CELLULAR RESPIRATION

Teacher's Guide

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 On- tario Educational Communications Authority. The series is available on videotape to educational institutions and nonprofit organizations.

The Series

Producer/Director . David Chamberlain Project Officers. John Amadio, David Way Writers: Susan Perry, David Way Consultant. Robert Whitney

The Guide

Writers: Randee Crisp, George Laundry, Robert Whitney Graphic Designer: Roswita Busskamp

Copyright 1990 by The Ontario Educational Communications Authority. All rijahts reserved.

Printed in Canada. 3626/90 Introduction 1

1. The and 3

2. 1 6

3. Glycolysis 2 9

4. The Krebs Cycle 12

5. Oxidative 14

6. and Nutrition 17

Glossary 19

Bibliography 21

This series of six 10-minute programs illustrates • discuss as the principal of cellular the complex world of biological respiration, at respiration and the involvement of ATP as the. both macro and molecular levels. Beginning with energy shuttle; a historical perspective and progressing to mod- ern research and theories, the programs examine • develop, in step-by-step fashion, the metabo- and coenzymes, phosphorylation, bio- lism of glucose through the processes of synthesis, glycolysis, and the Krebs cycle. glycolysis, the Krebs or citric cycle, and oxidative phosphorylation; Together, the Cellular Respiration video series and teacher's guide: • elucidate the role of in the controlled of glucose with the concomitant • describe the evolution of cellular respiration production of the respiratory waste product that presaged the development of present- ; and day forms; • explain the relationships of the three food • investigate the structure and function of the groups-, , and - organelle as the prime locus in nutrition. for the of (ATP);

1

After viewing this program and completing the Regardless of its source, energy for living things suggested activities, students should be able to: must be readily available at all times. Since inputs are irregular and unreliable, constant availability • name three major classes of molecules that necessitates some form of energy storage. A brief living things use to store energy, and designate overview of the mechanism of energy storage and carbohydrates as those most frequently em- release is the subject of this introductory program. ployed; The digestive system extracts from an 's food • explain the meanings of the following terms: the three major groups of macromolecules: pro- , mitochondrion, matrix, cristae, adeno- teins, fats, and carbohydrates.The most immedi- sine triphosphate (ATP), high-energy bond, ately available energy has been stored in carbohy- phosphoryl group, drates. This series assumes that most energy is (ADP), phosphorylation; provided to the cell in the form of glucose mole- cules. The release of chemical energy, to a form • describe the appearance of a mitochondrion as useful to living things, is called cellular respiration. seen through the transmission electron micro- scope; Cellular respiration is a complex series of chemical reactions that occur in both the cytosol and the • account for the theory that both mitochondria mitochondria of a cell. A mitochondrion consists of and evolved from independent a pair of membranes surrounding an amorphous ; interior, the matrix. The innermost membrane forms many inward-facing folds, the cristae, which • describe the structure of an ATP molecule and greatly increase the amount of membrane that can locate, within this structure, high-energy bonds; be packed within the mitochondrion. The similar- ity of a mitochondrion to a tiny cell suggests that the • explain the role of ATP in cell metabolism; mitochondria, like the chloroplasts, may have evolved from independent beings that invaded • name three interconnected phases of cellular larger cells as parasites. Over millions of years, they respiration.

3 became tolerated by, then vital to, their hosts. As a The reactions of cellular respiration, which provide consequence, there are many similarities between the ATP needed to drive life processes, are subdi- cellular respiration and . In fact, in vided into three phases: glycolysis, the Krebs cycle, many ways, cellular respiration can be considered and oxidative pbospborylation. All three phases the reverse of photosynthesis. will be covered, in turn, by this series.

Cellular respiration transfers most of the glucose molecules' energy into smaller "packages" of po- BEFORE VIEWING tential energy in molecules of adenosine triphosphate (ATP). ATP molecules contain enough energy to drive typical metabolic reactions. Some students may have little exposure to chem- istry. A short lesson on (or review of) the concepts of element, compound, atom, molecule, and cova- ATP is a complicated molecule consisting of por- lent bond should precede the program. Emphasize tions of a number of simpler, and more familiar, that a detailed knowledge of the structures of molecules linked by covalent bonds. The simple respiratory intermediates is not necessary. Instead, "building blocks" are a nitrogen-containing base the student should appreciate that molecules have (), a five-carbon (), and three unique and predictable shapes, and that cells molecules of phosphoric acid. The energy resides possess specialized agents (enzymes) that are able at one of two higb-energy bonds between the remanants of phosphoric acid molecules to select one type of molecule from among the (phosphoryl groups). When an ATP molecule multitude of other molecules present in the cell. A provides energy to a reactant, it transfers one of its quick review of a typical food chain and the place "high-energy bonds" to the reactant. Of course, of autotrophs and within it could also some atoms of the ATP are also transferred. be useful. Typically, the end phosphoryl group is transferred to the reactant, and adenosine dipbospbate (ADP) is left over. The reactant is now said to be AFTER VIEWING "phosphorylated" and the process of transferring a phosphoryl group to the reactant is called Activity l: phospborylation. Phosphorylation reactions are often employed in metabolism as a step in an How Carbohydrates Got Their energy-consuming reaction. 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 FIGURE 1.1 Structure of ATP damage to the desktop.

4 3, Be sure you are wearing safety goggles. Add hydroxyl groups (-OH) are in the correct just enough concentrated sulphuric acid to the positions above or below the ring. Use as few crucible to cover the sugar. shifts of atoms and/or bonds as possible. In 4. Note the color, odor, and appearance of the your notes, record the steps you followed in material left in the crucible. What do you think this conversion. Also record the number of it is? times you had to rotate a part of the molecule without shifting bonds or atoms. Compare Discussion your results with those of other students in the Concentrated sulphuric acid is a powerful dehy- class. Have your model evaluated by your drating agent which will withdraw from instructor before proceeding. Be sure to make other compounds, Assume that this will happen in any alterations suggested by the instructor this experiment. In terms of elements, what ap- before continuing. pears to be the composition of the sugar, based on the color of the resultant residue? Why, then, are 5. Evaluate the flexibility of the model. Is the this and other referred to as "carbohy- positioning of a hydroxyl group (-OH) on the drates"? top or bottom of the formula significant? Comment in your notes.

Activity 2: Discussion Visualizing Molecules 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 Apparatus isomer and isomerization and explain how molecular model kit these terms relate to this activity.

Method 2. Cellobiose is a formed during the 1. Examine the contents of the molecular model digestion of cellulose, and maltose is a disac- kit. Note that there are wood spheres of vari- charide formed during the digestion of starch. ous colors. These represent atoms of the Research the structures of these two sugars and elements. You will be using only carbon (black), relate them to this activity. Can enzymes distin- hydrogen (white), and oxygen (red) in this guish between these two ? 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 i nstructor for evaluation. Make any alterations suggested by your instructor before you con- tinue.

3. Evaluate the flexibility of the model. Is the positioning of a hydroxyl group (-OH) on the right or left of the formula significant? Com- ment 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 After viewing this program and completing the chemical potential energy supplied at ATP. suggested activities, students should be able to: Glucose arises principally from the hydrolysis of i dentify the major compound in which ani- glycogen, a polysaccharide stored in the liver and mals store energy; muscles. From the liver, glucose may be carried by the circulatory system to target cells which it outline the steps by which the first half of enters easily by membrane. Upon arrival in the glycolysis occurs; cytosol, the glucose is phosphorylated by ATP in an -catalyzed reaction. Its potential en- describe the change in chemical potential en- ergy is thereby increased; it also acquires a ergy accompanying the preparatory steps of negative charge which prevents its escape from glycolysis; the cell. The glucose is isomerized by an enzyme to fructose phosphate which then discuss the "coupling" of energy-consuming acquires a second phosphate group reaction with and energy-releasing reactions; a second ATP.The fructose diphosphate is then split into two parts: dihydroxyacetone phosphate identify the cytosol as the site of glycolysis. (DHAP) and phosphoglyceraldehyde (PGAL). The DHAP quickly undergoes isomerization to a sec- ond PGAL. Thus, a single 6-carbon glucose mole- cule has generated two 3-carbon PGAL mole- cules. Two ATP molecules have been sacrificed, but the two PGAL molecules have a higher potential energy than the original glucose mole- The energy that enters a cell in an energy-rich fuel cule. 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 descrip- tion 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 reac- Figure 2, 1 Endergonic. This program traces the first 5 tions. steps of glycolysis. In these steps, energy must be added to the system (endergonic)

6 The reaction of ATP with fructose phosphate Note: Numbers appearing in the following instruc- illustrates the important concept of reaction cou- tions are not given in the program. They indicate pling. The conversion of glucose phosphate to the number of the carbon atom(s) bearing phos- fructose phosphate has a small positive free phate groups or to be included in a product. The energy change. Fructose phosphate tends to numbers are those appearing in Figure 1.1 (Pro- spontaneously revert to glucose phosphate. gram 1) in glucose or derived from these in a However, any fructose phosphate that forms is . phosphorylated to fructose diphosphate in a reac- tion with a large negative free energy change. Method Thus, the net reaction changing glucose phos- Use the model of glucose (non-ring form) con- phate to fructose diphosphate (in two steps) has structed in Program 1, Activity 2, or construct a negative free energy change and proceeds another following the instructions in that activity. spontaneously. Many reactions in Using a single hole in an orange-colored atomic are driven against thermodynamic tendencies by model to represent phosphate, trace, with mod- being coupled to a simultaneous reaction having els, the conversion of glucose to glucose-6-phos- a large negative free energy change (often, the phate (you must discard a hydrogen atom to make hydrolysis of ATP). room for the phosphate), glucose-6-phosphate to fructose-6-phosphate (move the doubly bonded oxygen from carbon atom #1 to carbon atom #2 BEFORE VIEWING but do not discard any atoms), and fructose-6- phosphate to fructose- 1,6-diphosphate. Students should have some understanding of thermodynamic principles. These can be devel- Discussion oped to different levels, according to the needs 1. The change of glucose-6-phosphate to fruc- and expectations of the class. This could involve tose-6-phosphate is described as an isomeri- a discussion of the First Law of Thermodynamics. zation. By what feature is a reaction identified as an isomerization? A qualitative understanding is most easily derived from a consideration of the relative probabilities 2. You removed a hydrogen atom to make room of different distributions of energy and matter in for a phosphate during phosphorylation of a a chemical system (developed from a knowledge sugar. Hydrogen atoms cannot float about of the probabilities of different numbers arising freely in solution. Research and report on the when dice are rolled). This could be expanded actual fate of the hydrogen you had to re- into a discussion of the Second Law of Thermody- move. namics. With some classes, you might continue to discuss the chemical implications of the Third Law 3. Splitting fructose-1,6-phosphate into dihy- of Thermodynamics (often called the Nernst Heat droxyacetone phosphate and phosphopglyc- Theorem). eraldehyde requires the discarding of a car bon-carbon bond (between carbon atoms 3 and 4 of the fructose-1,6-phosphate). No AFTER VIEWING atomic nuclei are lost or gained, but one or more could change position. Using this infor- mation, predict the structural formula of dihy- ACtivity 1: droxyacetone phosphate (from carbon atoms Modelling the Reactions of Glycoly- 1, 2, and 3 of fructose-1,6-phosphate) and sis phosphoglyceraldehyde (from carbon atoms 4, 5, and 6 of fructose-1,6-phosphate).

Apparatus 4. The enzyme converting dihydroxyacetone molecular model kit phosphate into phosphoglyceraldehyde is called " phosphate isomerase." Discuss the appropriateness of this name.

7 3. is it possible to find a total measurement of the Activity 2: heat given off.? (Hint: consider the heat energy Measuring the Relative Quantities absorbed by the water.) 4. Is there a link between the molecular struc- of Energy Released from Different ture of a food substance and the energy Foods released? Discuss.

Apparatus assorted foods (nuts, sucrose cube, marshmallow, etc.) calorimeters needles corks Thermometer water (at room temperature) matches petri dishes balances clay triangles thermometers 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 Water tube. 4. Drive a pin through the centre of a cork and attach the food material to it at the top, as Soup can shown in the diagram. 5. Light the food material with a match, and fit Nut the calorimeter over it so that the bottom of the test tube is directly over the flame. Petri dish 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.

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 fulfill their energy After viewing this program and completing the requirements from what little useful energy glyco- suggested activities, students should be able to: lysis produces by linking it to . Fermentation converts pyruvate to acetaldehyde • trace the steps in the conversion of then to and in the process regenerates the phosphoglyceraldehyde to ; NAD molecule. The NAD then cycles back into glycolysis and maintains the production of ATP. • account for the net gain of ATP during glyco- The discussion of fermentation provides an im- lysis; portant example of biofeedback mechanisms.

• explain the difficulties encountered if a cell While glycolysis is able to meet the demands of reduces its entire complement of NAD; simple organisms, more complex organisms need additional reactions to harness the energy con- • describe the importance of anaerobic fermen- tained in the pyruvate and NADH molecules. The tation in ensuring the continued production program concludes by introducing the next stage of ATP as long as it is required and glucose is of cellular respiration-the Krebs Cycle-where available; pyruvate is used to make additional ATP mole- cules. • account for differences in identity and quan- tities of products of cellular respiration under aerobic and anaerobic conditions. BEFORE VIEWING

Review the concepts of oxidation and reduction: students should be able to identify these proc- esses by monitoring the transfers of electrons and/or hydrogen atoms during organic chemical This third program of the series completes the reactions. discussion of glycolysis by tracing the sequence of reactions from PGAL to the final product, pyruvate. It is useful to establish a series of 1-carbon The program illustrates the use of PGAL's poten- molecules arranged in order of decreased reduc- tial energy to synthesize ATP molecules and to tion status or increased oxidation state based reduce nicotinamide andenine dinucleotide (NAD) upon the hydrogen to oxygen ratio. The series to form the intermediate energy carrier molecule should include , methanol, formalde- NADH. hyde, formic acid, and carbon dioxide molecules. The relative positions of fats, carbohydrates, and The net energy production of glycolysis demon- a few amino in the reduction scale should be strates the inefficiency of this phase of cellular investigated.

9 Since texts vary in their naming of intermediates of cellular respiration, students should be made Glass U-tube 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 One-hole stopper to follow the fate of hydrogen atoms and the Two-hole stopper formation of water during biochemical reactions.

AFTER VIEWING

Activity 1: To Detect the Waste Products of Fermentation ()

Apparatus FIGURE 3.1 Apparatus to Detect the waste products of fermentation yeast packets (enough for number of pairs of students in class) 4. Set up a hot water bath using wire gauze and sucrose solution a ring on a ring stand above a Bunsen burner. bromthymol blue solution Heat 200 mL of water in a beaker until it comes Benedict's solution to a slow boil. Erylenmeyer flasks (125 mL) 5. Add 5 drops of Benedict's solution and 5 mL one-hole and two-hole rubber stoppers to fit of sucrose solution to a test tube, and label this flasks test tube A. graduated cylinders (50 mL) 6. Add 5 drops of Benedict's solution and 5 mL glass U-tubes of yeast and sucrose solution to a test tube, test tubes and label this test tube B. stirring rods 7. Heat test tubes A and B for 5 minutes in the marking pens water bath, and record any changes. test tube holders 8. Put 50 mL of bromthymol solution into a test tube racks second Erylenmeyer flask. Bunsen burners rings 9. Insert one end of a glass U tube into a one- hole rubber stopper and into a two-hole ring stands rubber stopper at the other end. Insert the wire gauze one-hole stopper into the flask with the yeast beakers mixture, and the two-hole stopper into the beaker tongs flask containing the bromthymol blue solu- flints tion (see Figure 3.1). Note: The longer end of safety goggles the U-tube should be below the level of bromthymol blue solution. If necessary, use Method glycerin as a lubricant. If the end of the tube 1. Pour 15 mL of warm water into a 125 mL Erylenmeyer flask. is not below the level of the solution, call your teacher for assistance. DO NOT adjust the 2. Add a packet of yeast to the water and mix tubing yourself. with a stirring rod. Add 50 mL of sucrose 10. Leave the apparatus setup in a warm place solution to the yeast mixture and mix well with a stirring rod. overnight. 3. Put on safety goggles.

1 0 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 obser- vations. 12. Record any observations after 48 hours.

Discussion 1. Discuss the reasons for the change or lack of change in: Chemical reaction • test tubes A and B after heating • the bromthymol blue after 24 hours FIGURE 3. 2 Exergonic. In the second series of events in • the bromthymol blue after 48 hours glycolysis, excess energy is released (exergonic) 2. Discuss the difference in products formed between anaerobic respiration which takes place in yeast cells (fermentation) and an- aerobic respiration which takes place in ani- mal muscle cells.

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 gener- ated by glycolysis, which is thought to have been one of the earliest of all biochemical processes to have evolved.

1. 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. 2. Identify and discuss the kinds of organisms that still survive today, using glycolytic reac- tions alone to produce the ATP needed to carry on their metabolic activities.

11 Pyruvate is a 3-carbon molecule. Through oxida- tive decarboxylation in the cytosol it is trans- formed into the 2-carbon molecule acetyl-CoA which enters the mitochondrion. Once inside the , acetyl-CoA transfers its After viewing this program and completing the energy into the Krebs cycle. The program follows suggested activities, students should be able to: the ten reactions of the Krebs cycle, focusing on the production of energy carriers. • appreciate that prehistoric life on land must have been preceded by the emergence of a A review of the Krebs Cycle shows that the energy glycolytic cycle and the accumulation of input from each acetyl-CoA creates one ATP atmospheric oxygen; molecule, one FADH Z , and three NADH mole- cules. Since each glucose molecule from glycoly- • recognize that glycolysis does not result in sis results in two molecules of acetyl-CoA, the sufficient energy for energetic life forms; cycle is considered to turn twice.

• trace and understand the sequences in the A summation of glycolysis, oxidative decarbox- Krebs cycle ( cycle); ylation, and the Krebs cycle together gives the total energy products from one glucose molecule • identify the end products of the cycle; as: four ATP molecules, ten NADH molecules, and two FADHZ molecules. The carbon atoms of the • state the energy products or carriers resulting glucose molecule have been expelled as six from this cycle; molecules of waste carbon dioxide. Most of the energy of glucose has been transferred to the • explain the fate of the glucose's carbon atoms. intermediate energy carriers NADH and FADH Z . The program concludes by setting up the final stage of cellular respiration, oxidative phosphoryla- tion, where the intermediate energy carriers are used to synthesize numerous ATP molecules.

Survival depends upon the availability of large The steps in the cycle-can be summarized as reserves of energy. Glycolysis, however, is an follows: inefficient source of energy and cannot supply 1. Acetyl-CoA reacts with oxoaloacetate to form these large reserves; therefore other phases of citric acid. energy production are required. This program 2. Citric acid loses a molecule of water to be- examines the second phase of cellular respiration, come aconitate. the Krebs cycle. The program follows the fate of 3. Aconitate adds water and is isomerized to become isocitrate. pyruvate from glycolysis as it is acted on as a substrate by enzymes within the mitochondria to 4. Isocitrate encounters NAD+, forming oxalo- generate ATP and intermediate energy carriers. succinate and NADH. 5. Oxalosuccinate loses a molecule of CO 2 to become ketoglutarate.

1 2 6. Ketoglutarate reacts with CoA to form suc- cinyl-CoA and a NADH molecule. BEFORE VIEWING 7. Succinyl-CoA joins with ADP and a phosphate to release CoA, an ATP molecule, and succi- Help the students to consolidate the previous nate. material by stressing the following points. 8. Succinate joins with an FAD molecule to form an FADH 2 molecule and fumarate. 1. Glycolysis is a very inefficient process: it 9. Fumarate adds water to become malate. yields only about 2% of the available energy 10. Malate reacts with NAD+ to become oxaloace- of glucose. Glycolysis alone, therefore, could tate and form a NADH molecule. not provide the energy needed to power energetic organisms. A summary of total energy products can be given as follows: 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. Divide the class into two main groups. One group, which can be subdivided into several In total, this process utilizes approximately 40% of research sections, is to write out the structural the available energy, whereas glycolysis utilizes formulae for all of the Krebs cycle intermedi- only about 2%. ary compounds, including high-energy trans- fer 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 mem- bers with an eye to presenting a detailed account of their results to the class.

2. Discuss the following points. NADH a. It is evident that glycolysis does not pro- duce 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? Figure 4. 1 Energy release from the Krebs Cycle (The c. What is the significance of the word cycle cycle can be considered to turn twice) 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?

1 3 This process takes place within the inner mito- chondrial membrane. Embedded within this membrane are four adjacent complexes that make up the .Three of these complexes act as proton (H+) pumps. After viewing this program and completing the Their function is to remove energy from the suggested activities, students should be able to: electrons as they move in pairs down an energy gradient. • trace how phase 1 and 2 of cellular respiration lead into oxidative phosphorylation; The process begins as NADH donates two elec- trons to the first complex. Two hydrogen • describe the process of the electron transport hitch a ride into the and the chains; two electrons transfer to the second complex and return to the matrix side of the membrane. Two • understand the role of oxygen in siphoning more hydrogen ions are moved into the third electrons from the electron transport chains; complex and are carried to the intermembrane space. Two electrons return down the fourth • explain how the energy gradient across the complex and two more hydrogen ions move into intermitochondrial membrane is created, and the intermembrane space. (Six hydrogen ions why this gradient is important; have now crossed.) Finally, an oxygen atom picks up two electrons and two hydrogen ions • follow the steps in ATP synthesis at the matrix and forms water. (It is the primary role of the side of the membrane; oxygen to siphon the electrons from the electron transfer chains.) • sum up the total production of ATP, NADH, and FADH2 from a single glucose molecule; The other energy carrier produced by the Krebs cycle, FADH 2 , enters the chain and results in four • state how many ATP molecules are produced more hydrogen ions being transferred to the at any step. 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 Cellular respiration in its first phase, glycolysis, activate an enzyme on the matrix side. This produces only two molecules of ATP. Phase 2, the enzyme catalyzes the reaction of ADP with a phosphate group to synthesize ATP. Krebs cycle, produces only two more ATP. However, phase 3, oxidative phosphorylation, produces an energy payload.

1 4 In summary, glycolysis results in two ATP mole- cules plus four more at the electron transport AFTER VIEWING chain, for a total of six ATP molecules. Oxidative decarboxylation and the Krebs cycle produce two Activity 1: ATP, eight NADH, and two FADHZ molecules. The eight NADH energy carriers produce 24 ATP The Energy of Carbohydrates molecules, and the two FADH Z produce another four ATP molecules. The net result is 36 molecules The catabolic metabolism of glucose could be of ATP. Therefore, cellular respiration results in 36 expressed as follows: ATP molecules from one glucose molecule; this represents about 41% of the available energy from the glucose molecule.

BEFORE VIEWING Note that 36 molecules of ATP are ultimately produced from 1 molecule of glucose. 1. Students should review the structure of the mitochondria., and consider such terms as cytosol, intermembrane space, cristae, matrix, 1. How many ATP molecules can be produced and electron transport chain. They should from 1 mole of glucose? (Recall that 1 mole review, as well, these processes: diffusion, contains approximately 6 x 10 23 molecules.) osmosis, and . 2. Each mole of ATP represents a capture of 31 kJ. Calculate the total energy available for the 2. Review the Krebs cycle in terms of where the 36 ATP molecules. intermediate energy carriers NADH and FADH Z are given off.

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 en-

ergy 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 elec- tron micrographs of mitochondria. Study photographs from 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 be- tween mitochondria from the different tis- sues, 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?

1 6 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 After viewing this program and completing the the circulatory system cannot provide the oxygen suggested activities, students should be able to: quickly enough. While glycolysis can provide a small quantity of ATP, not enough is synthesized • recognize that much of our knowledge about and this results in an energy shortage. cells comes from the development of models; The program describes how the process of cellu- • appreciate the immense turnover of ATP in lar respiration takes steps to overcome this short- the human body in a normal day; age. The pyruvate that normally heads off to the Krebs cycle follows a different path when oxygen • understand the basic operation of a muscle; is in short supply-a path that leads to the synthesis of . The steps in this sequence • describe how cells respond to an oxygen ensure the continuous production of ATP. There shortage caused by overexertion; is, of course, a debt to pay: a burning sensation within the muscles caused by the lactic acid • describe how an oversupply of ATP may be buildup. Fortunately, after a short rest, the return stored eventually as ""; of oxygen results in the metabolism of the lactic acid. • appreciate that the complexity and collective behavior of cells is a reaffirmation of life itself. 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 Scientists frequently develop models to explain the fat can be metabolized. This is done through the complexity of cellular respiration. These sequences that lead either to the glycolytic path- models, though, are often schematic diagrams way or directly into the Krebs cycle. and do not come close to revealing the magnifi- cence of the collective power of cells. Our bodies Throughout this series the programs have de- use and recycle about 40 kg of ATP each day, and picted how resourceful cells are and how the strenuous activity may cause them to use as much collective behavior of a cell is a reaffirmation of as 0.5 kg per minute. For all body movements, it the driving force of life itself. is ATP which provides the driving energy. This program examines the ability of cellular respira- tion to adjust to different conditions in the human body.

1 7 microscope slides FORE VIEWING cover slips dissecting needles 1. it could be advantageous for the student to medicine droppers recall or to look up the general structure of a forceps muscle. Recognition of such things as the protein layers of actin and myosin and the Method mechanics of muscle contraction would be 1. Obtain a piece of beef from your teacher. Pull helpful. the point of the dissecting needle across the long grain of the muscle several times until a 2. Review program 4 with special reference to small strand of tissue is removed. Caution: the section on the electron transport chain. Use the dissecting needle with care as it is very Review in particular the purpose of oxygen sharp. and the role of NAD+ and its development. 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 AFTER VIEWING the stain remain for 2 minutes, then add 2 drops of water to the slide. Cover the tissue with a cover slip. Activity 1: 4. Examine the tissue under the microscope at Muscle and Fat Energetics low power. Focus on a portion that is thin and lightly stained. Draw a portion of what you Discuss each of the following: see. 1. What role does each of the following play 5. Switch to high power. Look for the striated during the contraction of a muscle: pyruvate, appearance of the muscle cells. Muscle cells NADH, NAD+, lactic acid, ATP, ADP, glyco- are made of microfilaments called myofil gen, and oxygen? aments, which are composed of the proteins 2. Explain what happens when a muscle is actin and myosin. The portion of muscle from overexerted as during strenuous exercise and one stripe to the next is called a sarcomere. how this condition is alleviated. The darkly stained structures are the nuclei of 3. Discussthe conversion of energy during muscle the muscle cells. Locate the sarcomeres and contraction. the nuclei, and draw a diagram labelling these 4. What are some other uses of ATP by cells of structures. multicellular organisms? 6. Use high power to examine prepared slides of 5. Trace the of fatty acids through the and, if available, cardiac Krebs cycle. How does the ATP yield from a muscle. 6-carbon compare with the ATP yield from glucose? What problem occurs if fat Discussion catabolism is excessive? 1. From your observations, is a muscle fibre composed of several small cells, or one long Activity 2: cell containing many nuclei? 2. Observation of Skeletal Muscle Discuss the role of myosin, ATP, and actin during the contraction of a muscle cell. 3. What initiates contraction in vertebrate skele- Apparatus tal muscle? What other chemicals are in- beef toluidine-blue stain volved? prepared slides of skeletal muscle prepared slides of cardiac muscle, if available microscopes

1 8 decarboxylation the removal of the carboxyl group (COON) from an organic molecule

DH" dihydroxyacetone phosphate, one of the products of the splitting of fructose diphosphate acetylCoAthe main molecule of energy metabo- along with PGAL; the DHAP then undergoes lism; contains a high energy bond isomerization to become a second molecule of PGAL actin one of two proteins making up the microfil- aments of muscle tissue electron transport chain protein chain embed- ded within the mitochondrial membrane which adenine an organic base consisting of two car- facilitates the passage of electrons; third stage of bon-nitrogen rings respiration and principal site of ATP synthesis in the cell ADP adenosine diphosphate, a substance pro- duced when ATP gives up energy through the loss entropy refers to the unavailability of energy in of a phosphate radical a system, and a measure of a system's randomness or disorder; the basis of the Second Law of anaerobic fermentation fermentation is the Thermodynamics extraction of energy from organic compounds; anaerobic means that the process does not in- enzyme a protein that speeds up or slows down volve oxygen certain chemical reactions but does not, itself, change ATP adenosine triphosphate, a made up of adenine, ribose sugar, and three phosphate FAD+ the oxidized form of FADH 2 groups; this is the energy carrier in cell metabo- lism FADH2 flavin adenine dinucleotide; a carrier of lower energy electrons a compound containing carbon, hydrogen, and oxygen wherein the ratio of hydro- fatty acid an organic acid with a single carboxyl gen to oxygen is 2:1; carbohydrates include sugar, radical along with other carbon and hydrogen starch, etc. atoms cellular respiration, the production of energy glycogen a polysaccharide in which starch is through the process of oxidation; the energy is stored in animal cells produced through the Krebs cycle and phosphorylation glycolysis the process through which glucose is broken down to synthesis ATP see Krebs cycle Krebs cycle a cycle of oxidation and reduction coenzyme a that is a nonprotein organic and the decarboxylation reactions from which a molecule; a cofactor is an enzyme employing cell can derive ATP; also called the citric acid cycle metal ions to acquire electrons since the cycle which begins with pyruvate later forms citric acid which is oxidized to form CO 2 Coenzyme A organic molecule involved in en- zyme catalyzed process; this two-carbon mole- organic compound insoluble in water but cule is the main molecule of energy metabolism soluble in certain organic liquids such as fats, oils, water, phospholipids, etc. crista folded innner membrane of a mitochon- drion; the folds or cristae produce a large surface matrix the inner compartment of a mitochon- area in which are contained the electron transport drion chains

1 9 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 ; NAD+ is its oxidized form

NADP nicotinamide adenine dinucleotide phos- phate, 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 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 i n 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 129:125-40, May 87. Akeroyd, F. Michael. Teaching the Krebs cycle. Journal o fBiological Education 17:245-56, fall 83. Erickson, R. P.; Harper, K. J.; and Hopkin, S. R. Adenine and other factors indicative Alterthum, Flavio; Dombek, K. M.; and Ingram, of glycolytic metabolism in murine spermatozoa. L. O. Regulation of glycolytic flux and ethanol The journal of Heredity 78:407-09, Nov-Dec 87. production in : effects of intracellular adenine nucleotide concentrations Furth, Anna and Harding, John. Why sugar is bad on the in vitro activities of hexokinase, for you. New Scientist 123:44-7, S 23 89. A good phosphofructokinase, phosphoglycerate kinate, article for this series; deals with the evidence of and pyruvate . Applied and Environmental sugar-caused damage to long-lived proteins. Microbiology 55:1312-14, May 89. Milligan, L. P. and McBride, B. W. Energy costs of Bodner, George M. Metabolism: part 3. . pumping by animal tissues. The journal of Journal of Chemical Education 63:772-75, Sep 86. Nutrition 115:1374-82, Oct 85.

. Metabolism: glycolysis or the Embden- Poolman, Bert; Bosman, Boukje; and Kiers, Jan. Myerhoff pathway. Journal of Chemical Educa- Control of glycolysis by glyceraldehyde-3-phos- tion 63:566-70, Jl 86. An excellent article for this phate dehydrogenase in streptococcus cremoris series; the steps are clearly laid out complete with and streptococcus lactis. Journal of Bacteriology equations. 169:5887-90, Dec 87.

. Metabolism: part 2. The tricarboxylic acid Sherman, W. Mike. Carbohydrates, muscle glyco- (TCA), citric acid, or Krebs cycle. journal of gen, and improved performance. Physician and Chemical Education 63:673-77, Aug 86. Differen Sports Medicine 15:157-61, Feb 87. tiates the tricarboxylic acid (TCA) from glycolysis, and describes the connection between the two as Simard, Clermont et al. Effects of carbohydrate being the conversion of pyruvate into acetyl intake before and during an ice hockey game on coenzyme A. food and muscle energy substrates. Research Quarterly for Exercise and Sport 59:144-47, Brand, Martin D. and Murphy, Michael P. Control June 88. of electron flux through the respiratory chain in mitochondria and cells. Biological Reviews of the Wright, Russell G. and Bottino, Paul J. Mitochon- Cambridge Philosophical Society 62:141-93, drial DNA. Science Teacher 53:27-31, Apr 86. May 87.

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