CELLULAR RESPIRATION

Teacher's Guide

Teacher's GuideThis teacher's guide is designed for use with theCellularRespiration series of programs producedby TVOntario, the television service of the On-tario Educational Communications Authority. Theseries is available on videotape to educationalinstitutions and nonprofit organizations.

The Series

Producer/Director . David Chamberlain

Project Officers. John Amadio, David WayWriters: Susan Perry, David WayConsultant. Robert Whitney

The Guide

Writers: Randee Crisp,George Laundry,Robert Whitney

Graphic Designer: Roswita Busskamp

Printed in Canada. 3626/90

Cellular Respiration

Copyright 1990 by The Ontario EducationalCommunications Authority.All rijahts reserved.

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 illustratesthe complex world of biological respiration, atboth macro and molecular levels. Beginning witha historical perspective and progressing to mod-ern research and theories, the programs examineenzymes and coenzymes, phosphorylation, bio-synthesis, glycolysis, and the Krebs cycle.

Together, the Cellular Respiration video seriesand teacher's guide:

• describe the evolution of cellular respirationthat presaged the development of present-day life forms;

• investigate the structure and function of themitochondrion organelle as the prime locusfor the biosynthesis of adenosine triphosphate(ATP);

• discuss glucose as the principal fuel of cellularrespiration and the involvement of ATP as the.energy shuttle;

• develop, in step-by-step fashion, the metabo-lism of glucose through the processes ofglycolysis, the Krebs or citric acid cycle, andoxidative phosphorylation;

• elucidate the role of oxygen in the controlledcombustion of glucose with the concomitantproduction of the respiratory waste productcarbon dioxide; and

• explain the relationships of the three foodgroups-proteins, fats, and carbohydrates-in nutrition.

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After viewing this program and completing thesuggested activities, students should be able to:

• name three major classes of molecules thatliving things use to store energy, and designatecarbohydrates as those most frequently em-ployed;

• explain the meanings of the following terms:cytosol, mitochondrion, matrix, cristae, adeno-sine triphosphate (ATP), high-energy bond,phosphoryl group, adenosine diphosphate(ADP), phosphorylation;

• describe the appearance of a mitochondrion asseen through the transmission electron micro-scope;

• account for the theory that both mitochondriaand chloroplasts evolved from independentorganisms;

• describe the structure of an ATP molecule andlocate, within this structure, high-energy bonds;

• explain the role of ATP in cell metabolism;

• name three interconnected phases of cellularrespiration.

Regardless of its source, energy for living thingsmust be readily available at all times. Since inputsare irregular and unreliable, constant availabilitynecessitates some form of energy storage. A briefoverview of the mechanism of energy storage andrelease is the subject of this introductory program.

The digestive system extracts from an animal's foodthe three major groups of macromolecules: pro-teins, fats, and carbohydrates.The most immedi-ately available energy has been stored in carbohy-drates. This series assumes that most energy isprovided to the cell in the form of glucose mole-cules. The release of chemical energy, to a formuseful to living things, is called cellular respiration.

Cellular respiration is a complex series of chemicalreactions that occur in both the cytosol and themitochondria of a cell. A mitochondrion consists ofa pair of membranes surrounding an amorphousinterior, the matrix. The innermost membraneforms many inward-facing folds, the cristae, whichgreatly increase the amount of membrane that canbe packed within the mitochondrion. The similar-ity of a mitochondrion to a tiny cell suggests that themitochondria, like the chloroplasts, may haveevolved from independent beings that invadedlarger cells as parasites. Over millions of years, they

Crista

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became tolerated by, then vital to, their hosts. As aconsequence, there are many similarities betweencellular respiration and photosynthesis. In fact, inmany ways, cellular respiration can be consideredthe reverse of photosynthesis.

The reactions of cellular respiration, which providethe ATP needed to drive life processes, are subdi-vided into three phases: glycolysis, the Krebs cycle,and oxidative pbospborylation. All three phaseswill be covered, in turn, by this series.

Cellular respiration transfers most of the glucosemolecules' energy into smaller "packages" of po-tential energy in molecules of adenosinetriphosphate (ATP). ATP molecules contain enoughenergy to drive typical metabolic reactions.

ATP is a complicated molecule consisting of por-tions 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 threemolecules of phosphoric acid. The energy residesat one of two higb-energy bonds between theremanants of phosphoric acid molecules(phosphoryl groups). When an ATP moleculeprovides 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 transferredto the reactant, and adenosine dipbospbate (ADP)is left over. The reactant is now said to be"phosphorylated" and the process of transferring aphosphoryl group to the reactant is calledphospborylation. Phosphorylation reactions areoften employed in metabolism as a step in anenergy-consuming reaction.

BEFORE VIEWING

Some students may have little exposure to chem-istry. A short lesson on (or review of) the conceptsof element, compound, atom, molecule, and cova-lent bond should precede the program. Emphasizethat a detailed knowledge of the structures ofrespiratory intermediates is not necessary. Instead,the student should appreciate that molecules haveunique and predictable shapes, and that cellspossess specialized agents (enzymes) that are ableto select one type of molecule from among themultitude of other molecules present in the cell. Aquick review of a typical food chain and the placeof autotrophs and heterotrophs within it could alsobe useful.

AFTER VIEWING

Activity l:How Carbohydrates Got TheirName

FIGURE 1.1 Structure of ATP

Apparatussugar cubesconcentrated sulphuric acid

(Caution: highly corrosive)cruciblemortar and pestleprotective cover for desktopsafety goggleslaboratory coat or apronNote: This activity maybeperformed as a demonstration.

Method1. Grind a sugar cube to a powder

using a mortar and pestle.2. Transfer the powdered sugar to a

crucible which has been placedon a protective cover to preventdamage to the desktop.

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3, Be sure you are wearing safety goggles. Addjust enough concentrated sulphuric acid to thecrucible to cover the sugar.

4. Note the color, odor, and appearance of thematerial left in the crucible. What do you thinkit is?

DiscussionConcentrated sulphuric acid is a powerful dehy-drating agent which will withdraw water fromother compounds, Assume that this will happen inthis experiment. In terms of elements, what ap-pears to be the composition of the sugar, based onthe color of the resultant residue? Why, then, arethis and other sugars referred to as "carbohy-drates"?

hydroxyl groups (-OH) are in the correctpositions above or below the ring. Use as fewshifts of atoms and/or bonds as possible. Inyour notes, record the steps you followed inthis conversion. Also record the number oftimes you had to rotate a part of the moleculewithout shifting bonds or atoms. Compareyour results with those of other students in theclass. Have your model evaluated by yourinstructor before proceeding. Be sure to makeany alterations suggested by the instructorbefore continuing.

5. Evaluate the flexibility of the model. Is thepositioning of a hydroxyl group (-OH) on thetop or bottom of the formula significant?Comment in your notes.

Activity 2:Visualizing Molecules

Apparatusmolecular model kit

Discussion1. The formula of glucose is given in textbooks as

C6H1z06. To which of the structures in Figure1.2 does it apply? Research the meaning ofisomer and isomerization and explain howthese terms relate to this activity.

Method1. Examine the contents of the molecular model

kit. Note that there are wood spheres of vari-ous colors. These represent atoms of theelements. You will be using only carbon (black),hydrogen (white), and oxygen (red) in thisexercise.

2. Cellobiose is a disaccharide formed during thedigestion of cellulose, and maltose is a disac-charide formed during the digestion of starch.Research the structures of these two sugars andrelate them to this activity. Can enzymes distin-guish between these two disaccharides?

2. Construct a model of a glucose molecule. Usethe structural formula on the left of Figure 1.2for guidance. When the model has beencompleted to your satisfaction, take it to yourinstructor for evaluation. Make any alterationssuggested by your instructor before you con-tinue.

3. Evaluate the flexibility of the model. Is thepositioning of a hydroxyl group (-OH) on theright or left of the formula significant? Com-ment in your notes.

4. Now, attempt to convert your model into thering form depicted on the right of Figure 1.2,Consider the ring to be perpendicular to thepage and be sure that the hydrogens (-H) and FIGURE 1.2 Two Glucose Formulae (Ring Structure)

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After viewing this program and completing thesuggested activities, students should be able to:

identify the major compound in which ani-mals store energy;

outline the steps by which the first half ofglycolysis occurs;

describe the change in chemical potential en-ergy accompanying the preparatory steps ofglycolysis;

discuss the "coupling" of energy-consumingand energy-releasing reactions;

identify the cytosol as the site of glycolysis.

The energy that enters a cell in an energy-rich fuelsuch as glucose must be used to synthesize ATPmolecules in order to be used effectively. Theprocess begins with a series of reactions knowncollectively as glycolysis These reactions musthave evolved a very long time ago since theyexist, in identical form, in all living things.

The series devotes two programs to the descrip-tion of glycolysis. This first program on the subjectshows that energy (ATP) must first be sacrificed inorder to prepare the way for its later extraction. Italso indicates the importance of thermodynamicprinciples in explaining the progress of the reac-tions.

In the first half of glycolysis, the 6-carbon sugarglucose, is broken into two 3-carbon molecules ofphosphoglyceraldehyde (PGAL). This requiresthe addition to the original glucose molecule ofchemical potential energy supplied at ATP.

Glucose arises principally from the hydrolysis ofglycogen, a polysaccharide stored in the liver andmuscles. From the liver, glucose may be carriedby the circulatory system to target cells which itenters easily by membrane. Upon arrival in thecytosol, the glucose is phosphorylated by ATP inan enzyme-catalyzed reaction. Its potential en-ergy is thereby increased; it also acquires anegative charge which prevents its escape fromthe cell. The glucose phosphate is isomerized byan enzyme to fructose phosphate which thenacquires a second phosphate group reaction witha second ATP.The fructose diphosphate is thensplit into two parts: dihydroxyacetone phosphate(DHAP) and phosphoglyceraldehyde (PGAL). TheDHAP 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 higherpotential energy than the original glucose mole-cule.

Figure 2, 1 Endergonic. This program traces the first 5steps of glycolysis. In these steps, energy must be addedto the system (endergonic)

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The reaction of ATP with fructose phosphateillustrates the important concept of reaction cou-pling. The conversion of glucose phosphate tofructose phosphate has a small positive freeenergy change. Fructose phosphate tends tospontaneously revert to glucose phosphate.However, any fructose phosphate that forms isphosphorylated to fructose diphosphate in a reac-tion with a large negative free energy change.Thus, the net reaction changing glucose phos-phate to fructose diphosphate (in two steps) hasa negative free energy change and proceedsspontaneously. Many reactions in biochemistryare driven against thermodynamic tendencies bybeing coupled to a simultaneous reaction havinga large negative free energy change (often, thehydrolysis of ATP).

BEFORE VIEWING

Note: Numbers appearing in the following instruc-tions are not given in the program. They indicatethe number of the carbon atom(s) bearing phos-phate groups or to be included in a product. Thenumbers are those appearing in Figure 1.1 (Pro-gram 1) in glucose or derived from these in achemical reaction.

MethodUse the model of glucose (non-ring form) con-structed in Program 1, Activity 2, or constructanother following the instructions in that activity.Using a single hole in an orange-colored atomicmodel to represent phosphate, trace, with mod-els, the conversion of glucose to glucose-6-phos-phate (you must discard a hydrogen atom to makeroom for the phosphate), glucose-6-phosphate tofructose-6-phosphate (move the doubly bondedoxygen from carbon atom #1 to carbon atom #2but do not discard any atoms), and fructose-6-phosphate to fructose- 1,6-diphosphate.

Students should have some understanding ofthermodynamic principles. These can be devel-oped to different levels, according to the needsand expectations of the class. This could involvea discussion of the First Law of Thermodynamics.

Discussion1. The change of glucose-6-phosphate to fruc-

tose-6-phosphate is described as an isomeri-zation. By what feature is a reaction identifiedas an isomerization?

A qualitative understanding is most easily derivedfrom a consideration of the relative probabilitiesof different distributions of energy and matter ina chemical system (developed from a knowledgeof the probabilities of different numbers arisingwhen dice are rolled). This could be expandedinto a discussion of the Second Law of Thermody-namics. With some classes, you might continue todiscuss the chemical implications of the Third Lawof Thermodynamics (often called the Nernst HeatTheorem).

AFTER VIEWING

ACtivity 1:Modelling the Reactions of Glycoly-sis

2. You removed a hydrogen atom to make roomfor a phosphate during phosphorylation of asugar. Hydrogen atoms cannot float aboutfreely in solution. Research and report on theactual fate of the hydrogen you had to re-move.

3. Splitting fructose-1,6-phosphate into dihy-droxyacetone phosphate and phosphopglyc-eraldehyde requires the discarding of a carbon-carbon bond (between carbon atoms 3and 4 of the fructose-1,6-phosphate). Noatomic nuclei are lost or gained, but one ormore could change position. Using this infor-mation, predict the structural formula of dihy-droxyacetone phosphate (from carbon atoms1, 2, and 3 of fructose-1,6-phosphate) andphosphoglyceraldehyde (from carbon atoms4, 5, and 6 of fructose-1,6-phosphate).

Apparatusmolecular model kit

4. The enzyme converting dihydroxyacetonephosphate into phosphoglyceraldehyde iscalled "triose phosphate isomerase." Discussthe appropriateness of this name.

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Activity 2:Measuring the Relative Quantitiesof Energy Released from DifferentFoods

is it possible to find a total measurement of theheat given off.? (Hint: consider the heat energyabsorbed by the water.)

4. Is there a link between the molecular struc-ture of a food substance and the energyreleased? Discuss.

3.

Apparatusassorted foods (nuts, sucrose cube, marshmallow,etc.)calorimetersneedlescorkswater (at room temperature)matchespetri dishesbalancesclay trianglesthermometers

Method1. Put 10 mL of water at room temperature into

a test tube. Fit the test tube into the top of thecalorimeter, 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, asshown in the diagram.

5. Light the food material with a match, and fitthe calorimeter over it so that the bottom ofthe test tube is directly over the flame.

6. When the flame has gone out (after about 2min of heating), record the final temperatureof the water.

7. Reweigh the food material and record itsmass.

8. Repeat this procedure for each of the otherfood substances.

9. Calculate the temperature change caused byeach food substance per unit mass of thatsubstance burned.

Thermometer

Test tube

Water

Soup can

Nut

Petri dish

Discussion1. Compare each of the food materials used with

respect to their energy per unit mass. Whichfood types contain the most energy per unitmass?

2. Describe the possible sources of error in thisexperiment.

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After viewing this program and completing thesuggested activities, students should be able to:

• trace the steps in the conversion ofphosphoglyceraldehyde to pyruvic acid;

• account for the net gain of ATP during glyco-lysis;

respiration: the entire sequence of ten reactionstransfers only about two percent of the chemicalpotential energy of a glucose molecule to theproduction of ATP. The program shows howsimple organisms like yeast fulfill their energyrequirements from what little useful energy glyco-lysis produces by linking it to fermentation.

Fermentation converts pyruvate to acetaldehydethen to ethanol and in the process regenerates theNAD molecule. The NAD then cycles back intoglycolysis and maintains the production of ATP.

The discussion of fermentation provides an im-portant example of biofeedback mechanisms.

• explain the difficulties encountered if a cellreduces its entire complement of NAD;

• describe the importance of anaerobic fermen-tation in ensuring the continued productionof ATP as long as it is required and glucose isavailable;

• account for differences in identity and quan-

tities of products of cellular respiration underaerobic and anaerobic conditions.

While glycolysis is able to meet the demands ofsimple organisms, more complex organisms needadditional reactions to harness the energy con-tained in the pyruvate and NADH molecules. Theprogram concludes by introducing the next stageof cellular respiration-the Krebs Cycle-wherepyruvate is used to make additional ATP mole-cules.

BEFORE VIEWING

This third program of the series completes thediscussion of glycolysis by tracing the sequence ofreactions from PGAL to the final product, pyruvate.The program illustrates the use of PGAL's poten-tial energy to synthesize ATP molecules and toreduce nicotinamide andenine dinucleotide (NAD)to form the intermediate energy carrier moleculeNADH.

The net energy production of glycolysis demon-strates the inefficiency of this phase of cellular

Review the concepts of oxidation and reduction:students should be able to identify these proc-esses by monitoring the transfers of electronsand/or hydrogen atoms during organic chemicalreactions.

It is useful to establish a series of 1-carbonmolecules arranged in order of decreased reduc-tion status or increased oxidation state basedupon the hydrogen to oxygen ratio. The seriesshould include methane, methanol, formalde-hyde, formic acid, and carbon dioxide molecules.The relative positions of fats, carbohydrates, anda few amino acids in the reduction scale should beinvestigated.

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Since texts vary in their naming of intermediatesof cellular respiration, students should be madeaware that acids may be named as if they were notionized (e.g., pyruvic acid) or as their anions (e.g.,pyruvate). The latter represents the form found atphysiological pH, but the former makes it easierto follow the fate of hydrogen atoms and theformation of water during biochemical reactions.

AFTER VIEWING

Activity 1:To Detect the Waste Products ofFermentation(Anaerobic Respiration)

Glass U-tube

One-hole stopper

Two-hole stopper

Apparatusyeast packets (enough for number of pairs of

students in class)sucrose solutionbromthymol blue solutionBenedict's solutionErylenmeyer flasks (125 mL)one-hole and two-hole rubber stoppers to fit

flasksgraduated cylinders (50 mL)glass U-tubestest tubesstirring rodsmarking penstest tube holderstest tube racksBunsen burnersringsring stands

wire gauzebeakersbeaker tongsflintssafety goggles

Method1. 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 sucrosesolution to the yeast mixture and mix wellwith a stirring rod.

3. Put on safety goggles.

FIGURE 3.1 Apparatus to Detect the waste products offermentation

4. Set up a hot water bath using wire gauze anda ring on a ring stand above a Bunsen burner.Heat 200 mL of water in a beaker until it comesto a slow boil.

5. Add 5 drops of Benedict's solution and 5 mLof sucrose solution to a test tube, and label thistest tube A.

6. Add 5 drops of Benedict's solution and 5 mLof 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 thewater bath, and record any changes.

8. Put 50 mL of bromthymol solution into asecond Erylenmeyer flask.

9. Insert one end of a glass U tube into a one-hole rubber stopper and into a two-hole

rubber stopper at the other end. Insert theone-hole stopper into the flask with the yeastmixture, and the two-hole stopper into theflask containing the bromthymol blue solu-tion (see Figure 3.1). Note: The longer end ofthe U-tube should be below the level ofbromthymol blue solution. If necessary, useglycerin as a lubricant. If the end of the tubeis not below the level of the solution, call yourteacher for assistance. DO NOT adjust thetubing yourself.

10. Leave the apparatus setup in a warm placeovernight.

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11. After 24 hours, replace the flask containing50 mL of bromthymol blue solution withanother flask containing 50 mL of freshbromthymol blue solution. Record any obser-

vations.12. Record any observations after 48 hours.

Discussion1. 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 glycolysis, excess energy is released (exergonic)

2. Discuss the difference in products formedbetween anaerobic respiration which takesplace in yeast cells (fermentation) and an-aerobic respiration which takes place in ani-mal muscle cells.

Chemical reaction

FIGURE 3. 2 Exergonic. In the second series of events in

Activity 2:Early Anaerobic Biochemistry

Organisms that emerged during the first billionyears of the development of life on earth used noatmospheric oxygen to fuel their activities. Theycould fuel their metabolism only by ATP gener-ated by glycolysis, which is thought to have beenone of the earliest of all biochemical processes tohave evolved.

1. Discuss the kind of organisms that wouldhave probably been alive at that time. Explainand 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 tocarry on their metabolic activities.

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After viewing this program and completing thesuggested activities, students should be able to:

• appreciate that prehistoric life on land musthave been preceded by the emergence of aglycolytic cycle and the accumulation ofatmospheric oxygen;

• recognize that glycolysis does not result insufficient energy for energetic life forms;

Pyruvate is a 3-carbon molecule. Through oxida-tive decarboxylation in the cytosol it is trans-formed into the 2-carbon molecule acetyl-CoAwhich enters the mitochondrion. Once inside themitochondrial matrix, acetyl-CoA transfers itsenergy into the Krebs cycle. The program followsthe ten reactions of the Krebs cycle, focusing on

the production of energy carriers.

A review of the Krebs Cycle shows that the energyinput from each acetyl-CoA creates one ATPmolecule, one FADH Z , and three NADH mole-

cules. Since each glucose molecule from glycoly-sis results in two molecules of acetyl-CoA, thecycle is considered to turn twice.

• trace and understand the sequences in theKrebs cycle (citric acid cycle);

• identify the end products of the cycle;

• state the energy products or carriers resultingfrom this cycle;

• explain the fate of the glucose's carbon atoms.

A summation of glycolysis, oxidative decarbox-ylation, and the Krebs cycle together gives thetotal energy products from one glucose moleculeas: four ATP molecules, ten NADH molecules, andtwo FADHZ molecules. The carbon atoms of theglucose molecule have been expelled as sixmolecules of waste carbon dioxide. Most of theenergy of glucose has been transferred to theintermediate energy carriers NADH and FADH Z .The program concludes by setting up the finalstage of cellular respiration, oxidative phosphoryla-tion, where the intermediate energy carriers areused to synthesize numerous ATP molecules.

The steps in the cycle-can be summarized asfollows:1. Acetyl-CoA reacts with oxoaloacetate to form

citric acid.2. Citric acid loses a molecule of water to be-

come aconitate.3. Aconitate adds water and is isomerized to

become isocitrate.4. Isocitrate encounters NAD+, forming oxalo-

succinate and NADH.5. Oxalosuccinate loses a molecule of CO 2 to

become ketoglutarate.

Survival depends upon the availability of largereserves of energy. Glycolysis, however, is aninefficient source of energy and cannot supplythese large reserves; therefore other phases ofenergy production are required. This program

examines the second phase of cellular respiration,the Krebs cycle. The program follows the fate ofpyruvate from glycolysis as it is acted on as asubstrate by enzymes within the mitochondria togenerate ATP and intermediate energy carriers.

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6.

7.

8.

9.10.

A summary of total energy products can be givenas follows:

In total, this process utilizes approximately 40% ofthe available energy, whereas glycolysis utilizesonly about 2%.

Figure 4. 1 Energy release from the Krebs Cycle (Thecycle can be considered to turn twice)

Ketoglutarate reacts with CoA to form suc-cinyl-CoA and a NADH molecule.Succinyl-CoA joins with ADP and a phosphateto release CoA, an ATP molecule, and succi-

nate.Succinate joins with an FAD molecule to form

an FADH 2 molecule and fumarate.Fumarate adds water to become malate.Malate reacts with NAD+ to become oxaloace-tate and form a NADH molecule.

NADH

BEFORE VIEWING

Help the students to consolidate the previousmaterial by stressing the following points.

1. Glycolysis is a very inefficient process: ityields only about 2% of the available energyof glucose. Glycolysis alone, therefore, couldnot provide the energy needed to powerenergetic organisms.

2. Pyruvate formation was the end process of theglycolytic pathway; this pyruvate will be thestarting point of the Krebs cycle.

AFTER VIEWING

1. Divide the class into two main groups. Onegroup, which can be subdivided into severalresearch sections, is to write out the structuralformulae for all of the Krebs cycle intermedi-ary compounds, including high-energy trans-fer compounds (NADH and FADH) and otherproducts such as CO 2.

The other group is to build the intermediariesfrom atomic model kits, using the standard colorcodes to represent different kinds of atoms.

Both groups should thoroughly brief their mem-bers with an eye to presenting a detailed accountof their results to the class.

2. Discuss the following points.a. It is evident that glycolysis does not pro-

duce enough ATP energy for higher lifeforms to carry out their activities,

b. Why would glycolysis and the Krebs cyclefunctioning together in tandem still notprovide enough energy to fuel complexorganisms?

c. What is the significance of the word cyclein the term Krebs cycle? What substance isregenerated at the end of the cycle and isused at the beginning of the next one?Why is this cycle gone through twice forthe complete respiration of each glucosemolecule?

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After viewing this program and completing thesuggested activities, students should be able to:

• trace how phase 1 and 2 of cellular respirationlead into oxidative phosphorylation;

• describe the process of the electron transportchains;

• understand the role of oxygen in siphoningelectrons from the electron transport chains;

• explain how the energy gradient across theintermitochondrial membrane is created, andwhy this gradient is important;

• follow the steps in ATP synthesis at the matrixside of the membrane;

• sum up the total production of ATP, NADH,and FADH2 from a single glucose molecule;

• state how many ATP molecules are producedat any step.

Cellular respiration in its first phase, glycolysis,produces only two molecules of ATP. Phase 2, theKrebs cycle, produces only two more ATP.However, phase 3, oxidative phosphorylation,produces an energy payload.

This process takes place within the inner mito-chondrial membrane. Embedded within thismembrane are four adjacent protein complexesthat make up the electron transport chain.Threeof these complexes act as proton (H+) pumps.Their function is to remove energy from the

electrons as they move in pairs down an energygradient.

The process begins as NADH donates two elec-trons to the first complex. Two hydrogen ionshitch a ride into the intermembrane space and thetwo electrons transfer to the second complex andreturn to the matrix side of the membrane. Twomore hydrogen ions are moved into the thirdcomplex and are carried to the intermembranespace. Two electrons return down the fourthcomplex and two more hydrogen ions move intothe intermembrane space. (Six hydrogen ionshave now crossed.) Finally, an oxygen atompicks up two electrons and two hydrogen ionsand forms water. (It is the primary role of theoxygen to siphon the electrons from the electrontransfer chains.)

The other energy carrier produced by the Krebscycle, FADH 2 , enters the chain and results in fourmore hydrogen ions being transferred to theintermembrane space. The concentration of H+ ismuch higher in the intermembrane space than onthe matrix side. This concentration results in apotential energy gradient, and this energy will beused to synthesize ATP. Pairs of protons (H+) aremoved down special channels; these protonsactivate an enzyme on the matrix side. Thisenzyme catalyzes the reaction of ADP with aphosphate group to synthesize ATP.

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In summary, glycolysis results in two ATP mole-cules plus four more at the electron transportchain, for a total of six ATP molecules. Oxidativedecarboxylation 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 anotherfour ATP molecules. The net result is 36 moleculesof ATP. Therefore, cellular respiration results in 36ATP molecules from one glucose molecule; thisrepresents about 41% of the available energy fromthe glucose molecule.

AFTER VIEWING

Activity 1:The Energy of Carbohydrates

The catabolic metabolism of glucose could beexpressed as follows:

BEFORE VIEWING

1. Students should review the structure of themitochondria., and consider such terms ascytosol, intermembrane space, cristae, matrix,and electron transport chain. They shouldreview, as well, these processes: diffusion,osmosis, and active transport.

2. Review the Krebs cycle in terms of where theintermediate energy carriers NADH and FADH Z

are given off.

Note that 36 molecules of ATP are ultimatelyproduced from 1 molecule of glucose.

1. How many ATP molecules can be producedfrom 1 mole of glucose? (Recall that 1 molecontains approximately 6 x 10 23 molecules.)

2. Each mole of ATP represents a capture of 31kJ. Calculate the total energy available for the36 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 differenttextbooks). From your answer to question 2above, 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 of100% glucose. Theoretically, how much en-

ergy could it release in kilojoules? Theoreti-cally, how many molecules of ATP could beproduced?

Activity 2:Mitochondria Morphology

1. Consult a suitable text containing large elec-tron micrographs of mitochondria. Studyphotographs from muscle tissue and from atleast two other types of tissue (e.g., liver,pancreas, kidney, digestive tract, etc.) andobtain 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 theend of the electron transport chain? Whatdrives the protons across the inner membraneand what is their ultimate fate? Discuss.

5. During fermentation (anaerobic respiration)what is the fate of the electron generatedduring the glycolysis of glucose?

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After viewing this program and completing thesuggested activities, students should be able to:

• recognize that much of our knowledge aboutcells comes from the development of models;

• appreciate the immense turnover of ATP inthe human body in a normal day;

• understand the basic operation of a muscle;

• describe how cells respond to an oxygenshortage caused by overexertion;

• describe how an oversupply of ATP may bestored eventually as "fat";

• appreciate that the complexity and collectivebehavior of cells is a reaffirmation of life itself.

Scientists frequently develop models to explainthe complexity of cellular respiration. Thesemodels, though, are often schematic diagramsand do not come close to revealing the magnifi-cence of the collective power of cells. Our bodiesuse and recycle about 40 kg of ATP each day, andstrenuous activity may cause them to use as muchas 0.5 kg per minute. For all body movements, itis ATP which provides the driving energy. Thisprogram examines the ability of cellular respira-tion to adjust to different conditions in the humanbody.

The program begins with modelling the role ofATP in the contraction of a muscle. The action ofATP is shown on the two proteins in muscle cellsactin and myosin. In time of overexertion, thebody may suffer a temporary oxygen shortage asthe circulatory system cannot provide the oxygenquickly enough. While glycolysis can provide asmall quantity of ATP, not enough is synthesizedand this results in an energy shortage.

The program describes how the process of cellu-lar respiration takes steps to overcome this short-age. The pyruvate that normally heads off to theKrebs cycle follows a different path when oxygenis in short supply-a path that leads to thesynthesis of lactic acid. The steps in this sequenceensure the continuous production of ATP. Thereis, of course, a debt to pay: a burning sensationwithin the muscles caused by the lactic acidbuildup. Fortunately, after a short rest, the returnof oxygen results in the metabolism of the lacticacid.

Too much glucose intake, on the other hand, canresult in the production of too much ATP. Thissurplus triggers a sequence of events wherebyacetyl-CoA produces fatty acids that are stored asfat.

This process can be reversed by dieting, in whichthe fat can be metabolized. This is done throughsequences that lead either to the glycolytic path-way or directly into the Krebs cycle.

Throughout this series the programs have de-picted how resourceful cells are and how thecollective behavior of a cell is a reaffirmation ofthe driving force of life itself.

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2.

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, glyco-gen, and oxygen?

2. Explain what happens when a muscle isoverexerted as during strenuous exercise andhow this condition is alleviated.

3. Discuss the conversion of energy during musclecontraction.

4. What are some other uses of ATP by cells ofmulticellular organisms?

5. Trace the catabolism of fatty acids through theKrebs cycle. How does the ATP yield from a6-carbon fatty acid compare with the ATPyield from glucose? What problem occurs if fatcatabolism is excessive?

Activity 2:Observation of Skeletal Muscle

Apparatusbeef toluidine-blue stainprepared slides of skeletal muscleprepared slides of cardiac muscle, if availablemicroscopes

FORE VIEWING

1. it could be advantageous for the student torecall or to look up the general structure of amuscle. Recognition of such things as theprotein layers of actin and myosin and themechanics of muscle contraction would behelpful.

Review program 4 with special reference tothe section on the electron transport chain.Review in particular the purpose of oxygen

and the role of NAD+ and its development.

microscope slidescover slipsdissecting needlesmedicine droppersforceps

Method1. Obtain a piece of beef from your teacher. Pull

the point of the dissecting needle across thelong grain of the muscle several times until asmall strand of tissue is removed. Caution:Use the dissecting needle with care as it is verysharp.

2. Using forceps, transfer the strand of beef tothe centre of a clean slide.

3. Put 2 drops of toluidine-blue on the tissue. Letthe stain remain for 2 minutes, then add 2

drops of water to the slide. Cover the tissuewith a cover slip.

4. Examine the tissue under the microscope atlow power. Focus on a portion that is thin andlightly stained. Draw a portion of what you

see.5. Switch to high power. Look for the striated

appearance of the muscle cells. Muscle cellsare made of microfilaments called myofilaments, which are composed of the proteinsactin and myosin. The portion of muscle fromone stripe to the next is called a sarcomere.The darkly stained structures are the nuclei ofthe muscle cells. Locate the sarcomeres andthe nuclei, and draw a diagram labelling thesestructures.

6. Use high power to examine prepared slides ofskeletal muscle and, if available, cardiacmuscle.

Discussion1. From your observations, is a muscle fibre

composed of several small cells, or one longcell containing many nuclei?Discuss the role of myosin, ATP, and actinduring the contraction of a muscle cell.What initiates contraction in vertebrate skele-tal muscle? What other chemicals are in-volved?

2.

3.

1 8

acetylCoAthe main molecule of energy metabo-lism; contains a high energy bond

actin one of two proteins making up the microfil-aments of muscle tissue

adenine an organic base consisting of two car-

bon-nitrogen rings

ADP adenosine diphosphate, a substance pro-duced when ATP gives up energy through the lossof a phosphate radical

anaerobic fermentation fermentation is theextraction of energy from organic compounds;anaerobic means that the process does not in-volve oxygen

ATP adenosine triphosphate, a nucleotide madeup of adenine, ribose sugar, and three phosphategroups; this is the energy carrier in cell metabo-

lism

carbohydrate a compound containing carbon,hydrogen, and oxygen wherein the ratio of hydro-gen to oxygen is 2:1; carbohydrates include sugar,

starch, etc.

decarboxylation the removal of the carboxylgroup (COON) from an organic molecule

DH" dihydroxyacetone phosphate, one of theproducts of the splitting of fructose diphosphatealong with PGAL; the DHAP then undergoesisomerization to become a second molecule ofPGAL

electron transport chain protein chain embed-ded within the mitochondrial membrane whichfacilitates the passage of electrons; third stage ofrespiration and principal site of ATP synthesis inthe cell

entropy refers to the unavailability of energy ina system, and a measure of a system's randomnessor disorder; the basis of the Second Law ofThermodynamics

enzyme a protein that speeds up or slows downcertain chemical reactions but does not, itself,change

FAD+ the oxidized form of FADH 2

FADH2 flavin adenine dinucleotide; a carrier oflower energy electrons

fatty acid an organic acid with a single carboxyl

radical along with other carbon and hydrogenatoms

cellular respiration, the production of energythrough the process of oxidation; the energy isproduced through the Krebs cycle andphosphorylation

citric acid cycle see Krebs cycle

glycogen a polysaccharide in which starch isstored in animal cells

glycolysis the process through which glucose isbroken down to synthesis ATP

coenzyme a cofactor that is a nonprotein organicmolecule; a cofactor is an enzyme employingmetal ions to acquire electrons

Coenzyme A organic molecule involved in en-zyme catalyzed process; this two-carbon mole-cule is the main molecule of energy metabolism

crista folded innner membrane of a mitochon-drion; the folds or cristae produce a large surfacearea in which are contained the electron transportchains

Krebs cycle a cycle of oxidation and reductionand the decarboxylation reactions from which acell can derive ATP; also called the citric acid cyclesince the cycle which begins with pyruvate laterforms citric acid which is oxidized to form CO 2

lipid organic compound insoluble in water butsoluble in certain organic liquids such as fats, oils,water, phospholipids, etc.

matrix the inner compartment of a mitochon-drion

1 9

mitochondrion cytoplasmic organelle; each onerepresents a complete mechanism that producesenergy (plural: mitochondria)

myosin one of the muscle proteins

NAD nicotinamide andenine dinucleotide, acoenzyme that acts as an electron acceptor; NAD+is its oxidized form

NADP nicotinamide adenine dinucleotide phos-phate, an electron acceptor in the process ofrespiration

phosphoglyceraldehyde shortened to PGAL, athree-carbon molecule; a six-carbon molecule of

glucose is broken into two molecules of PGALwith the input of ATP

photosynthesis the formation of carbohydratesfrom carbon dioxide and water in the presence oflight and chlorophyll

protein a chain of amino acids joined by peptidebonds

pyruvate a three-carbon (3C) compound; theend product of glycolysis and the material withwhich the Krebs cycle begins

ribose a sugar of the five-carbon type

sarcomere the fundamental unit of contractionin muscle tissue

20

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 ethanolproduction in saccharomyces cerevisiae: effectsof intracellular adenine nucleotide concentrationson the in vitro activities of hexokinase,phosphofructokinase, phosphoglycerate kinate,and pyruvate kinase. Applied and Environmental

Microbiology 55:1312-14, May 89.

Bodner, George M. Metabolism: part 3. Lipids.

Journal of Chemical Education 63:772-75, Sep 86.

Dobson, G. P. and Hochachka, P. W. Role ofglycolysis in adenylate depletion and repletionduring work and recovery in teleost white muscle.The journal of Experimental Biology 129:125-40,May 87.

Erickson, R. P.; Harper, K. J.; and Hopkin, S. R.Adenine nucleotides and other factors indicativeof glycolytic metabolism in murine spermatozoa.The journal of Heredity 78:407-09, Nov-Dec 87.

Furth, Anna and Harding, John. Why sugar is badfor you. New Scientist 123:44-7, S 23 89. A goodarticle for this series; deals with the evidence ofsugar-caused damage to long-lived proteins.

Milligan, L. P. and McBride, B. W. Energy costs ofion pumping by animal tissues. The journal ofNutrition 115:1374-82, Oct 85.

. Metabolism: glycolysis or the Embden-Myerhoff pathway. Journal of Chemical Educa-tion 63:566-70, Jl 86. An excellent article for thisseries; the steps are clearly laid out complete withequations.

Poolman, Bert; Bosman, Boukje; and Kiers, Jan.

Control of glycolysis by glyceraldehyde-3-phos-phate dehydrogenase in streptococcus cremorisand streptococcus lactis. Journal of Bacteriology169:5887-90, Dec 87.

. Metabolism: part 2. The tricarboxylic acid(TCA), citric acid, or Krebs cycle. journal ofChemical Education 63:673-77, Aug 86. Differentiates the tricarboxylic acid (TCA) from glycolysis,and describes the connection between the two asbeing the conversion of pyruvate into acetylcoenzyme A.

Brand, Martin D. and Murphy, Michael P. Controlof electron flux through the respiratory chain in

mitochondria and cells. Biological Reviews of the

Cambridge Philosophical Society 62:141-93,May 87.

Sherman, W. Mike. Carbohydrates, muscle glyco-gen, and improved performance. Physician andSports Medicine 15:157-61, Feb 87.

Simard, Clermont et al. Effects of carbohydrateintake before and during an ice hockey game onfood and muscle energy substrates. ResearchQuarterly for Exercise and Sport 59:144-47,June 88.

Wright, Russell G. and Bottino, Paul J. Mitochon-drial DNA. Science Teacher 53:27-31, Apr 86.

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Program TitleThe Cell and EnergyGlycolysis 1Glycolysis 2The Krebs CycleOxidative PhosphorylationMetabolism and Nutrition

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