1 Enzymes and Metabolic Pathways Un-lecture.part II NOTE: number corresponds to slides posted on the website. 44. ETS and Oxidative phosphorylation: When we completed the Krebs cycle, all of the bonds between carbons in the glucose molecule were broken and all that remained was CO 2, H 2 O and energy captured by energy transfer molecules (ATP/GTP, NADH, FADH 2 ). Now we have to remove the electrons that were captured by NADH and FADH 2, returning them to their oxidized state (NAD +, FAD + ), so that they can go back and continue to pick up energy when other glucose molecules are broken down. NAD+ and FAD+ are derived from vitamin B. Vitamin B is a water soluble vitamin that cannot be stored in the body, so your cells must conserve the NAD+ and FAD+ so that there will be sufficient transfer molecules present in the cell during cellular respiration so that energy is not lost. If you recall, in fermentation, when we didn t have any oxygen present, these electrons were picked up by pyruvic acid, which was the final electron acceptor. In this case, oxygen is present and oxygen will become the final electron acceptor. This process takes place on the folds of the inner membrane of the mitochondria, called the cristae. A series of iron containing molecules, called the cytochromes, are embedded in the cristae. This series of molecules is called the electron transport system, or ETS. When the NADH and FADH 2 enter the ETS, the cytochromes receive a pair of electrons from them, reducing the iron for a moment. The electrons are then passed to the next cytochrome. At the end of the chain, the electrons are passed to oxygen, the final electron acceptor. While the electrons are being passed from one cytochrome to the next, the energy that was captured by the NADH and FADH 2 drives a reaction in the cell that generates ATP. This is called oxidative phosphorylation, because the cell is phosphorylating ATP (adding a phosphate to ADP so that it becomes ATP) in the presence of oxygen. The actual process by which the ATP is made is called chemiosmosis. The next few slides will go into a little more detail. 45. Electron transport system. This illustration shows the sequence of iron containing molecules that comprise the electron transport system. As the electrons are passed from one molecule to the other sequentially, ATP is produced, and the electrons and hydrogen are passed to oxygen, the final electron acceptor, producing water. 46. Electron transport: More explanations, same stuff. and no, you don t have to know the exact sequence of the cytochromes! Just understand the process.
2 47. Chemiosmosis: This slide describes the technicalities of chemiosmosis, the process by which ATP is generated as the electrons move down the ETS. Here is a simpler explanation of this and the next slide. First, think about the structure of a mitochondrion. Remember that it is an organelle that has two membranes, an outer membrane, and an inner membrane that is heavily folded. The center of the mitochondrion is called the matrix. The space between the folded membrane on the inside, and the outer membrane is called the inner membranous space. The Krebs cycle occurs in the mitochondrial matrix, and the ETS occurs on the cristae. As the electron pairs are passed from one cytochrome to the next, proton pumps are opened and hydrogen ions are pumped into the inner membranous space from the matrix. These hydrogen ions want to move back into the matrix, but the only way they can get back in is to move through special pores that contain an ATPase enzyme. As the hydrogen moves back through the pore, ATP is produced. NADH and FADH 2 enter the electron transport chain in a different spot. NADH enters earlier, so it opens more proton pumps than FADH 2. Therefore, each NADH generates 3ATP, while each FADH 2 generates 2 ATP. 48. Chemiosmosis: More technical explanation. Read the paragraph above. 49. This is an illustration of the process of chemiosmosis. 50. The final electron acceptor, oxygen: More of the same. 51. Illustration showing the electron transport chain and where some poisons act. Rotenone is an ingredient found in certain pesticides. It is poisonous because it prevents the production of ATP by blocking one of the proton pumps in chemiosmosis. Cyanide and carbon monoxide also act on the ETS by blocking the transfer of electrons to oxygen. Oligomycin is a macrolide antibiotic produced by Streptomyces species that blocks the ATPase enzyme and prevents the production of ATP. 52. Summary of aerobic cellular respiration: Make sure you understand this table and that you are able to fill out a similar one, even if the columns are not the same! You should know all of the steps of aerobic cellular respiration, how much CO2 is produced in each step, how much ATP is generated gross and net in each step, how much energy is used (or added), and what the end products of each step are. Please notice that for one glucose molecule, a total of 38 ATP is produced. In the electron transport system, the 34 ATP that are shown come from ETS and chemiosmosis. Look at the total number of NADH produced. There are 2 in glycolysis, 2 in the intermediate step, and 6 in the Krebs cycle = 10 NADH. From slide number 47 above, you see that every NADH that moves down the ETS generates 3 ATP. Therefore 10 x 3 = 30 ATP from
3 the NADH. For every FADH 2, 2 ATP are produced. You produce 2 FADH 2 in the Krebs cycle, so 2 x 2 = 4 ATP from the FADH 2. This is a total of 34 ATP from the conversion of NADH and FADH 2 to ATP in chemiosmosis. The additional 4ATP are made directly (2 ATP net in glycolysis and 2 ATP in the Krebs cycle). Therefore, you have a total of 34 ATP + 4 ATP = 38ATP. 53. Summary of aerobic cellular respiration: Illustration of the entire process. 54. Metabolism of lipids and proteins 55. Where nutrients enter the cycle to produce energy: This flow chart shows that carbohydrates enter cycle to produce energy at the beginning, in glycolysis, while fats and proteins can also be used for energy, but enter the process later, in the Krebs cycle. 56. Other energy sources: Triglycerides and amino acids can both be used as sources of energy. In fact, the American diet is so high in protein, that amino acids are often converted, and stored in the body as fat. The flow chart at the bottom shows that if you increase the amount of food that you eat, you go through glycolysis, but when you enter the intermediate step, if the body already has produced sufficient ATP, cellular respiration is inhibited and the glucose is stored as glycogen and fat. 57. Contributions: Where does the energy we use come from? The largest reserve of fuel in our bodies is the fat that we already have, our endogenous (built in) triglycerides. In a lean male, 17,500 milimoles of triglycerides are stored as adipose tissue, 300 milimoles are stored in the skeletal muscle, and 0.l5 milimoles of triglyceride are carried as very low density lipoproteins (VLDL) in the plasma. The total amount of energy stored as triglycerides amounts to about 135,000 kilocalories. (kilo = 1000). The total amount of energy that is stored as glycogen is 2000 kilocalories. When you exercise, your skeletal muscle uses fatty acid as the main energy source, with anywhere from 5-50% coming from the triglycerides (made up of glycerol and 3 fatty acids, remember?) found in the muscle and very little from the lipoproteins circulating in the blood. Why is the body using triglycerides instead of glucose for energy? Because there is so much more energy in triglycerides! In a few slides, I ll show you how to calculate the amount of energy produced with triglycerides are broken down and you can see for yourself! 58. Fuel content of 70kg person: This chart summarizes the contributions of energy sources in the average person. Notice that the smallest contribution comes from carbohydrates.
4 59. Branch point: At what point in cellular respiration does the cell decide if you should continue to break glucose down completely, and at what point is fat made instead? When sugar enters the cell, it goes through glycolysis and then enters the intermediate step. It is in the intermediate step when acetyl coa is made that we have a branch point, and the acetyl coa can go in several directions. As you learned previously, if there is no oxygen, then energy production ends with fermentation at this point. If oxygen is present, and energy is needed, then the acetyl coa goes to the Krebs cycle and on to the ETS to produce energy. If there is plenty of ATP at this point, then the acetyl coa is linked together to produce lipids. So, the branch point in cellular respiration is acetyl coa. 60. Lipid anabolism: Let s take a look first at how and why lipids are made. Lipids, such as triglycerides, are used to store large amounts of energy. If you look at the structure of a triglyceride, it s easy to see why. Triglycerides are made up of a 3 carbon molecule called glycerol, which is produced during glycolysis, and 3 fatty acid chains, made up of long chains of carbons. Fatty acids are produced at the branch point by linking together acetyl coa s (remember these are two carbon molecules that are produced in the intermediate step by removing a carbon from pyruvic acid). The same technique is used to produce other types of lipids as well. So, linking together 10 acetyl coa will make a fatty acid chain that is 20 carbons long. The three fatty acid chains are attached to the glycerol by dehydration synthesis. This is called lipogenesis. 61. Lipogenesis: This slide shows the different types of lipids that can be produced in adipose tissue and the liver from acetyl coa. 62. Lipid catabolism: This slide describes how lipids are broken down. The first step is lipolysis, which is the hydrolysis of triglycerides into 3 fatty acids and glycerol. The next step is to break the fatty acid chains down. Remember that they were made by linking together acetyl coa. So, the reverse occurs. The fatty acid chains are broken down into acetyl coa beginning from the carboxyl end (COOH) in a process called betaoxidation. The rest is easy! The acetyl coa and glycerol will be sent through the Krebs cycle, producing ATP. 63. beta-oxidation: This slide shows the chemical reactions that occur in the breakdown of a fatty acid chain and the amount of energy released at each point. 64. How much ATP is formed: We are going to figure out how to calculate the amount of ATP produced in a triglyceride made up of three 18-carbon fatty acid chains.
5 65. For each 18 C fatty acid chain: Shows the first steps to determine the amount of ATP produced. 1) If you have an 18 carbon fatty acid chain, it will produce 9 acetyl coa (remember, each acetyl coa contains 2 carbons, so you divide 18/2 = 9. The acetyl coa in a fatty acid are linked by 9-1 bonds = 8 bonds. For each bond broken 1 NADH and 1 FADH 2 are produced. For one fatty acid chain in our triglyceride then, we will produce 8 x (1 NADH + 1 FADH 2 ) = 8 NADH and 8 FADH 2. Now let s figure out how much energy is made when the 9 acetyl coa are broken down. The acetyl coa is going to go into the Krebs cycle. Each acetyl coa is broken down in one turn of the Krebs cycle, producing 1 ATP, 3 NADH, and 1 FADH 2. Since we have 9 acetyl, this will result in 9 ATP, 27 NADH, and 9 FADH Total ATP so far: Now let s calculate how much ATP we have produced so far. First of all, we made 9 ATP in the Krebs cycle. Then, we made 27 NADH for the 9 acetyl + 8 NADH for the bonds we broke when we chopped the fatty acid chain into acetyl. The total number of NADH for one 18-C fatty acid chain is 27+8 = 35. For each NADH, we will produce 3 ATP. Therefore, there will be 35x3 = 105 ATP produced. We also made 9 FADH2 from the break down of the acetyl, and 8 FADH 2 for the bonds broken when we chopped up the fatty acid into acetyl = 17 FADH 2 Each FADH 2 generates 2 ATP, therefore, we will produce 17 x 2 = 34 ATP for the FADH 2. So, for one fatty acid chain, a total of ATP = 148 ATP is produced. We must subtract 1 ATP from each fatty acid chain for the activation energy it takes to start the break down. Therefore, our net ATP for one 18-C fatty acid chain = How much ATP for the whole triglyceride? OK. Each triglyceride, you will recall, is composed of one glycerol and three fatty acids. Glycerol is derived from PGAL, an intermediary product of glycolysis. It is sent through the Krebs cycle resulting in 20 ATP. Now let s add together all the ATP we made. We have 147 x 3 fatty acid chains = 441 ATP + 20 ATP (from the glycerol) =461 ATP. So now you know why triglyceride is a better energy storage molecule than glucose. It makes a lot more energy! You should be able to calculate the amount of energy produced in a number of different triglyceride and glucose combinations. In this example, all three fatty acid chains had the same number of carbons. However, that isn t always the case. If you are given a problem like this, pay attention to the number of carbons in each fatty acid chain. 68. Amino acid metabolism: The concept of nitrogen balance refers to whether or not someone is using protein to build muscle or to grow (positive nitrogen balance), or if the protein they are eating is being broken down and used as a source of energy, as in people who are starving of cachexic (wasting away) due to cancer or some other illness (negative nitrogen balance). Proteins are made up of amino acids, and
6 amino acids can be used as an energy source. The bottom of the slide reminds you of the molecular structure of an amino acid, which contains a carboxyl end, and amine end and one of 20 radicals (because there are 20 different amino acids. 69. Transamination: Pay attention to the purpose! This is a process whereby the amine group of an amino acid is transferred to a keto acid (such as pyruvic acid) and converted into an amino acid. This happens when your cells are translating and do not have a particular amino acid that they need to continue. If they have the correct radical available, they will make the one they need. There are some amino acids, however, that are essential and these cannot be made. They have to be in the diet. 70. Transamination: This shows the two molecules, pyruvate (pyruvic acid) and alanine. Alanine is formed by transamination of pyruvate. 71. Oxidative deamination: Pay attention to the purpose! This process is used by the cells to convert an amino acid either directly to energy or to convert it to glucose or store it as fat. It is the opposite of transamination, in that you are removing an amine group, which is sent out of the body as ammonia in the urine, and the resulting keto acid is then sent to the Krebs cycle. If energy is needed, aerobic respiration is completed. If there is sufficient energy, acetyl coa is linked at the branch point to produce fats. If any of you have ever worked in a convalescent home where there are cachexic elderly patients, you know that the ammonia in the urine is often so strong that your eyes feel like they are burning. 72. Additional energy sources: Energy containing nutrients are found in the blood and include glucose, ketone bodies, fatty acids, lactic acids, and amino acids. Some parts of the body use certain energy sources preferentially. One of these is the brain. Due to the extreme ph sensitivity of the brain, it cannot function using ketone bodies for long. So, the rest of the body will spare glucose for the brain so that the brain has what it needs. This is called glucose sparing. You already know about oxygen debt. When insufficient oxygen is available during strenuous exercise, pyruvic acid is converted to lactic acid in the muscle. This can then be converted back to pyruvic acid and go through the Krebs cycle when sufficient oxygen becomes available. 73. The role of the liver: In the liver, glucose can be produced from certain substances in the body (gluconeogenesis), glycogen can be produced (glycogenesis), and glycogen can be broken down (glycogenolysis). Substances can only be converted to free glucose in the liver. 74. Gluconeogenesis: This is also called the Cori cycle. The liver contains an enzyme called lactic acid dehydrogenase. This enzyme catalyzes the
7 conversion of lactic acid to glucose-6-phosphate. The final product in this process is the production of glucose. Glucose can be made by this method using lactic acid, keto acids, and glycerol. 75. Glycogenesis and glycogenolysis: Remember that glycogen is a polymer that acts as a storage form of glucose in vertebrates. This is necessary because glucose cannot be stored in large quantities in the cell. Glucose is put into a metabolic pathway almost immediately. If it were to accumulate in the cell, it would cause glycosylation of other molecules and structures that could damage or kill the cell. Also, as more sugar accumulates in the cell, the inside of the cell becomes hyperosmotic and draws in water from the ECF, causing the cell to swell and burst. Therefore, organs that need lots of energy quickly can store glucose in the form of glycogen. Glycogenesis is the formation of glycogen from glucose, while glycogenolysis is the hydrolysis of glycogen to produce glucose-6-phosphate. 76. Glycogenesis: This slide shows the pathway of glycogenesis. Notice that glucose becomes glucose-6-phosphate, but instead of being rearranged to fructose-6-phosphate (which happens in glycolysis), it is converted to glucose-1-phosphate. These molecules are then linked together (polymerized) in the presence of an enzyme called glycogen synthetase to produce glycogen. 77. Glycogenolysis: This is the opposite of glycogenesis. Glycogen is converted to glucose-1-phosphate in the presence of an enzyme called glycogen phosphorylase. The glu-1-phosphate is then converted to glu- 6-phosphate. 78. Glucose-6-phosphate: Because the plasma membrane is hydrophobic by nature, this molecule cannot leave the cells in which it is formed. When it is made in skeletal muscle, the skeletal muscle uses it as glucose-6-phosphate. But, the only place in which it can be converted into free glucose, which can leave the cell and move into the blood, is in the liver. Here the phosphate group is removed, and this allows glucose to move about freely. 79. Essential nutrients: This slide defines what we consider essential. Notice that it something that we need, but cannot make in the body. A short list of some of the essential nutrients follows. 80. Vitamins: Why do we need vitamins? You already know about the importance of NAD+ and FAD+. Other vitamins are needed as coenzymes and cofactors for enzymes. So, they are required for the enzyme to be the correct shape to function properly. A list of water soluble and fat soluble vitamins is given.