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Transcript
CELLULAR RESPIRATION Teacher's Guide Cellular Respiration Teacher's Guide This teacher's guide is designed for use with the CellularRespiration series of programs produced by TVOntario, the television service of the Ontario Educational Communications Authority. The series is available on videotape to educational institutions and nonprofit organizations. The Series Producer/Director . Project Officers. Writers: Consultant. David Chamberlain John Amadio, David Way Susan Perry, David Way Robert Whitney The Guide Writers: Graphic Designer: Randee Crisp, George Laundry, Robert Whitney Roswita Busskamp Copyright 1990 by The Ontario Educational Communications Authority. All rijahts reserved. Printed in Canada. 3626/90 Introduction 1 1. The Cell and Energy 3 2. Glycolysis 1 6 3. Glycolysis 2 9 4. The Krebs Cycle 12 5. Oxidative Phosphorylation 14 6. Metabolism and Nutrition 17 Glossary 19 Bibliography 21 This series of six 10-minute programs illustrates the complex world of biological respiration, at both macro and molecular levels. Beginning with a historical perspective and progressing to modern research and theories, the programs examine enzymes and coenzymes, phosphorylation, biosynthesis, glycolysis, and the Krebs cycle. Together, the Cellular Respiration video series and teacher's guide: • describe the evolution of cellular respiration that presaged the development of presentday life forms; • investigate the structure and function of the mitochondrion organelle as the prime locus for the biosynthesis of adenosine triphosphate (ATP); 1 • discuss glucose as the principal fuel of cellular respiration and the involvement of ATP as the. energy shuttle; • develop, in step-by-step fashion, the metabolism of glucose through the processes of glycolysis, the Krebs or citric acid cycle, and oxidative phosphorylation; • elucidate the role of oxygen in the controlled combustion of glucose with the concomitant production of the respiratory waste product carbon dioxide; and • explain the relationships of the three food groups-proteins, fats, and carbohydratesin nutrition. After viewing this program and completing the suggested activities, students should be able to: • name three major classes of molecules that living things use to store energy, and designate carbohydrates as those most frequently employed; • explain the meanings of the following terms: cytosol, mitochondrion, matrix, cristae, adenosine triphosphate (ATP), high-energy bond, phosphoryl group, adenosine diphosphate (ADP), phosphorylation; • describe the appearance of a mitochondrion as seen through the transmission electron microscope; Regardless of its source, energy for living things must be readily available at all times. Since inputs are irregular and unreliable, constant availability necessitates some form of energy storage. A brief overview of the mechanism of energy storage and release is the subject of this introductory program. The digestive system extracts from an animal's food the three major groups of macromolecules: proteins, fats, and carbohydrates.The most immediately available energy has been stored in carbohydrates. This series assumes that most energy is provided to the cell in the form of glucose molecules. The release of chemical energy, to a form useful to living things, is called cellular respiration. • account for the theory that both mitochondria and chloroplasts evolved from independent organisms; • describe the structure of an ATP molecule and locate, within this structure, high-energy bonds; • explain the role of ATP in cell metabolism; • name three interconnected phases of cellular respiration. Crista 3 Cellular respiration is a complex series of chemical reactions that occur in both the cytosol and the mitochondria of a cell. A mitochondrion consists of a pair of membranes surrounding an amorphous interior, the matrix. The innermost membrane forms many inward-facing folds, the cristae, which greatly increase the amount of membrane that can be packed within the mitochondrion. The similarity of a mitochondrion to a tiny cell suggests that the mitochondria, like the chloroplasts, may have evolved from independent beings that invaded larger cells as parasites. Over millions of years, they became tolerated by, then vital to, their hosts. As a consequence, there are many similarities between cellular respiration and photosynthesis. In fact, in many ways, cellular respiration can be considered the reverse of photosynthesis. The reactions of cellular respiration, which provide the ATP needed to drive life processes, are subdivided into three phases: glycolysis, the Krebs cycle, and oxidative pbospborylation. All three phases will be covered, in turn, by this series. Cellular respiration transfers most of the glucose molecules' energy into smaller "packages" of potential energy in molecules of adenosine triphosphate (ATP). ATP molecules contain enough energy to drive typical metabolic reactions. BEFORE VIEWING Some students may have little exposure to chemistry. A short lesson on (or review of) the concepts of element, compound, atom, molecule, and covalent bond should precede the program. Emphasize that a detailed knowledge of the structures of respiratory intermediates is not necessary. Instead, the student should appreciate that molecules have unique and predictable shapes, and that cells possess specialized agents (enzymes) that are able to select one type of molecule from among the multitude of other molecules present in the cell. A quick review of a typical food chain and the place of autotrophs and heterotrophs within it could also be useful. ATP is a complicated molecule consisting of portions of a number of simpler, and more familiar, molecules linked by covalent bonds. The simple "building blocks" are a nitrogen-containing base (adenine), a five-carbon sugar (ribose), and three molecules of phosphoric acid. The energy resides at one of two higb-energy bonds between the remanants of phosphoric acid molecules (phosphoryl groups). When an ATP molecule provides energy to a reactant, it transfers one of its "high-energy bonds" to the reactant. Of course, some atoms of the ATP are also transferred. Typically, the end phosphoryl group is transferred to the reactant, and adenosine dipbospbate (ADP) is left over. The reactant is now said to be "phosphorylated" and the process of transferring a phosphoryl group to the reactant is called phospborylation. Phosphorylation reactions are often employed in metabolism as a step in an energy-consuming reaction. AFTER VIEWING Activity l: How Carbohydrates Got Their Name Apparatus sugar cubes concentrated sulphuric acid (Caution: highly corrosive) crucible mortar and pestle protective cover for desktop safety goggles laboratory coat or apron Note: This activity maybe performed as a demonstration. Method 1. Grind a sugar cube to a powder using a mortar and pestle. 2. Transfer the powdered sugar to a crucible which has been placed on a protective cover to prevent damage to the desktop. FIGURE 1.1 Structure of ATP 4 3, Be sure you are wearing safety goggles. Add just enough concentrated sulphuric acid to the crucible to cover the sugar. 4. Note the color, odor, and appearance of the material left in the crucible. What do you think it is? hydroxyl groups (-OH) are in the correct positions above or below the ring. Use as few shifts of atoms and/or bonds as possible. In your notes, record the steps you followed in this conversion. Also record the number of times you had to rotate a part of the molecule without shifting bonds or atoms. Compare your results with those of other students in the class. Have your model evaluated by your instructor before proceeding. Be sure to make any alterations suggested by the instructor before continuing. Discussion Concentrated sulphuric acid is a powerful dehydrating agent which will withdraw water from other compounds, Assume that this will happen in this experiment. In terms of elements, what appears to be the composition of the sugar, based on the color of the resultant residue? Why, then, are this and other sugars referred to as "carbohydrates"? 5. Evaluate the flexibility of the model. Is the positioning of a hydroxyl group (-OH) on the top or bottom of the formula significant? Comment in your notes. Activity 2: Visualizing Molecules Discussion 1. The formula of glucose is given in textbooks as C6H1z06. To which of the structures in Figure 1.2 does it apply? Research the meaning of isomer and isomerization and explain how these terms relate to this activity. Apparatus molecular model kit Method 2. Cellobiose is a disaccharide formed during the digestion of cellulose, and maltose is a disaccharide formed during the digestion of starch. Research the structures of these two sugars and relate them to this activity. Can enzymes distinguish between these two disaccharides? 1. Examine the contents of the molecular model kit. Note that there are wood spheres of various colors. These represent atoms of the elements. You will be using only carbon (black), hydrogen (white), and oxygen (red) in this exercise. 2. Construct a model of a glucose molecule. Use the structural formula on the left of Figure 1.2 for guidance. When the model has been completed to your satisfaction, take it to your instructor for evaluation. Make any alterations suggested by your instructor before you continue. 3. Evaluate the flexibility of the model. Is the positioning of a hydroxyl group (-OH) on the right or left of the formula significant? Comment in your notes. 4. Now, attempt to convert your model into the ring form depicted on the right of Figure 1.2, Consider the ring to be perpendicular to the page and be sure that the hydrogens (-H) and FIGURE 1.2 Two Glucose Formulae (Ring Structure) 5 In the first half of glycolysis, the 6-carbon sugar glucose, is broken into two 3-carbon molecules of phosphoglyceraldehyde (PGAL). This requires the addition to the original glucose molecule of chemical potential energy supplied at ATP. After viewing this program and completing the suggested activities, students should be able to: Glucose arises principally from the hydrolysis of glycogen, a polysaccharide stored in the liver and muscles. From the liver, glucose may be carried by the circulatory system to target cells which it enters easily by membrane. Upon arrival in the cytosol, the glucose is phosphorylated by ATP in an enzyme-catalyzed reaction. Its potential energy is thereby increased; it also acquires a negative charge which prevents its escape from the cell. The glucose phosphate is isomerized by an enzyme to fructose phosphate which then acquires a second phosphate group reaction with a second ATP.The fructose diphosphate is then split into two parts: dihydroxyacetone phosphate (DHAP) and phosphoglyceraldehyde (PGAL). The DHAP quickly undergoes isomerization to a second PGAL. Thus, a single 6-carbon glucose molecule has generated two 3-carbon PGAL molecules. Two ATP molecules have been sacrificed, but the two PGAL molecules have a higher potential energy than the original glucose molecule. identify the major compound in which animals store energy; outline the steps by which the first half of glycolysis occurs; describe the change in chemical potential energy accompanying the preparatory steps of glycolysis; discuss the "coupling" of energy-consuming and energy-releasing reactions; identify the cytosol as the site of glycolysis. The energy that enters a cell in an energy-rich fuel such as glucose must be used to synthesize ATP molecules in order to be used effectively. The process begins with a series of reactions known collectively as glycolysis These reactions must have evolved a very long time ago since they exist, in identical form, in all living things. The series devotes two programs to the description of glycolysis. This first program on the subject shows that energy (ATP) must first be sacrificed in order to prepare the way for its later extraction. It also indicates the importance of thermodynamic principles in explaining the progress of the reactions. Figure 2, 1 Endergonic. This program traces the first 5 steps of glycolysis. In these steps, energy must be added to the system (endergonic) 6 The reaction of ATP with fructose phosphate illustrates the important concept of reaction coupling. The conversion of glucose phosphate to fructose phosphate has a small positive free energy change. Fructose phosphate tends to spontaneously revert to glucose phosphate. However, any fructose phosphate that forms is phosphorylated to fructose diphosphate in a reaction with a large negative free energy change. Thus, the net reaction changing glucose phosphate to fructose diphosphate (in two steps) has a negative free energy change and proceeds spontaneously. Many reactions in biochemistry are driven against thermodynamic tendencies by being coupled to a simultaneous reaction having a large negative free energy change (often, the hydrolysis of ATP). Note: Numbers appearing in the following instructions are not given in the program. They indicate the number of the carbon atom(s) bearing phosphate groups or to be included in a product. The numbers are those appearing in Figure 1.1 (Program 1) in glucose or derived from these in a chemical reaction. Method Use the model of glucose (non-ring form) constructed in Program 1, Activity 2, or construct another following the instructions in that activity. Using a single hole in an orange-colored atomic model to represent phosphate, trace, with models, the conversion of glucose to glucose-6-phosphate (you must discard a hydrogen atom to make room for the phosphate), glucose-6-phosphate to fructose-6-phosphate (move the doubly bonded oxygen from carbon atom #1 to carbon atom #2 but do not discard any atoms), and fructose-6phosphate to fructose- 1,6-diphosphate. BEFORE VIEWING Students should have some understanding of thermodynamic principles. These can be developed to different levels, according to the needs and expectations of the class. This could involve a discussion of the First Law of Thermodynamics. Discussion 1. The change of glucose-6-phosphate to fructose-6-phosphate is described as an isomerization. By what feature is a reaction identified as an isomerization? A qualitative understanding is most easily derived from a consideration of the relative probabilities of different distributions of energy and matter in a chemical system (developed from a knowledge of the probabilities of different numbers arising when dice are rolled). This could be expanded into a discussion of the Second Law of Thermodynamics. With some classes, you might continue to discuss the chemical implications of the Third Law of Thermodynamics (often called the Nernst Heat Theorem). 2. You removed a hydrogen atom to make room for a phosphate during phosphorylation of a sugar. Hydrogen atoms cannot float about freely in solution. Research and report on the actual fate of the hydrogen you had to remove. 3. Splitting fructose-1,6-phosphate into dihydroxyacetone phosphate and phosphopglyceraldehyde requires the discarding of a car bon-carbon bond (between carbon atoms 3 and 4 of the fructose-1,6-phosphate). No atomic nuclei are lost or gained, but one or more could change position. Using this information, predict the structural formula of dihydroxyacetone phosphate (from carbon atoms 1, 2, and 3 of fructose-1,6-phosphate) and phosphoglyceraldehyde (from carbon atoms 4, 5, and 6 of fructose-1,6-phosphate). AFTER VIEWING ACtivity 1: Modelling the Reactions of Glycolysis Apparatus molecular model kit 4. The enzyme converting dihydroxyacetone phosphate into phosphoglyceraldehyde is called "triose phosphate isomerase." Discuss the appropriateness of this name. 7 3. is it possible to find a total measurement of the heat given off.? (Hint: consider the heat energy absorbed by the water.) 4. Is there a link between the molecular structure of a food substance and the energy released? Discuss. Activity 2: Measuring the Relative Quantities of Energy Released from Different Foods Apparatus assorted foods (nuts, sucrose cube, marshmallow, etc.) calorimeters needles corks water (at room temperature) matches petri dishes balances clay triangles thermometers Thermometer Test tube Method 1. Put 10 mL of water at room temperature into a test tube. Fit the test tube into the top of the calorimeter, as shown in the diagram. 2. Weigh and record the mass of the food. 3. Record the temperature of the water in the test tube. 4. Drive a pin through the centre of a cork and attach the food material to it at the top, as shown in the diagram. 5. Light the food material with a match, and fit the calorimeter over it so that the bottom of the test tube is directly over the flame. 6. When the flame has gone out (after about 2 min of heating), record the final temperature of the water. 7. Reweigh the food material and record its mass. 8. Repeat this procedure for each of the other food substances. 9. Calculate the temperature change caused by each food substance per unit mass of that substance burned. Water Soup can Nut Petri dish Discussion 1. Compare each of the food materials used with respect to their energy per unit mass. Which food types contain the most energy per unit mass? 2. Describe the possible sources of error in this experiment. 8 respiration: the entire sequence of ten reactions transfers only about two percent of the chemical potential energy of a glucose molecule to the production of ATP. The program shows how simple organisms like yeast fulfill their energy requirements from what little useful energy glycolysis produces by linking it to fermentation. Fermentation converts pyruvate to acetaldehyde then to ethanol and in the process regenerates the NAD molecule. The NAD then cycles back into glycolysis and maintains the production of ATP. The discussion of fermentation provides an important example of biofeedback mechanisms. After viewing this program and completing the suggested activities, students should be able to: • trace the steps in the conversion of phosphoglyceraldehyde to pyruvic acid; • account for the net gain of ATP during glycolysis; • explain the difficulties encountered if a cell reduces its entire complement of NAD; • describe the importance of anaerobic fermentation in ensuring the continued production of ATP as long as it is required and glucose is available; • account for differences in identity and quantities of products of cellular respiration under aerobic and anaerobic conditions. While glycolysis is able to meet the demands of simple organisms, more complex organisms need additional reactions to harness the energy contained in the pyruvate and NADH molecules. The program concludes by introducing the next stage of cellular respiration-the Krebs Cycle-where pyruvate is used to make additional ATP molecules. BEFORE VIEWING Review the concepts of oxidation and reduction: students should be able to identify these processes by monitoring the transfers of electrons and/or hydrogen atoms during organic chemical reactions. This third program of the series completes the discussion of glycolysis by tracing the sequence of reactions from PGAL to the final product, pyruvate. The program illustrates the use of PGAL's potential energy to synthesize ATP molecules and to reduce nicotinamide andenine dinucleotide (NAD) to form the intermediate energy carrier molecule NADH. It is useful to establish a series of 1-carbon molecules arranged in order of decreased reduction status or increased oxidation state based upon the hydrogen to oxygen ratio. The series should include methane, methanol, formaldehyde, formic acid, and carbon dioxide molecules. The relative positions of fats, carbohydrates, and a few amino acids in the reduction scale should be investigated. The net energy production of glycolysis demonstrates the inefficiency of this phase of cellular 9 Since texts vary in their naming of intermediates of cellular respiration, students should be made aware that acids may be named as if they were not ionized (e.g., pyruvic acid) or as their anions (e.g., pyruvate). The latter represents the form found at physiological pH, but the former makes it easier to follow the fate of hydrogen atoms and the formation of water during biochemical reactions. Glass U-tube One-hole stopper Two-hole stopper AFTER VIEWING Activity 1: To Detect the Waste Products of Fermentation (Anaerobic Respiration) Apparatus yeast packets (enough for number of pairs of students in class) sucrose solution bromthymol blue solution Benedict's solution Erylenmeyer flasks (125 mL) one-hole and two-hole rubber stoppers to fit flasks graduated cylinders (50 mL) glass U-tubes test tubes stirring rods marking pens test tube holders test tube racks Bunsen burners rings ring stands wire gauze beakers beaker tongs flints safety goggles Method 1. Pour 15 mL of warm water into a 125 mL Erylenmeyer flask. 2. Add a packet of yeast to the water and mix with a stirring rod. Add 50 mL of sucrose solution to the yeast mixture and mix well with a stirring rod. 3. Put on safety goggles. FIGURE 3.1 Apparatus to Detect the waste products of fermentation 4. Set up a hot water bath using wire gauze and a ring on a ring stand above a Bunsen burner. Heat 200 mL of water in a beaker until it comes to a slow boil. 5. Add 5 drops of Benedict's solution and 5 mL of sucrose solution to a test tube, and label this test tube A. 6. Add 5 drops of Benedict's solution and 5 mL of yeast and sucrose solution to a test tube, and label this test tube B. 7. Heat test tubes A and B for 5 minutes in the water bath, and record any changes. 8. Put 50 mL of bromthymol solution into a second Erylenmeyer flask. 9. Insert one end of a glass U tube into a onehole rubber stopper and into a two-hole rubber stopper at the other end. Insert the one-hole stopper into the flask with the yeast mixture, and the two-hole stopper into the flask containing the bromthymol blue solution (see Figure 3.1). Note: The longer end of the U-tube should be below the level of bromthymol blue solution. If necessary, use glycerin as a lubricant. If the end of the tube is not below the level of the solution, call your teacher for assistance. DO NOT adjust the tubing yourself. 10. Leave the apparatus setup in a warm place overnight. 10 11. After 24 hours, replace the flask containing 50 mL of bromthymol blue solution with another flask containing 50 mL of fresh bromthymol blue solution. Record any observations. 12. Record any observations after 48 hours. Discussion 1. Discuss the reasons for the change or lack of change in: • test tubes A and B after heating • the bromthymol blue after 24 hours • the bromthymol blue after 48 hours 2. Discuss the difference in products formed between anaerobic respiration which takes place in yeast cells (fermentation) and anaerobic respiration which takes place in animal muscle cells. Chemical reaction FIGURE 3. 2 Exergonic. In the second series of events in glycolysis, excess energy is released (exergonic) Activity 2: Early Anaerobic Biochemistry Organisms that emerged during the first billion years of the development of life on earth used no atmospheric oxygen to fuel their activities. They could fuel their metabolism only by ATP generated by glycolysis, which is thought to have been one of the earliest of all biochemical processes to have evolved. 1. 2. Discuss the kind of organisms that would have probably been alive at that time. Explain and give the range of variability of possible life forms. Identify and discuss the kinds of organisms that still survive today, using glycolytic reactions alone to produce the ATP needed to carry on their metabolic activities. 1 1 After viewing this program and completing the suggested activities, students should be able to: • appreciate that prehistoric life on land must have been preceded by the emergence of a glycolytic cycle and the accumulation of atmospheric oxygen; • recognize that glycolysis does not result in sufficient energy for energetic life forms; • trace and understand the sequences in the Krebs cycle (citric acid cycle); • identify the end products of the cycle; • state the energy products or carriers resulting from this cycle; • explain the fate of the glucose's carbon atoms. Survival depends upon the availability of large reserves of energy. Glycolysis, however, is an inefficient source of energy and cannot supply these large reserves; therefore other phases of energy production are required. This program examines the second phase of cellular respiration, the Krebs cycle. The program follows the fate of pyruvate from glycolysis as it is acted on as a substrate by enzymes within the mitochondria to generate ATP and intermediate energy carriers. Pyruvate is a 3-carbon molecule. Through oxidative decarboxylation in the cytosol it is transformed into the 2-carbon molecule acetyl-CoA which enters the mitochondrion. Once inside the mitochondrial matrix, acetyl-CoA transfers its energy into the Krebs cycle. The program follows the ten reactions of the Krebs cycle, focusing on the production of energy carriers. A review of the Krebs Cycle shows that the energy input from each acetyl-CoA creates one ATP molecule, one FADH Z , and three NADH molecules. Since each glucose molecule from glycolysis results in two molecules of acetyl-CoA, the cycle is considered to turn twice. A summation of glycolysis, oxidative decarboxylation, and the Krebs cycle together gives the total energy products from one glucose molecule as: four ATP molecules, ten NADH molecules, and two FADHZ molecules. The carbon atoms of the glucose molecule have been expelled as six molecules of waste carbon dioxide. Most of the energy of glucose has been transferred to the intermediate energy carriers NADH and FADH Z . The program concludes by setting up the final stage of cellular respiration, oxidative phosphorylation, where the intermediate energy carriers are used to synthesize numerous ATP molecules. The steps in the cycle-can be summarized as follows: 1. Acetyl-CoA reacts with oxoaloacetate to form citric acid. 2. Citric acid loses a molecule of water to become aconitate. 3. Aconitate adds water and is isomerized to become isocitrate. 4. Isocitrate encounters NAD+, forming oxalosuccinate and NADH. 5. Oxalosuccinate loses a molecule of CO 2 to become ketoglutarate. 12 6. Ketoglutarate reacts with CoA to form succinyl-CoA and a NADH molecule. 7. Succinyl-CoA joins with ADP and a phosphate to release CoA, an ATP molecule, and succinate. 8. Succinate joins with an FAD molecule to form an FADH 2 molecule and fumarate. 9. Fumarate adds water to become malate. 10. Malate reacts with NAD+ to become oxaloacetate and form a NADH molecule. A summary of total energy products can be given as follows: BEFORE VIEWING Help the students to consolidate the previous material by stressing the following points. 1. Glycolysis is a very inefficient process: it yields only about 2% of the available energy of glucose. Glycolysis alone, therefore, could not provide the energy needed to power energetic organisms. 2. Pyruvate formation was the end process of the glycolytic pathway; this pyruvate will be the starting point of the Krebs cycle. AFTER VIEWING 1. In total, this process utilizes approximately 40% of the available energy, whereas glycolysis utilizes only about 2%. Divide the class into two main groups. One group, which can be subdivided into several research sections, is to write out the structural formulae for all of the Krebs cycle intermediary compounds, including high-energy transfer compounds (NADH and FADH) and other products such as CO 2. The other group is to build the intermediaries from atomic model kits, using the standard color codes to represent different kinds of atoms. Both groups should thoroughly brief their members with an eye to presenting a detailed account of their results to the class. 2. NADH Figure 4. 1 Energy release from the Krebs Cycle (The cycle can be considered to turn twice) 13 Discuss the following points. a. It is evident that glycolysis does not produce enough ATP energy for higher life forms to carry out their activities, b. Why would glycolysis and the Krebs cycle functioning together in tandem still not provide enough energy to fuel complex organisms? c. What is the significance of the word cycle in the term Krebs cycle? What substance is regenerated at the end of the cycle and is used at the beginning of the next one? Why is this cycle gone through twice for the complete respiration of each glucose molecule? After viewing this program and completing the suggested activities, students should be able to: • trace how phase 1 and 2 of cellular respiration lead into oxidative phosphorylation; • describe the process of the electron transport chains; • understand the role of oxygen in siphoning electrons from the electron transport chains; • explain how the energy gradient across the intermitochondrial membrane is created, and why this gradient is important; • follow the steps in ATP synthesis at the matrix side of the membrane; • sum up the total production of ATP, NADH, and FADH2 from a single glucose molecule; • state how many ATP molecules are produced at any step. Cellular respiration in its first phase, glycolysis, produces only two molecules of ATP. Phase 2, the Krebs cycle, produces only two more ATP. However, phase 3, oxidative phosphorylation, produces an energy payload. This process takes place within the inner mitochondrial membrane. Embedded within this membrane are four adjacent protein complexes that make up the electron transport chain.Three of these complexes act as proton (H+) pumps. Their function is to remove energy from the electrons as they move in pairs down an energy gradient. The process begins as NADH donates two electrons to the first complex. Two hydrogen ions hitch a ride into the intermembrane space and the two electrons transfer to the second complex and return to the matrix side of the membrane. Two more hydrogen ions are moved into the third complex and are carried to the intermembrane space. Two electrons return down the fourth complex and two more hydrogen ions move into the intermembrane space. (Six hydrogen ions have now crossed.) Finally, an oxygen atom picks up two electrons and two hydrogen ions and forms water. (It is the primary role of the oxygen to siphon the electrons from the electron transfer chains.) The other energy carrier produced by the Krebs cycle, FADH 2, enters the chain and results in four more hydrogen ions being transferred to the intermembrane space. The concentration of H+ is much higher in the intermembrane space than on the matrix side. This concentration results in a potential energy gradient, and this energy will be used to synthesize ATP. Pairs of protons (H+) are moved down special channels; these protons activate an enzyme on the matrix side. This enzyme catalyzes the reaction of ADP with a phosphate group to synthesize ATP. 14 In summary, glycolysis results in two ATP molecules plus four more at the electron transport chain, for a total of six ATP molecules. Oxidative decarboxylation and the Krebs cycle produce two ATP, eight NADH, and two FADHZ molecules. The eight NADH energy carriers produce 24 ATP molecules, and the two FADH Z produce another four ATP molecules. The net result is 36 molecules of ATP. Therefore, cellular respiration results in 36 ATP molecules from one glucose molecule; this represents about 41% of the available energy from the glucose molecule. BEFORE VIEWING 1. 2. AFTER VIEWING Activity 1: The Energy of Carbohydrates The catabolic metabolism of glucose could be expressed as follows: Note that 36 molecules of ATP are ultimately produced from 1 molecule of glucose. Students should review the structure of the mitochondria., and consider such terms as cytosol, intermembrane space, cristae, matrix, and electron transport chain. They should review, as well, these processes: diffusion, osmosis, and active transport. 1. 2. Review the Krebs cycle in terms of where the intermediate energy carriers NADH and FADH Z are given off. How many ATP molecules can be produced from 1 mole of glucose? (Recall that 1 mole contains approximately 6 x 10 23 molecules.) Each mole of ATP represents a capture of 31 kJ. Calculate the total energy available for the 36 ATP molecules. Cytoplasm Mitochondrion Figure 5.1 An overview of oxidative respiration 1 5 3. One mole of glucose represents about 2831 kj (this value might differ slightly in different textbooks). From your answer to question 2 above, calculate the overall efficiency. 4. Given that glucose has a formula of C6 H12 06, calculate its molecular mass. 5. Suppose a candy bar contained 90 grams of 100% glucose. Theoretically, how much energy could it release in kilojoules? Theoreti-cally, how many molecules of ATP could be produced? Activity 2: Mitochondria Morphology 1. Consult a suitable text containing large electron micrographs of mitochondria. Study photographs from muscle tissue and from at least two other types of tissue (e.g., liver, pancreas, kidney, digestive tract, etc.) and obtain clear photocopies of them. 2, Discuss the differences and similarities between mitochondria from the different tissues, and relate this to their tissue function. 3. Identify the outer and inner membranes, cristae, and matrix of a mitochondrion. 4. Where are the respiratory proteins located? What is the ultimate fate of the electrons at the end of the electron transport chain? What drives the protons across the inner membrane and what is their ultimate fate? Discuss. 5. During fermentation (anaerobic respiration) what is the fate of the electron generated during the glycolysis of glucose? 16 After viewing this program and completing the suggested activities, students should be able to: • recognize that much of our knowledge about cells comes from the development of models; • appreciate the immense turnover of ATP in the human body in a normal day; • understand the basic operation of a muscle; • describe how cells respond to an oxygen shortage caused by overexertion; • describe how an oversupply of ATP may be stored eventually as "fat"; • appreciate that the complexity and collective behavior of cells is a reaffirmation of life itself. Scientists frequently develop models to explain the complexity of cellular respiration. These models, though, are often schematic diagrams and do not come close to revealing the magnificence of the collective power of cells. Our bodies use and recycle about 40 kg of ATP each day, and strenuous activity may cause them to use as much as 0.5 kg per minute. For all body movements, it is ATP which provides the driving energy. This program examines the ability of cellular respiration to adjust to different conditions in the human body. The program begins with modelling the role of ATP in the contraction of a muscle. The action of ATP is shown on the two proteins in muscle cells actin and myosin. In time of overexertion, the body may suffer a temporary oxygen shortage as the circulatory system cannot provide the oxygen quickly enough. While glycolysis can provide a small quantity of ATP, not enough is synthesized and this results in an energy shortage. The program describes how the process of cellular respiration takes steps to overcome this shortage. The pyruvate that normally heads off to the Krebs cycle follows a different path when oxygen is in short supply-a path that leads to the synthesis of lactic acid. The steps in this sequence ensure the continuous production of ATP. There is, of course, a debt to pay: a burning sensation within the muscles caused by the lactic acid buildup. Fortunately, after a short rest, the return of oxygen results in the metabolism of the lactic acid. Too much glucose intake, on the other hand, can result in the production of too much ATP. This surplus triggers a sequence of events whereby acetyl-CoA produces fatty acids that are stored as fat. This process can be reversed by dieting, in which the fat can be metabolized. This is done through sequences that lead either to the glycolytic pathway or directly into the Krebs cycle. Throughout this series the programs have depicted how resourceful cells are and how the collective behavior of a cell is a reaffirmation of the driving force of life itself. 17 FORE VIEWING 1. 2. it could be advantageous for the student to recall or to look up the general structure of a muscle. Recognition of such things as the protein layers of actin and myosin and the mechanics of muscle contraction would be helpful. Review program 4 with special reference to the section on the electron transport chain. Review in particular the purpose of oxygen and the role of NAD+ and its development. AFTER VIEWING Activity 1: Muscle and Fat Energetics Discuss each of the following: 1. What role does each of the following play during the contraction of a muscle: pyruvate, NADH, NAD+, lactic acid, ATP, ADP, glycogen, and oxygen? 2. Explain what happens when a muscle is overexerted as during strenuous exercise and how this condition is alleviated. 3. Discuss the conversion of energy during muscle contraction. 4. What are some other uses of ATP by cells of multicellular organisms? 5. Trace the catabolism of fatty acids through the Krebs cycle. How does the ATP yield from a 6-carbon fatty acid compare with the ATP yield from glucose? What problem occurs if fat catabolism is excessive? Activity 2: Observation of Skeletal Muscle Apparatus beef toluidine-blue stain prepared slides of skeletal muscle prepared slides of cardiac muscle, if available microscopes microscope slides cover slips dissecting needles medicine droppers forceps Method 1. Obtain a piece of beef from your teacher. Pull the point of the dissecting needle across the long grain of the muscle several times until a small strand of tissue is removed. Caution: Use the dissecting needle with care as it is very sharp. 2. Using forceps, transfer the strand of beef to the centre of a clean slide. 3. Put 2 drops of toluidine-blue on the tissue. Let the stain remain for 2 minutes, then add 2 drops of water to the slide. Cover the tissue with a cover slip. 4. Examine the tissue under the microscope at low power. Focus on a portion that is thin and lightly stained. Draw a portion of what you see. 5. Switch to high power. Look for the striated appearance of the muscle cells. Muscle cells are made of microfilaments called myofil aments, which are composed of the proteins actin and myosin. The portion of muscle from one stripe to the next is called a sarcomere. The darkly stained structures are the nuclei of the muscle cells. Locate the sarcomeres and the nuclei, and draw a diagram labelling these structures. 6. Use high power to examine prepared slides of skeletal muscle and, if available, cardiac muscle. Discussion 1. From your observations, is a muscle fibre composed of several small cells, or one long cell containing many nuclei? 2. Discuss the role of myosin, ATP, and actin during the contraction of a muscle cell. 3. What initiates contraction in vertebrate skeletal muscle? What other chemicals are involved? 18 decarboxylation the removal of the carboxyl group (COON) from an organic molecule acetylCoAthe main molecule of energy metabolism; contains a high energy bond actin one of two proteins making up the microfilaments of muscle tissue adenine an organic base consisting of two carbon-nitrogen rings ADP adenosine diphosphate, a substance produced when ATP gives up energy through the loss of a phosphate radical anaerobic fermentation fermentation is the extraction of energy from organic compounds; anaerobic means that the process does not involve oxygen ATP adenosine triphosphate, a nucleotide made up of adenine, ribose sugar, and three phosphate groups; this is the energy carrier in cell metabolism carbohydrate a compound containing carbon, hydrogen, and oxygen wherein the ratio of hydrogen to oxygen is 2:1; carbohydrates include sugar, starch, etc. cellular respiration, the production of energy through the process of oxidation; the energy is produced through the Krebs cycle and phosphorylation citric acid cycle see Krebs cycle coenzyme a cofactor that is a nonprotein organic molecule; a cofactor is an enzyme employing metal ions to acquire electrons Coenzyme A organic molecule involved in enzyme catalyzed process; this two-carbon molecule is the main molecule of energy metabolism crista folded innner membrane of a mitochondrion; the folds or cristae produce a large surface area in which are contained the electron transport chains DH" dihydroxyacetone phosphate, one of the products of the splitting of fructose diphosphate along with PGAL; the DHAP then undergoes isomerization to become a second molecule of PGAL electron transport chain protein chain embedded within the mitochondrial membrane which facilitates the passage of electrons; third stage of respiration and principal site of ATP synthesis in the cell entropy refers to the unavailability of energy in a system, and a measure of a system's randomness or disorder; the basis of the Second Law of Thermodynamics enzyme a protein that speeds up or slows down certain chemical reactions but does not, itself, change FAD+ the oxidized form of FADH 2 FADH2 flavin adenine dinucleotide; a carrier of lower energy electrons fatty acid an organic acid with a single carboxyl radical along with other carbon and hydrogen atoms glycogen a polysaccharide in which starch is stored in animal cells glycolysis the process through which glucose is broken down to synthesis ATP Krebs cycle a cycle of oxidation and reduction and the decarboxylation reactions from which a cell can derive ATP; also called the citric acid cycle since the cycle which begins with pyruvate later forms citric acid which is oxidized to form CO 2 lipid organic compound insoluble in water but soluble in certain organic liquids such as fats, oils, water, phospholipids, etc. matrix the inner compartment of a mitochondrion 19 mitochondrion cytoplasmic organelle; each one represents a complete mechanism that produces energy (plural: mitochondria) myosin one of the muscle proteins NAD nicotinamide andenine dinucleotide, a coenzyme that acts as an electron acceptor; NAD+ is its oxidized form NADP nicotinamide adenine dinucleotide phosphate, an electron acceptor in the process of respiration phosphoglyceraldehyde shortened to PGAL, a three-carbon molecule; a six-carbon molecule of glucose is broken into two molecules of PGAL with the input of ATP photosynthesis the formation of carbohydrates from carbon dioxide and water in the presence of light and chlorophyll protein a chain of amino acids joined by peptide bonds pyruvate a three-carbon (3C) compound; the end product of glycolysis and the material with which the Krebs cycle begins ribose a sugar of the five-carbon type sarcomere the fundamental unit of contraction in muscle tissue 20 Dobson, G. P. and Hochachka, P. W. Role of glycolysis in adenylate depletion and repletion during work and recovery in teleost white muscle. The journal of Experimental Biology 129:125-40, May 87. Akeroyd, F. Michael. Teaching the Krebs cycle. Journal o fBiological Education 17:245-56, fall 83. Alterthum, Flavio; Dombek, K. M.; and Ingram, L. O. Regulation of glycolytic flux and ethanol production in saccharomyces cerevisiae: effects of intracellular adenine nucleotide concentrations on the in vitro activities of hexokinase, phosphofructokinase, phosphoglycerate kinate, and pyruvate kinase. Applied and Environmental Microbiology 55:1312-14, May 89. Erickson, R. P.; Harper, K. J.; and Hopkin, S. R. Adenine nucleotides and other factors indicative of glycolytic metabolism in murine spermatozoa. The journal of Heredity 78:407-09, Nov-Dec 87. Furth, Anna and Harding, John. Why sugar is bad for you. New Scientist 123:44-7, S 23 89. A good article for this series; deals with the evidence of sugar-caused damage to long-lived proteins. Bodner, George M. Metabolism: part 3. Lipids. Journal of Chemical Education 63:772-75, Sep 86. Milligan, L. P. and McBride, B. W. Energy costs of ion pumping by animal tissues. The journal of Nutrition 115:1374-82, Oct 85. . Metabolism: glycolysis or the EmbdenMyerhoff pathway. Journal of Chemical Education 63:566-70, Jl 86. An excellent article for this series; the steps are clearly laid out complete with equations. Poolman, Bert; Bosman, Boukje; and Kiers, Jan. Control of glycolysis by glyceraldehyde-3-phosphate dehydrogenase in streptococcus cremoris and streptococcus lactis. Journal of Bacteriology 169:5887-90, Dec 87. . Metabolism: part 2. The tricarboxylic acid (TCA), citric acid, or Krebs cycle. journal of Chemical Education 63:673-77, Aug 86. Differen tiates the tricarboxylic acid (TCA) from glycolysis, and describes the connection between the two as being the conversion of pyruvate into acetyl coenzyme A. Sherman, W. Mike. Carbohydrates, muscle glycogen, and improved performance. Physician and Sports Medicine 15:157-61, Feb 87. Brand, Martin D. and Murphy, Michael P. Control of electron flux through the respiratory chain in mitochondria and cells. Biological Reviews of the Cambridge Philosophical Society 62:141-93, May 87. Simard, Clermont et al. Effects of carbohydrate intake before and during an ice hockey game on food and muscle energy substrates. Research Quarterly for Exercise and Sport 59:144-47, June 88. Wright, Russell G. and Bottino, Paul J. Mitochondrial DNA. Science Teacher 53:27-31, Apr 86. 21 Ordering Information To order this publication or videotapes of the Cellular Respiration series, or to obtain further information, please contact one of the following: Ontario TVOntario Sales and Licensing Box 200, Station Q Toronto, Ont. M4T 2T1 (416) 484-2613 United States TVOntario U.S. Sales Office 901 Kildaire Farm Road Building A Cary, North Carolina 27511 Phone: 800-331-9566 Fax: 919-380-0961 E-mail: [email protected] Cellular Respiration Videotapes Program Title The Cell and Energy Glycolysis 1 Glycolysis 2 The Krebs Cycle Oxidative Phosphorylation Metabolism and Nutrition BPN 296401 296402 296403 296404 296405 296406