because cells do not want to store lots of energy as ATP as it will be broken down and wasted. If this step is stopped or slowed, citrate will build up, which feeds back and turns off glycolysis to stop more pyruvate and acetyl CoA from entering the cycle. α-Ketogluterate Dehydrogenase NADH+H+ is produced followed by the release of carbon dioxide. Therefore, like the last reaction, there must be an important redox reaction followed by a decarboxylation reaction. Additionally, a group transfer takes place. Regulatory Point of α-Ketogluterate Dehydrogenase Again, useful substrates for the electron transport chain have been produced. It is downregulated by succinyl coA (competitively switched off by its product), switched off by NADH (again competitively by its product) and allosterically downregulated by ATP, for the same reasons as the last regulatory point. NB. Excess α-ketogluterate can be used to form amino acids. Succinyl CoA Synthetase Again, a thioester bond on succinyl CoA is broken to produce the energy for this reaction. This converts succinyl CoA to succinate and also forms GTP from GDP and Pi, which goes off to make a molecule of ATP: ↔ This ATP producing reaction is performed by nucleoside diphosphokinase (again, kinase showing that phosphoryl transfer is involved). Next Three Steps These have been grouped together to begin with because this sequence of oxidation, hydration, followed by oxidation is a reoccurring theme and is seen quite often in metabolism (such as in fatty acid oxidation, fatty acid synthesis and amino acid breakdown).
  44. 44. 43 Succinate Dehydrogenase This is the first of the three steps. It is a dehydrogenase, meaning that it is a redox reaction and that it also produces something useful for the electron transport chain, in this case FADH2. FADH2 forms a direct physical link with the electron transport chain – it is in complex II of the electron transport chain, which we will see in the next lecture. It is an intermediate which passes its electrons to the next step in the electron transport chain, coenzyme Q. The reaction itself has turned succinate into fumerate. Fumerase This reaction turns fumerate into maltate. The reaction is not given away by its name, but water can be seen to be entering the reaction, making it a hydration reaction. The point of this step is to add H+ and OH- to prepare for the next step by destabilising the molecule. Malate Dehydrogenase This is a redox reaction, turning malate into oxaloacetate. It produces NADH+H+ from NAD+ , which is used in the electron transport chain. This step has a positive ΔG but is driven forwards due to the cycle (especially the next step [due to the thioester bond], the first step of the cycle again) having a very negative ΔG. It is also driven because the products are taken away (and used in the electron transport chain and Kreb’s cycle) and the equilibrium is shifted. Clinical Relevance If pyruvate dehydrogenase (in the link reaction) is inhibited (by mercury for example), the patient becomes unable to respire aerobically. Therefore, the cells which store little energy (due to having few mitochondria) and are therefore affected first are neurons. Therefore, these patients very often have neurological symptoms.
  45. 45. 44 Electron Transport Chain Mitochondria The electron transport chain takes place in mitochondria of cells. Mitochondria have two membranes. The outer one is generally freely permeable because it has porins in it (beta barrel proteins that cross a cellular membrane and act as a pore through which molecules can diffuse). The inner membrane is impermeable to polar molecules. This integrity is required for oxidative ATP production. The inner membrane is highly folded (cristae) and has lots of transporters (as it is impermeable). Shuttles These transporters are called mitochondrial shuttles. We will be learning three in some detail. NADH Movement: Glycerol 3-Phosphate Shuttle This is the glycerol 3-phosphate shuttle. It predominates in muscle, though most cells use it. It transports NADH+H+ by transferring the high energy part of it to make FADH2, meaning that it will enter a complex II and produce 1.5 ATP rather than 2.5. This may seem counterintuitive, but left to its own devices, the concentration of NADH+H+ will build up in muscles, inhibiting the transport of further NADH in. Therefore, muscles sacrifice efficiency for quantity, as it makes more ATP in total (this is important when thinking of the fight or flight response).
  46. 46. 45 NADH Movement: Malate-Aspartate Shuttle NADH+H+ is entering the mitochondrial matrix in this shuttle. This is a more efficient shuttle as it will then enter at complex I and produce 2.5 ATP. Note that the arrows are “wiggly S arrows”, meaning, for example that α-ketogluterate goes to glutamate. Note that you only need to learn one side of the shuttle as the top is mirrored by the bottom. ATP-ADP Translocase
  47. 47. 46 ATP moves out of the mitochondrial matrix and ADP moves in. As protons have been pumped into the cytosolic side via the electron transport chain, there is a positive net charge. Comparatively, the inside of the membrane is negative. ATP is more negative than ADP as it has an extra phosphate. ATP is therefore attracted to the cytosolic side of the membrane. As shown in the diagram above, the enzyme changes shape throughout the transfer. 25% of the energy produced in the electron transport chain goes into powering this pump because it saps the electrical gradient produced by the electron transport chain. Standard Reduction Potential = E° This is a measure of a compound’s affinity for e- ; its oxidising potential; its potential to become reduced. The more positive this number, the greater the compound’s potential to accept e- (become reduced and act as an oxidising agent). Oppositely to ΔG, the more positive ΔE° is, the more likely a reaction is to happen. The Electron Transport Chain: A Chain of Redox Reactions Electrons move through a chain of donors and acceptors (along a gradient of compounds with increasing ΔE°). The reaction with the highest ΔE° is the recombination with oxygen to form water. Overall, energy is generated; ΔG = -220kJ/mole NADH (n=2). Since ATP requires 30.5kJ/mole to form from ADP, more than enough energy is available. However, only about 2.5 ATP are generated because the chain is not efficient (energy is lost in processes such as heat). The Electron Transport Chain: NADH Shown here are complexes I, III and IV of the electron transport chain. Complex I oxidises NADH then reduces coenzyme Q (as implied by the name of complex I). Two electrons enter complex I, are passed to coenzyme Q, and protons are pumped across the membrane. These electrons are passed to complex III. Coenzyme Q is oxidised, cytochrome C is reduced (again, as implied by the name of complex III) and protons are pumped across the membrane. The electrons are then passed on to complex IV which accepts the electrons and passes them onto oxygen, pumping more protons as it does. Water is then formed. By convention, the final equation forms one molecule of H2O (using ½O2).
  48. 48. 47 Complex I: NADH-Coenzyme Oxidoreductase Complex I is NADH-Coenzyme Oxidoreductase. It uses two bound cofactors to accomplish its electron transfer from NADH to coenzyme Q, an enzyme carrier. The first cofactor is flavin mononucleotide (FMN) which is similar to FAD but only accepts one electron at a time. The other cofactors are iron- sulphur centres. Iron is a key feature of the electron transport chain as it has different oxidation states and can switch between them through redox reactions. CoQ is lipid based and so can move freely within the membrane. It is an electron carrier and is based within the membrane, meaning that it is hydrophobic. Complex III: Coenzyme Q-Cytochrome C Oxidoreductase This complex passes electrons from CoQ to cytochrome C via an Fe-S complex followed by either b or c1 type cytochromes, ending up at cytochrome C. The electrons pass one at a time to cytochrome C. Cytochrome C This is a mobile, water soluble cytochrome which is loosely bound to the inner mitochondrial membrane. Like haemoglobin and myoglobin, cytochromes contain haem proteins (the complexed metal ion here is Fe). Cytochrome C carries one electron at a time to complex IV.
  49. 49. 48 Complex IV: Cytochrome C Oxidase This enzyme also contains cytochromes, some of which are copper containing (not just iron as with the others). Copper can also be used to transport electrons. Once cytochrome C has transferred two electrons (one at a time) to complex IV, they go through these cytochromes (don’t learn the names) and are recombined with oxygen. Complex II: Succinate-Coenzyme Q Oxidoreductase This complex contains the enzyme succinate dehydrogenase, which is part of the citric acid cycle. It contains FAD (another physical link to the Kreb’s cycle) and uses iron sulphur complexes. Additionally, it uses a cytochrome which contains a haem like group which can carry electrons. Mutations of this complex cause a wide variety of disorders. Shown below are the processes involved in complex II. It then passes on the 2e- in the same way as complex I, so this does not need to be shown again. Unlike the other complexes, this complex is not a hydrogen pump, so less ATP is produced overall. Efficiency About 220 kJ/mole is produced from NADH oxidation. To produce ATP requires 30.5kJ/mole. However, only around 2.5 ATP are produced per NADH. Therefore, the efficiency is again about 40%.
  50. 50. 49 Reactive Oxygen Species (ROS) These are also known as free radicals and lots are produced by the electron transport chain. They are caused by the incomplete reduction of oxygen in this pathway. They are harmful because they are very reactive (they react with molecules such as proteins and enzymes, changing their functions by breaking them down or modifying them). Cells strive to avoid having reactive oxygen species. Some cells do use them for digesting things (e.g. macrophages), but the mechanisms we will cover are aimed at preventing them. → → → → Defence against ROS – Enzymes The following equations and enzyme names should be learned. Superoxide dismutase This enzyme takes one form of ROS and recombines them with some protons to form oxygen and hydrogen peroxide. ↔ Catalase This enzyme is very abundant in living organisms and breaks down hydrogen peroxide to form oxygen and water. ↔ Inhibition This pathway has specific inhibitors which should be learned. The first complex of the electron transport chain, NADH-CoQ oxidoreductase, is inhibited by rotenone (an insecticide), and anytal (a barbiturate). Complex III, CoQ-cytochrome C oxidoreductase, is inhibited by antimycin. Complex IV, cytochrome c oxidase is inhibited by several things which should be learned. Cyanide binds to a ferric form (Fe3+ ) on cyt a3, meaning that electrons cannot be bound there; and carbon monoxide binds to a ferrous form (Fe2+ ) on cyt a3. These two are the most important inhibitors of this complex, but others include azide (which binds to a ferric form of cyt a3 again) and nitrite (Fe in haemoglobin to ferric form). Oxidative Phosphorylation It was originally thought that ATP generation took place at complexes I, III and IV. It is now known that the coupling of electron transport to ATP is indirect in that a proton gradient is generated across the inner mitochondrial membrane, driving ATP synthesis. This is known as the chemiosmotic theory. The
  51. 51. 50 protons have a thermodynamic tendency to return to the matrix, known as the proton-motive force (the analogy of a water wheel is often used). ATP Synthase Knowledge of certain parts of this complex is required. The parts marked with the letter C rotate. This movement additionally rotates the axle in the centre. However, the cluster of units around the top (the head) does not rotate and is held in place (by a part in the membrane), whilst the axle rotates within it.
  52. 52. 51 Shown below is a top down view of this complex. Each α and β pair of the head of the complex form a catalytic site. The γ unit (axle) rotates and breaks the symmetry of the α β hexamer. Essentially, as the γ unit rotates, it changes the shape of the three catalytic sites. It changes them from an open state when ADP and Pi freely move in and out, into a state where the two are loosely held and are therefore trapped. As it rotates further, it tightly binds ADP and Pi and this is when the reaction happens to form ATP. This occurs as long as there is a flow of protons. Rotation of γ Subunit The subunit rotates 50-100 times a second and is driven by a proton motive force. H+ enters and leaves via 2 half channels, which prevent the immediate release of H+ . One subunit channel contains aspartic acid (an amino acid) which is negatively charged and therefore attracts H+ into the channel. This rotates the c subunits, as displayed below.
  53. 53. 52 Control of Oxidative Phosphorylation Oxidative phosphorylation is tightly controlled by ATP demand; if more ATP is required, more is produced. The electron transport chain and ATP synthase are tightly coupled together, meaning that if one isn’t working, nor does the other. Therefore, the electron transport chain must be functioning in order for ATP to be produced. The process is also very tightly controlled by ADP. If lots of ADP is supplied, the reaction goes faster until the ADP has been used up and converted to ATP. Mitochondrial States Mitochondria exist in one of five states, depending on the tissue and what the tissue does. Mitochondria in fatigued skeletal muscle is in state 5; mitochondria in heart muscle is in state 3; and mitochondria in rested skeletal muscle is in state 4. Electron Transport is coupled to ATP Production in Oxidative Phosphorylation As mentioned previously, most mitochondria are said to be tightly coupled. There is no electron flow without ATP production (phosphorylation) and vice versa. This is because there are no protons flowing through meaning that the concentration of protons will build up between the membranes, inhibiting the pumps (complexes). If there is no ADP, then ATP cannot be made and the electron transport chain will not happen either. If there is no oxygen, there will be no electron transport chain because it is coupled to ATP synthase. Unless substrates are available for both mechanisms, neither will occur.
  54. 54. 53 Uncoupling Proteins Thermogenesis There are different ways in which we are able to maintain body temperature. One is exercise induced, another is diet induced, another is shivering and the last is non-shivering thermogenesis. Non-Shivering Thermogenesis This is heat generation without shivering. This is useful in neonates as they cannot shiver. There are different ways of generating this heat, one of which is futile cycling and the other is a specialised form of fat called brown adipose tissue. This lecture will focus upon a pathway within brown adipose tissue. The differences between white and brown adipocytes are shown below. Brown adipose tissue has many smaller vacuole droplets (resulting in a larger surface area and therefore a faster reaction) and many more mitochondria. The mitochondria also have more cristae (infoldings of the mitochondrial membrane). UCP is the Key Within brown adipocytes is a specialised uncoupling protein called UCP1. This protein responds to noradrenaline, a neurotransmitter. It is under sympathetic nervous system control. It uncouples oxidative phosphorylation from the electron transport chain, meaning that one reaction may occur without the other (see page 54). These cells use fat/lipid as their fuel (this process will be explained in a future lecture on β-oxidation).
  55. 55. 54 The above diagrams show an alternative pathway (through an uncoupling protein [UCP1]) which protons can take when UCP is turned on, resulting in the production of lots of heat and dissipation of the proton gradient. This therefore inhibits complex V (ATP synthase). Brown adipose tissue can produce more energy than any other type of tissue in the body. Small amounts of ATP are still generated in the usual way at some points on the membrane.
  56. 56. 55 Brown Adipose Tissue Diminishes With Age Neonatal humans have well developed brown adipose tissue, whereas adult humans have little. Some people have more than others, and it turns on when it is cold. Thin males seem to have more of it. Dinitrophenol 2,4-dinitrophenol can cross the inner mitochondrial membrane very easily because it is hydrophobic and lipid soluble. It is able to accept and donate protons and is therefore able to move them across the membrane much like UCP1. It can therefore dissipate the proton motive force and uncouple oxidative phosphorylation. This releases energy and results in an increased O2 and NADH consumption because the electron transport chain has to work faster to try and restore the proton motive force. 2,4-dinitrophenol is found in herbicides and fungicides and is very toxic because when it uncouples the electron transport chain from ATP synthesis, it forces cells to respire anaerobically. In most organisms, this results in death. It is also used in explosives manufacture and it seems to cause weight loss and fevers such as hyperthermia.
  57. 57. 56 Gluconeogenesis Gluconeogenesis is not a simple reversal of glycolysis. The irreversible steps in glycolysis must be bypassed: hexokinase, phosphofructokinase and pyruvate kinase. The rest of the pathway is a simple reversal. Gluconeogenesis occurs mostly in the liver, but also in the kidney. It is regulated in almost the opposite way to glycolysis (reciprocal regulation). Pyruvate Carboxylase This is attached to the mitochondria and converts pyruvate to oxaloacetate. It is a carboxylation reaction, the reverse of the opposite reaction in glycolysis. In the same way, rather than producing ATP, it uses it. It takes place within the mitochondria. Regulatory Point of Pyruvate Carboxylase Pyruvate carboxylase is upregulated by acetyl CoA and downregulated by ADP. Note that the equivalent step in glycolysis is reciprocally regulated in that ATP downregulates it. An Example of Oxaloacetate Export
  58. 58. 57 This shuttle was explored in a previous lecture and reoccurs here to move oxaloacetate in and out of the mitochondria. Phosphoenolpyruvate Carboxykinase This uses GTP and therefore requires energy. It is a decarboxylation reaction as well as a phosphorylation and both reactions are equally important. Regulatory Point of Phosphoenolpyruvate Carboxykinase This is regulated in the same way as the last enzyme in that it is downregulated by ADP. Pyruvate kinase is reciprocally regulated. Fructose 1,6-Bisphosphatase This is a hydrolysis reaction and breaks off a phosphoryl group. It is the last step of gluconeogenesis in nearly all tissues (most do not go back to glucose, but stop at glucose 6-phosphate as this keeps it within the cell). Glucose 6-phosphate is then used for processes within the cell. The final step of glucose 6-phosphate to glucose only occurs in specialised tissues responsible for blood glucose homeostasis as they release glucose into the blood. Primarily, this occurs in the liver. Regulatory Point of Fructose 1,6-Bisphosphatase Fructose 1,6-bisphosphatase does the opposite job (is reciprocally regulated) to PFK (the key regulatory point of glycolysis). It is upregulated by citrate, downregulated by AMP and downregulated by fructose 2,6-bisphosphate. It is the last unique step in most tissues. Glucose 6-Phosphatase This is the final step and occurs primarily in the liver and also in the kidneys. The enzyme itself is embedded in the membrane. It exports glucose into the blood to raise blood glucose levels; regulation of this enzyme regulates blood glucose. It is shown below:
  59. 59. 58 Fructose 2,6-Bisphosphate This regulates glycolysis and gluconeogenesis. It stimulates glycolysis and inhibits gluconeogenesis. Two enzymes regulate its levels (it is made by phosphofructokinase 2 and degraded by fructose bisphosphatase 2) and they are both found on the same polypeptide chain. Entry of Glycerol Fat is stored as triacylglycerol, which is comprised of three fatty acids and one glycerol. The entry of glycerol comes from the breakdown of triacylglycerol. This is carried out because fatty acids cannot be used for gluconeogenesis. Glycerol enters at dihydroxyacetone phosphate (DHAP). Cori Cycle (Removing Lactic Acid) The simplified diagram below represents a cell. The type of cell varies with the stages in the cycle. A red blood cell or an anaerobically respiring muscle cell will be taking in glucose from blood, as shown in the top left of the diagram. Under normal conditions, these cells (with few mitochondria) are making lots of lactic acid, shown in the bottom left of the diagram. If this builds up in the cell, it will inhibit glycolysis. Therefore, it is exported from these cells back into the blood, where it goes primarily
  60. 60. 59 to the liver, but also to the kidneys. The lactate is converted back to pyruvate. Through gluconeogenesis, this is converted to glucose 6-phosphate, then to glucose (in the liver and kidneys). This is then exported back into the blood and used by respiring cells.
  61. 61. 60 Pentose Phosphate Pathway The pentose phosphate pathway primarily produces 5 carbon sugars, such as ribulose 5-phosphate (a phosphorylated pentose) from the oxidation of glucose 6-phosphate. The first step is regulated and two NADPH are produced. NADPH is used to produce cholesterol, fatty acids and amino acids, as well as helping with detoxification in the liver and protection against oxidative stress (reactive oxygen species), particularly in red blood cells. The pentose phosphate pathway has an oxidative phase (the regulatory point) followed by a non-oxidative phase. It occurs in the cytoplasm. Glucose 6-Phosphate Dehydrogenase The name of this enzyme and the fact that an enzyme carrier is involved shows that this is a redox reaction. NADPH is shown being produced – an important product of this pathway. Regulatory Point of Glucose 6-Phosphate Dehydrogenase This is the rate limiting step of this pathway. It is primarily regulated by the concentration of its substrate and products. It is primarily under the control of the concentration of NADP+ and NADPH (Le Chatelier’s principle). High substrate concentration (NADP+ ) drives the reaction and high product (NADPH) slows down the reaction (and vice versa). Lactonase This is a hydrolysis reaction (shown by the addition of water) which prepares the molecule for the next step.
  62. 62. 61 6-Phosphogluconate Dehydrogenase Again, the enzyme name shows that this is a redox reaction. NADPH, an important product, is shown being made. Additionally, CO2 is removed (decarboxylation) which changes the 6 carbon molecule into a 5 carbon molecule: one of the points of this pathway. Non-Oxidative Phase This is the rest of the pathway and produces many useful products, some of which are interconverted. It produces sugar molecules stretching between 3 and 7 carbons. These include important molecules such as nucleotides, ribulose, DNA and RNA.
  63. 63. 62 Lipid Metabolism Triacylglycerols (TAG) Although they are the same thing, avoid calling these ‘tryglycerides’. Triacylglycerols provide building blocks to make other molecules such as phospholipids (membranes), glycolipids (signalling proteins and embedded in membranes), hormones (lipid based and intracellular receptors) and intracellular messengers (such as IP3 and DAG). Triacylglycerols are a highly concentrated fuel source. This hydrophobic molecule is not hydrated as opposed to 1g of glycogen which binds to a “shell” of 2g of water. This means that only a third of the weight of glycogen in the body is useable as energy, whereas almost 100% of a lipid’s weight is. Therefore, lipids (triacylglycerols) produce about 7 times more energy per gram than carbohydrate (glycogen). Typically, the body stores about enough energy in glycogen for 24 hours; about ten times that amount is stored in protein, found mostly in muscle; and enough energy to last several weeks is stored in triacylglycerols. This storage of energy as fat enables survival for extended periods of time and is a very efficient way of storing large amounts of energy. A major store of TAG is white adipocytes, where it is found in big lipid vacuoles (see page 55). Using TAG for Energy Hormone signalling can switch on a lipase enzyme by phosphorylating it, breaking down lipases into glycerol and free fatty acids, which can be used in the cell or exported to be used as energy elsewhere
  64. 64. 63 in the body. Hormones which perform this include glucagon, epinephrine (adrenaline) and cortisol. Triacylglycerol is broken down into three fatty acids and one glycerol. Fatty Acid Metabolism There are two pathways involved in fatty acid metabolism: β- oxidation and fatty acid synthesis. They mirror each other in their reaction types and in their reaction order. However, they are different pathways with different enzymes and occur in different compartments of the cell. This prevents them from cycling. The important sequence of oxidation, hydration, oxidation, lysis has been mentioned in previous lectures and is seen throughout metabolism. β-Oxidation: Fatty Acid Activation The pathway starts with an Acyl CoA. The R in the molecule represents a continuing chain; it could be a long or short fatty acid. The fatty acid must be joined to coenzyme A before β-oxidation can be performed. This process is called fatty acid activation and is the process by which a fatty acid is joined to coenzyme A prior to oxidation in the cytoplasm. This requires ATP. Both phosphate groups are stripped from each ATP, requiring the equivalent of two ATP to phosphorylate again fully (to reform the ATP). Therefore, this has a net effect of putting two ATP into the reaction. The enzyme which performs this is acyl CoA synthetase. In theory, this is a reversible reaction, but it is made irreversible by the rapid hydrolysis of pyrophosphate (PPi, the product of the dephosphorylation) into two individual phosphates. This means that the concentration of the product (PPi) is very low, which drags the reaction forwards. A high energy thioester bond is formed (this is the reason ATP is required). Breaking this bond powers future reactions in the pathway (and ultimately the Kreb’s cycle). This enzyme is regulated by substrate concentrations (the more fatty acid substrate going in, the faster β-Oxidation goes). Tracing this back, if more hormones are available (such as glucagon) β-Oxidation goes faster.
  65. 65. 64 β-Oxidation: Getting Acyl CoA into the Mitochondria The point of β-Oxidation is to feed fats into the citric acid cycle. It occurs in the mitochondria. As the fatty acid activation step occurred outside the mitochondria, acyl CoA needs to be transported into the mitochondria by a mitochondrial shuttle. A simplified version of the shuttle is shown below. Acyl is removed from coenzyme A and attached to carnitine. It is then transported across the membrane via translocase, a carrier protein. Following this, acyl is broken off and reattached to CoA, reforming acyl CoA inside the mitochondria. This is done because CoA is a large molecule and cannot cross the membrane. This step is downregulated by malonyl Coenzyme A (malonyl-CoA). This is important as malonyl-CoA is a product of fatty acid synthesis, meaning that this step is downregulated by the pathway’s eventual products, as less acyl CoA is required inside the cell. Malonyl will be covered in fatty acid synthesis. Acyl CoA Dehydrogenase This is the first step in β-Oxidation. It is a redox reaction, as suggested by the enzyme name. Importantly, FAD is reduced into FADH2 (which can enter the electron transport chain) and the fatty acid substrate is oxidised. The energy to drive the reaction is derived from the thioester bond made previously. NB. Despite the name, the product of this reaction is not a trans-fat.
  66. 66. 65 Enoyl CoA Hydratase This is a hydration reaction (water can be seen entering and the enzyme name suggests this). This step is preparation for the 2nd oxidation in the series. L-3 Hydroxyacyl CoA Dehydrogenase This is an oxidation reaction which produces NADH (which goes to the electron transport chain to make 2.5 ATP) and H+ . The formed product is simply a substrate for the next enzyme.
  67. 67. 66 ß-Ketothiolase The enzyme name reveals that this reaction is a thiolysis. Acetyl CoA is split from the rest of the molecule and is the input into the citric acid cycle. The CoA is recycled and used again. The remaining molecule is acyl CoA, shortened by two carbon atoms. It is run through ß-oxidation again in a loop until it becomes 4C-ketoacyl CoA, a four carbon molecule, which produces two molecules of acetyl CoA. These enter the citric acid cycle. ß-Ketothiolase is inhibited by its product. Therefore, the whole of ß-oxidation is regulated by the substrate going in at the beginning and the product coming out at the end.
  68. 68. 67 Unsaturated Fatty Acids Technically, this is another pathway in itself, but it will not be explored in great depth. Unsaturated fatty acids cannot be dealt with just using the pathway already covered (ß-oxidation) as it cannot deal with double bonds. Therefore, there are two extra steps required. Whilst knowledge of the enzymes involved in these steps is not required, the reaction types should be known. The first extra step is an isomerisation (using the enzyme cis-∆3 -enoyl isomerase), and the second is a reduction (using 2,4-dienoyl CoA reductase). The reduction step is involved to remove the double bond(s) in unsaturated fatty acids and the isomerisation step prepares the molecule for this reaction. Odd Chain Fatty Acids These are another type of fatty acid which ß-oxidation cannot deal with. This is because the final step leaves a five carbon fatty acid, meaning that following the last thiolysis step, a three carbon molecule (propionyl CoA) will remain. This three carbon molecule goes directly into the citric acid cycle as succinate. Propionyl CoA is converted into succinate in a couple of extra steps, the first of which is a carboxylation (requiring ATP), turning it into a four carbon molecule. The second step is an isomerisation. Enzyme knowledge is not required, but what occurs and the net result must be learned. This reaction requires vitamin B12 because one of the enzymes involved uses it as a cofactor. Oxidation in Peroxisomes ß-oxidation is not the only type of oxidation. This is another form which will not be explored in great detail. Peroxisomes are organelles found in cells which are also able to oxidise fatty acids. This occurs in normal situations a lot less than ß-oxidation, but occurs more in locations where the ß-oxidation pathway is damaged. It is also common with very long chained fatty acids, not a lot of which are found in human diets. The reaction sequence is essentially the same: oxidation-hydration-oxidation- thiolysis. However, the first oxidation mechanism is different (and performed with a different enzyme). It yields FADH2, which is the same as the first step of ß-oxidation. However, because peroxisomes are outside the mitochondria, rather than the electrons being passed onto the electron transport chain, they are combined with other reactants to produce hydrogen peroxide (H2O2), which is dealt with by catalase (covered in an earlier lecture; see page 51). Therefore, the FADH2 produced does not produce any ATP.
  69. 69. 68 Ketone Bodies Oxaloacetate is required for the entry of acetyl CoA into the citric acid cycle. However, during a fasting state or if a person is suffering from diabetes, oxaloacetate can also be used for gluconeogenesis. The oxaloacetate is taken out of the mitochondria and used to make new glucose. The link reaction is irreversible, so glucose cannot be generated from acetyl CoA directly, meaning that glucose cannot be generated from fatty acids. As the oxaloacetate has been removed from the mitochondria, it is not present in the citric acid cycle anymore and so this stops working. Therefore, the acetyl CoA that would enter the citric acid cycle has to be used somewhere else. Remembering that ß-oxidation is controlled by the concentrations of its products and substrates shows that it is important for acetyl CoA to be used up in some way in order to prevent the inhibition of ß-oxidation. In a starvation state, the body is reliant of the breaking down of fat to generate energy; some energy is generated through ß-oxidation so it is important that this process continues. Therefore, acetyl CoA is turned into ketones for a similar reason to why lactic acid is produced during anaerobic respiration. The steps in this pathway are explored below. Ketones: 3-Ketothiolase Two molecules of acetyl CoA enter this step and are joined together by through the energy produced when breaking one of the thioester bonds. The reaction type is a thiolysis and essentially reverses what was done in ß-oxidation, to produce a four carbon molecule.
  70. 70. 69 Ketones: Hydroxymethylglutaryl CoA Synthase This is a condensation reaction; water can be seen entering and another two carbon molecule can be seen joining, producing a six carbon molecule. The reaction is again driven by the breaking of a thioester bond from acetyl CoA. The produced molecule is relatively unstable and is produced solely as a substrate for the next step. Ketones: Hydroxymethylglutaryl CoA Cleavage Enzyme This is an irreversible lyase reaction and it can be seen to produce acetoacetate. This is the first of three produced ketones which will be transported out of the cell. It is a water soluble molecule and can be exported by acetyl transport. Tissues such as the brain use these molecules for energy. Ketones: ß-Hydroxybutyrate Dehydrogenase This reaction reduces acetoacetate. Observe that NADH is consumed and the enzyme name includes the word “dehydrogenase”, showing that this is a redox reaction. This is a reversible reaction which forms an equilibrium of acetate:butyrate. The equilibrium depends of the concentration ratio of
  71. 71. 70 NADH+H+ to NAD+ in the mitochondria. Acetone, the other product, causes the breath to smell of pear drops and is useless. It is an irreversible, spontaneous reaction which does not require an enzyme. Acetone is mostly “excreted” through the lungs as it is very volatile, which is what leads to the pear drop smell on an individual’s breath (this is known as ketoacidosis). Ketones as Fuel Some cells use ketones in preference to glucose, such as those found in heart muscle, the renal cortex (the outer portion of the kidney between the renal capsule and the renal medulla) and the brain. Normally the brain uses around 75% glucose for its power, but during starvation or fasting, it gradually alters its energy supply so that 75% of it comes from ketones. During starvation, these organs are the principle consumers of the remaining glucose in order to keep their oxaloacetate levels high in order to run the citric acid cycle, which the ketones are fed back into. Therefore, glucose is conserved for these tissues, whilst other tissues produce ketones for these tissues. As starvation proceeds, glucose levels drop and ketone levels rise, leading to ketoacidosis. Most free fatty acids are not used very well in the brain due to the presence of a blood brain barrier which they do not cross very well. Therefore, even when not starving, the brain still uses some ketones produced elsewhere for energy. The brain is therefore reliant on ketones during starvation. Ketones: CoA Transferase This is the step which forms the loop turning acetoacetate back into acetoacetyl CoA. It is used to liberate the energy stored in ketones for cells using them for energy. It uses energy from a thioester bond which drives a group transfer reaction. Therefore, fat is used to make ketones in a tissue, which are transported by the blood to organs such as the brain or heart, which use this enzyme to undo the reactions and feed the acetyl CoA back into the citric acid cycle. Essentially, this pathway is used to make sure the important organs and mechanisms of the body still function. This will be recapped later in whole body metabolism lectures.
  72. 72. 71 Ketosis Ketosis is a state of elevated levels of ketone bodies in the body. This is seen in starving individuals, or in diabetics (mostly type I). In this case, no insulin is produced meaning that no glucose is taken into the liver. Therefore, no oxaloacetate is made to process fatty acids (the citric acid cycle does not run). Adipose tissue will continue to mobilise fatty acids to attempt to provide energy as the fatty acids are not being stored. The fatty acids are taken up by the liver (which has no glucose) and are converted to ketones. These ketones are acidic and are exported by the liver into the blood as to avoid a build up of acetyl CoA which would stop ß-oxidation and therefore energy production. The blood pH gradually falls, impairing the central nervous system function (amongst other things) which causes the symptoms seen in behaviour, followed by a loss of consciousness (coma) and eventually death. Fatty Acid Synthesis Acetyl CoA Carboxylase This pathway simply involves the reciprocal steps to ß-oxidation and can therefore be covered quite quickly. However, prior to this, acetyl CoA is carboxylated to form malonyl CoA. This is performed by the enzyme acetyl CoA carboxylase and is the committed step in fatty acid synthesis. Essentially, a two carbon molecule is turned into a three carbon molecule, using ATP. Once this step has been performed, the pathway must be followed until its completion; it is essentially irreversible, making it a regulatory point.
  73. 73. 72 Regulatory Point of Acetyl CoA Carboxylase This enzyme is regulated both by covalent modification (phosphorylation) and allosteric control. The diagram below shows the different states of this enzyme. The form on the far left is fully active and working fast. The middle one is switched off, and the state on the right is partially active, where it is able to catalyse the reaction, but slowly. The top shape on the enzyme represents the active site and how open it is. Following phosphorylation, the enzyme is switched off. This reaction is performed by a kinase enzyme which requires ATP and is regulated by adrenaline and glucagon (as the body does not need to store more fat). In the opposite way, a phosphatase enzyme dephosphorylates the enzyme and switches it on. This is activated when adrenaline and glucagon are not present, and when insulin is present. The other regulation type is an allosteric mechanism which responds to citrate. When there is lots of citrate present, the enzyme is partially reactivated. This is because lots of citrate indicates that the cell has lots of building blocks and precursors from the citric acid cycle. Acyl Carrier Protein The intermediates of fatty acid synthesis, malonyl and acetyl, are covalently bonded to an acyl carrier protein (ACP). This shares structural similarities with coenzyme A and functions in the same way. The series of reactions in fatty acid synthesis react with intermediates attached to ACP. The two enzymes which perform the bonding of acetyl and malonyl to ACP are acetyl transacylase and malonyl transacylase respectively.
  74. 74. 73 Fatty Acid Synthase Fatty acid synthase is one big molecule which contains the enzymes involved in fatty acid synthesis on one polypeptide chain. It makes fatty acids up to a reasonably long length and is located in the cytoplasm. Due to this, substrates need to be transported out into the cytoplasm using a mitochondrial shuttle. Carnitine based molecules cannot be used as carnitine only transports long chain fatty acids. A different shuttle is required to move acetyl CoA out of the matrix. It uses ATP and NADH and produces NADPH. In the same way as the citric acid cycle, oxaloacetate is made from pyruvate and citrate from oxaloacetate. Citrate can be transported across the membrane into the cytoplasm. The top almost mirrors the bottom; apart from the fact that oxaloacetate is turned into malate before pyruvate. Overall, acetyl CoA has moved across the cytoplasm.
  75. 75. 74 Acyl-Malonyl ACP Condensing Enzyme This is a condensation reaction which increases the chain length. Malonyl (3C) and Acetyl (2C) are joined and a carbon dioxide is lost (decarboxylation), leaving a 4 carbon molecule, acetoacetyl ACP. The reaction is powered by the decarboxylation of malonyl ACP. Reciprocally to β-oxidation, acetyl has effectively gained two carbons. β-Ketoacyl ACP Reductase This is a reduction, as opposed to the oxidation in β-oxidation. The reducing agent is NADPH and is a key reason for its production. A developing theme that can be observed is that NADH is generated in energy yielding conditions and NADPH is required in biosynthesis pathways. 3-Hydroxyacyl ACP Dehydratase This is a dehydration reaction which prepares the molecule for the next step. This pathway is aiming for a saturated fatty acid. Enoyl ACP Reductase This is a reduction reaction as opposed to the oxidation seen in β-oxidation, and NADPH is again the reducing agent. These types of pathway are often targeted by antibacterial agents or antibiotics; this step is inhibited by the antibacterial triclosan. This ends the pathway, which begins again when butryl ACP condenses with another malonyl ACP to make a six carbon fatty acid. This cycle repeats, increasing the fatty acid chain length by two carbons every time. This continues until the molecule reaches 16 carbons in length. This is because this complex of enzymes (acyl carrier protein) cannot produce fatty acids longer than this length.

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