“Big-Picture” Worksheets To Help Students Learn and Understand the

Activity pubs.acs.org/jchemeduc

“Big-Picture” Worksheets To Help Students Learn and Understand the Pentose Phosphate Pathway and the Calvin Cycle Paul A. Sims* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, United States S Supporting Information *

ABSTRACT: Two worksheets designed to help biochemistry instructors teach the pentose phosphate pathway and the Calvin cycle are presented. The worksheets avoid excessive detail, but portray the pathways in such a manner that the fates of all molecules involved are depicted; this format allows students to follow the interconversions without having to wonder where a particular intermediate originated or what final compound it is destined to become. When combined with knowledge of the structures of some basic monosaccharides and of key enzymatic steps (e.g., transketolase and transaldolase), the use of these worksheets can help students learn and understand the pathways rather than superficially memorize them.

KEYWORDS: Upper-Division Undergraduate, Biochemistry, Communication/Writing, Aldehydes/Ketones, Biosynthesis, Carbohydrates, Catalysis, Enzymes, Metabolism

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indicated). But textbook portrayals of the PPP and Calvin cycle are not easily amenable to such altering. Instead, textbook representations have tended to focus either on the big picture or on select steps in the pathway, and, as a result, students do not see the complete pathway in one figure. To address this concern, a search of the chemical and biochemical education literature was undertaken to determine if these pathways might be presented in a manner that included both the big-picture view and a depiction of all the chemical transformations. A search of this Journal revealed a number of outstanding articles that discussed the Calvin cycle,1−8 but only one article that discussed the PPP to a reasonable extent.9 In addition, two interesting articles presented lab activities centered on different enzymes that are common to both pathways,10,11 and another article advocated the use of a game to teach biochemical pathways, including the PPP.12 Although these articles provided good explanations of their particular points of emphasis, none presented the pathways in a manner that would allow students to follow easily the stoichiometry or to trace unambiguously the fates of all compounds involved in the pathways. But an article13 by the late Donald E. Nicholson held the key to presenting the PPP in a manner that would help students learn this pathway. The salient features of Nicholson’s portrayal of the PPP were his emphasis on the cyclic nature of this pathway and his inclusion of coefficients to indicate stoichiometry.13 The cyclic portrayal helped to clarify the fates of intermediate compounds, but the use of coefficients to indicate stoichiometry (e.g., 6

eaching metabolic pathways to undergraduate biochemistry students is challenging because some students may perceive the learning of pathways as an exercise in rote memorization. This perception is unfortunate because biochemical pathways can be taught in a manner that relies less on brute-force memorization and more on reasoning one’s way through a particular pathway using chemical logic. Teaching in this manner is facilitated when appropriate learning aids (e.g., worksheets) are incorporated into class discussions. Ideally, these learning aids should show the complete sequence of reactions and indicate the overall stoichiometry of the pathway. In other words, the appropriate learning aids should show the “big-picture” view, but also contain sufficient detail to convey the chemistry involved in the pathway. Two pathways for which the development of appropriate learning aids has been notoriously difficult are the pentose phosphate pathway (PPP) and the Calvin cycle. Part of the difficulty of constructing appropriate learning aids for the PPP and Calvin cycle stems from the inherent complexity of these pathways; it is difficult to strike a balance that shows the big picture but also includes all of the chemical transformations. When simpler pathways are considered, it is relatively easy to construct a worksheet that shows the entire pathway and includes all chemical steps. Oftentimes, worksheets can be made from textbook figures that have been altered or modified to show, for example, just the skeleton of the pathway. When students complete these worksheets by supplying the names and structures of the intermediates and the names of all enzymes involved, they have a view of the pathway that is reasonably complete (although interconnections with other pathways may or may not have been © 2014 American Chemical Society and Division of Chemical Education, Inc.

Published: February 24, 2014 541

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Figure 1. Transfers in the transketolase and transaldolase reactions: (A) transfer of the glycolaldehyde moiety from a ketose donor to an aldose acceptor in the transketolase reaction; (B) transfer of the dihydroxyacetone moiety from a ketose donor to an aldose acceptor in the transaldolase reaction. The tilt of the aldehyde carbonyls helps to indicate the stereochemistry of the reaction. Modified after Nicholson.13−15

that the names are in a sense opposite from what one should expect, then perhaps this fact will help them avoid confusion.

glucose 6-phosphate, 6 ribulose 5-phosphate), while helpful, did not allow students (especially more visually oriented students) to “see” the transformations that led to the overall stoichiometry. Thus, more detail was incorporated into Nicholson’s portrayal to help students keep track of the fates of the molecules involved, and this modification led to the development of the worksheets reported herein.

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USING THE WORKSHEETS A blank version of the PPP worksheet (with selected portions magnified and filled-in) is presented in Figure 2, and full versions, both completed and blank, are provided in the Supporting Information. In these worksheets, white circles were used to represent CHOH and CH2O− groups; gray circles were used to represent anomeric, aldehyde, ketone, carboxylate, or CO2 carbons; and capital P’s were used to represent phosphoryl (PO32−) groups. This symbolism made it possible to portray the molecules in a condensed form, which, in turn, allowed nearly all of the molecules involved in these pathways to be shown on the worksheets.17 The inclusion of nearly all reacting molecules made it possible for a student to keep track of the fates of the intermediates and to obtain, as mentioned, visual confirmation of the stoichiometry. Students were given blank versions of the worksheets during class (printed in landscape orientation with narrow margins to allow for ease of viewing), and they were encouraged to complete the worksheets by filling in the boxes that corresponded to the names of missing enzymes, intermediates, or cofactors. At the same time, blank versions of the worksheets were projected onto a screen to enable completion of the worksheets in a uniform and timely manner. The discussion of each pathway began in the upper, left-hand corner of each worksheet and proceeded clockwise. Students were reminded that “irreversible” enzymatic steps were indicated with unidirectional arrows and that reversible enzymatic steps were

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BEFORE THE WORKSHEETS Before using the worksheets, it is imperative that students know the structures of the component monosaccharides, especially their phosphorylated forms. It also is important that students know and understand the reactions, concepts, and intermediates of glycolysis and gluconeogenesis; these pathways are generally covered prior to discussions of the PPP and Calvin cycle. But two new reactions that are essential to understanding these pathways are the transketolase (both pathways) and transaldolase (PPP) reactions. The names of both enzymes are misnomers because transketolase catalyzes the transfer of an aldehyde moiety (glycolaldehyde) from a ketose donor to an aldose acceptor, and transaldolase catalyzes the transfer of a ketone moiety (dihydroxyacetone) from a ketose donor to an aldose acceptor13 (Figure 1). Although the issue of the unfortunate misnaming was raised on other occasions,14,15 the names were too firmly entrenched, and essentially all recent textbooks of biochemistry use the misnomers. The more accurate names glycolaldehydetransferase and dihydroxyacetonetransferase are recognized and accepted,16 but neither is widely used. From the student perspective, if they remember 542

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Figure 2. The pentose phosphate pathway: (A) blank version of the worksheet; (B) magnified portions of the worksheet (indicated with colored boxes) shown with filled-in blanks that list the correct, abbreviated names of the compounds and enzyme. Note that gray circles represent the anomeric, aldehyde, ketone, carboxylate, or CO2 carbons, and hatched circles (in part A) represent oxygens. Abbreviations: DHAP = dihydroxyacetone phosphate, GAP = glyceraldehyde 3-phosphate, R 5 P = ribose 5-phosphate, S 7 P = sedoheptulose 7-phosphate, and TIM = triose phosphate isomerase.

nucleotide biosynthesis. These discussions help to reinforce the notion that the pathway does not have to proceed in a cyclic manner, and that the manner in which it proceeds depends on the needs of the cell.14 Finally, the Calvin cycle shares many of the same enzymatic steps with the PPP, and these similarities were highlighted in a recent work that showed three-dimensional portrayals of these pathways.18 The portrayals were admirable, but both showed an erythrose 4-phosphate appearing part way through the pathways with no explanation of where or how this compound originated.18 The Calvin cycle worksheet (available in the Supporting Information) avoids this confusion and explicitly accounts for each intermediate of the cycle, including the net production of an organic biomolecule (glyceraldehyde 3phosphate) from an inorganic source (three CO2’s). In some textbook representations of the Calvin cycle, this net production is not always clear, and prior to using this worksheet in class, students would ask about the fate of the fixed carbon.

indicated with bidirectional, equilibrium arrows. They also were encouraged to use visual cues (e.g., the gray shading of the aldehyde and ketone carbons) to help them distinguish epimerization and isomerization reactions and to know which compounds are ketose donors and which are aldose acceptors in the transaldolase and transketolase reactions. After the worksheets were filled out, the projected versions remained on the screen while another writing surface (e.g., chalkboard) was used to facilitate detailed discussions of select reactions. For example, it was possible to note the oxidative part of the PPP (Figure 2, top, central portion of the worksheet) and to discuss simultaneously the two oxidative reactions (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) in more detail. The simultaneous portrayals of the big picture (i.e., overall pathway) and the detailed steps helped students place these reactions in context within the overall pathway. In a similar manner, discussions about the nonoxidative part of the PPP were used to help students appreciate how the glycolytic intermediates glyceraldehyde 3-phosphate and fructose 6-phosphate can be used to make erythrose 4-phosphate and xylulose 5-phosphate; how the xylulose 5-phosphate can, in turn, be epimerized to ribulose 5phosphate; and, finally, how the ribulose 5-phosphate can be isomerized to ribose 5-phosphate, which is necessary for

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EFFECTIVENESS OF THE WORKSHEETS The worksheets were made available to all students in an upper-division introductory biochemistry class. A formal 543

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Table 1. Average Point-Biserial Correlation Values and Average P-Values for Exam Questions That Were Asked Pre- and Postimplementation of the PPP and Calvin Cycle Worksheets Question Typea

Pre- or Postimplementation of the Worksheets

Average Point-Biserial Correlation Value

Average PValue

Pre Post Post

0.55(±0.06) 0.52(±0.07) 0.60(±0.09)

0.45(±0.04) 0.5(±0.1) 0.56(±0.03)

Generalrecognize broad features of the pathwayb Detailedrecognize structures of intermediates in the pathwayc Detailedidentify names of compounds or enzymes in the pathwayd

The “general, pre” question was from different versions of an exam given during the spring, 2012 semester, and the “detailed, post” questions were from different versions of exams given during the fall, 2012 semester. Values in parentheses represent standard deviations. bThis question was part of a 30-question exam and was answered by 168 students. cThese questions were part of a 28-question exam and were answered by 151 students. d These questions were part of a 33-question exam and were answered by 151 students. a

work and legacy of Donald E. Nicholson and (ii) a complete listing of the exam questions (and associated statistics) that formed the basis of Table 1. This material is available via the Internet at http://pubs.acs.org.

evaluation of their effectiveness as teaching aids, however, was somewhat limited because the nature of the exam questions, pre- and postincorporation of the worksheets, was so different. Prior to using the worksheets, exam questions over these topics tended to be very general because the corresponding textbook figures were not as helpful or clear as they might be. After the worksheets were implemented, the exam questions were considerably more detailed and challenging. Thus, it was not possible to evaluate responses to the same question pre- and postimplementation of the worksheets, but it was possible to examine some of the statistics associated with the different types of questions that were asked on exams. The statistics associated with different types of questions that were asked pre- and postincorporation of the worksheets are shown in Table 1; these statistics include the point-biserial correlation values and the p-values for different types of questions. The point-biserial correlation is a type of discrimination index; it is a measure of how well a question tends to be answered correctly by students who perform well overall versus those who tend to perform poorly overall, and its value should be greater than 0.2.19,20 The p-value represents the proportion of students that answered a particular question correctly, and its value should be in the range of ∼0.3−0.7.19,20 The average point-biserial correlation values are not dramatically different among the pre- and postimplementation questions, but the average p-values are slightly higher on the postimplementation questions. Thus, the inclusion of worksheets made it possible to ask more detailed questions that, on average, were answered correctly by about half the class. (A complete listing of the questions and statistics associated with each question is provided in the Supporting Information.)

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Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The author thanks students in the introductory biochemistry class at this university for their willingness to adapt to new teaching strategies. The author also thanks Paul F. Cook, Katie M. Branscum, and Alyssa C. Hill for reviewing the manuscript and providing many helpful comments. In particular, Paul Cook offered the suggestion to color gray the aldehyde, ketone, carboxylate, and CO2 carbons in the worksheets.

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REFERENCES

(1) Calvin, M. The Path of Carbon in Photosynthesis. J. Chem. Educ. 1949, 26, 639−657. (2) Benson, A. A. Photosynthesis: First Reactions. J. Chem. Educ. 1954, 31, 484−487. (3) Bassham, J. A.; Benson, A. A.; Calvin, M. Isotope Studies in Photosynthesis. J. Chem. Educ. 1953, 30, 274−283. (4) Park, R. B. Advances in Photosynthesis. J. Chem. Educ. 1962, 39, 424−429. (5) Bassham, J. A. New Aspects of Photosynthesis. J. Chem. Educ. 1961, 38, 151−155. (6) Calvin, M. The Nurture of Creative Science and the Men Who Make It. The Photosynthesis Story: A Case History. J. Chem. Educ. 1958, 35, 428−432. (7) Bassham, J. A. Photosynthesis. J. Chem. Educ. 1959, 36, 548−554. (8) Bishop, M. B.; Bishop, C. B. Photosynthesis and Carbon Dioxide Fixation. J. Chem. Educ. 1987, 64, 302−305. (9) Horecker, B. L. Pathways of Carbohydrate Metabolism and Their Physiological Significance. J. Chem. Educ. 1965, 42, 244−253. (10) Abernethy, J. L. Demonstration of Transketolase Activity in Plant Leaves: Synthesis of L-Glucoheptulose. J. Chem. Educ. 1965, 42, 286−288. (11) Jewett, K.; Sandwick, R. K. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory. J. Chem. Educ. 2011, 88, 1170−1174. (12) Ooi, B. G.; Sanger, M. J. “Which Pathway Am I?” Using a Game Approach To Teach Students about Biochemical Pathways. J. Chem. Educ. 2009, 86, 454−455. (13) Nicholson, D. E. Some Reflections on Metabolic Cartography. Biochem. Educ. 1972, 1, 6−7.

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SUMMARY Two worksheets were designed to facilitate teaching the PPP and Calvin cycle. The worksheets portrayed these pathways in a manner that retained an overall, big-picture view, but also included enough detail to allow students to follow the fates of all reacting molecules. It is hoped that instructors of biochemistry will find these worksheets to be helpful, and feedback regarding the effectiveness of these worksheets is, of course, welcomed. Adobe Illustrator files of the worksheets are included in the Supporting Information to allow instructors to modify the worksheets, and feedback regarding such modifications also is welcomed.

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AUTHOR INFORMATION

ASSOCIATED CONTENT

S Supporting Information *

TIF and Adobe Illustrator files of the filled-in and blank worksheets; a PDF that contains (i) a brief discussion of the 544

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(14) Nicholson, D. E. Further Reflections on Metabolic CartographyTwenty-Five Years On. Biochem. Educ. 1997, 25, 62−70. (15) Nicholson, D. E. Pentose Phosphate Pathway(s) Minimaps. Biochem. Mol. Biol. Educ. 2001, 29, 179−182. (16) International Union of Biochemistry and Molecular Biology (IUBMB) http://www.iubmb.org/ (accessed Dec 2012). (17) Separate ATP/ADP, NADP+/NADPH, 6-phosphoglucono-δlactone, and 6-phosphogluconate molecules are not explicitly shown; rather, coefficients are used to indicate the numbers of each of these compounds. (18) Sillero, A.; Selivanov, V. A.; Cascante, M. Pentose Phosphate and Calvin Cycles: Similarities and Three-Dimensional Views. Biochem. Mol. Biol. Educ. 2006, 34, 275−277. (19) Bodner, G. M. Statistical Analysis of Multiple-Choice Exams. J. Chem. Educ. 1980, 57, 188−190. (20) Eubanks, I. D., Eubanks, L. T., Eds. Writing Tests and Interpreting Test Statistics: A Practical Guide; American Chemical Society, Division of Chemical Education, Examinations Institute, Clemson University: Washignton, DC, 1995; pp 16−20.

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