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Keywords:

  • Alkaline phosphatase;
  • bioinformatics;
  • assessment;
  • enzyme kinetics

Abstract

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was developed and implemented. In this exercise, students examine the structure of alkaline phosphatase using the free, on-line bioinformatics protein-modeling program Protein Explorer. Specifically, students examine the active site residues of alkaline phosphatase and propose functions for these residues. Furthermore, by examining the mechanism of alkaline phosphatase and by using the published kinetic data, students propose specific roles for several active-site residues. Paired t-test analysis of pre- versus postexercise assessment data shows that the completion of the exercise improves student's ability to use kinetic data correctly thereby determining a probable catalytic function for an active site amino acid.

The American Society for Biochemistry and Molecular Biology (ASBMB) Recommended Biochemistry and Molecular Biology Undergraduate Curriculum lists kinetics as a core-content item that biochemistry and molecular biology students should master before receiving their baccalaureate degree1. Enzyme kinetics has traditionally been a major focus in both the lecture and laboratory curriculum for biochemistry courses. Laboratory exercises that utilize alkaline phosphatase as a model enzyme to illustrate steady-state kinetic properties of enzymes, including determining Km and Vmax, and the inhibition constants for various substances have been developed and used extensively in the undergraduate biochemistry laboratory2, 3. Alkaline phosphatase's stability, straightforward assay, and inexpensive assay reagents make it an ideal enzyme for teaching enzyme kinetics in the biochemistry laboratory. This traditional laboratory exercise not only helps students learn how to experimentally determine enzyme kinetic constants but also reinforces the enzyme kinetics material that is covered extensively in the lecture portion of a biochemistry course.

At the University of Wisconsin-La Crosse (UW-L), the one-semester Survey of Biochemistry course includes seven 55-minute lecture periods devoted to enzymes in which the following topics are covered: 1) general properties of enzymes, 2) effect of enzymes on activation energy and reaction coordinates, 3) catalytic mechanisms, specifically focusing on chymotrypsin, and 4) enzyme kinetics. The enzyme-kinetics section includes four lectures covering chemical kinetics, derivation of the Michaelis–Menten equation, analysis of kinetic data, and enzyme inhibition. The course also includes one 3-hour laboratory section in which the students experimentally determine the Km and Vmax values for the interaction of the enzyme, alkaline phosphatase, and the substrate, p-nitrophenyl phosphate. Students also determine the type of inhibition inorganic phosphate displays on alkaline phosphatase and the KI value for this interaction.

During the lecture enzyme-kinetics section, the concept of how scientists use enzyme-kinetic values to study enzyme active sites and mechanisms is highlighted, which emphasize the significance of kinetic values. For example, kinetic data for a native and an altered enzyme with a one amino acid substitution are compared, and through analyzing the kinetic data, plausible-specific functions for that particular active site amino acid are proposed. Evaluation of kinetic-assessment exercises, including homework questions, in-class problems, and exam questions, which were specifically designed to assess this concept, showed that many students did not understand this concept and thus could not properly use kinetic data to help elucidate the function of an amino acid.

To address this student-learning deficit, a computer laboratory exercise for the Survey of Biochemistry course was developed that complements the traditional “wet” alkaline phosphatase kinetics laboratory. The exercise allows the students to use a protein-modeling bioinformatics program to visualize alkaline phosphatase's structure and active site while also illustrating how kinetic data can be used to study the mechanism of alkaline phosphatase. This laboratory exercise was developed as part of the Bioinformatics Across Life Science Curricula program, a collaborative program, between instructors in the biology, chemistry, and microbiology departments that incorporates bioinformatics throughout all life science programs at UW-L4, 5.

This article describes the bioinformatics alkaline phosphatase structure/kinetics exercise that was developed and implemented in the Survey of Biochemistry course at UW-L. Improvement of student learning was documented using summative-assessment data from pre- and postexercise questions and also using student perceptions of their learning experience. (Note: A complete student exercise handout and set of instructor guidelines can be found in the on-line supplemental materials.)

ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The three major goals for the alkaline phosphatase bioinformatics exercise are for the students to examine 1) the structure of alkaline phosphatase using the free, on-line protein-modeling program Protein Explorer, 2) the active site residues of alkaline phosphatase and propose general functions for these residues, and 3) the mechanism of alkaline phosphatase function, and using published kinetic data, propose-specific roles for several of the active site residues in alkaline phosphatase.

At UW-L, the exercise is incorporated into the biochemistry laboratory curriculum during the laboratory session immediately following the wet-laboratory alkaline phosphatase kinetics exercise and previous to the lecture exam that covers the topic of enzyme kinetics. It is designed to be completed by students in a one 3-hour laboratory session, though with slight modification it can be carried out in a single 2-hour session. The exercise requires student access to computers with internet capability. Students work in groups of two; however, each student has their own individual computer and works through the protein-modeling component of the exercise individually. This ensures that each student has exposure to Protein Explorer and Chime and gains experience working with these important protein structure, bioinformatics tools. The exercise is carried out during a laboratory session, thus ∼20 students (10 groups) perform the exercise at the same time. This number allows for adequate instructor assistance for each group throughout the exercise.

The overall exercise is divided into two parts. In Part I, students work in groups of two and proceed through a series of exercises in which they first explore the overall structure and then the active site structure of alkaline phosphatase. At the end of this part of the exercise, students are asked to propose plausible functions for several specific active site amino acids. Students become familiar with using the program Protein Explorer and manipulating the protein structure using the plug-in Chime. In Part II, the instructor begins by presenting the published catalytic mechanism of alkaline phosphatase to the class. Then, by utilizing the structural information gained in Part I, the presented general mechanism, and the published kinetic data, students are asked to refine their earlier ideas regarding the role for several active site amino acids. The full exercise handout provided to students is posted on the UW-L Bioinformatics Across the Curriculum website (http://bioweb.uwlax.edu/GenWeb/Molecular/CHM325.doc) or can be found in the on-line supplemental materials. A full set of instructor guidelines that facilitate the adoption of this exercise in the classroom can also be found in the on-line supplemental materials.

Part I: Structure of Alkaline Phosphatase

Students begin by accessing the free, on-line program Protein Explorer and Chime (http://www.umass.edu/microbio/chime/pe_beta/pe/protexpl/frntdoor.htm) to examine the structure of alkaline phosphatase. The alkaline phosphatase PDB Identification Code of 1ED8 was selected for use in this exercise because it displays a phosphate group bound within the active site and thus helps students visualize substrate binding. The student-exercise handout provides explicit directions on how to use Protein Explorer for this specific exercise and warns students that the directions must be followed very carefully to ensure that they obtain all the information necessary to complete this exercise. Screen-captured images of what students should be seeing in Protein Explorer are shown periodically in the handout so that students can confirm that they are at the right part of the exercise (Fig. 1 for sample images).

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Figure 1. Screen capture images of Protein Explorer that display various images of alkaline phosphatase studied during the bioinformatics exercise. The images of alkaline phosphatase illustrate a) the homodimer quaternary structure, the disulfide bonds, and the two active sites; b) the secondary structural elements; c) the active site with the cofactors (two Zn and one Mg), the phosphate molecule (which mimics substrate binding), and Glu 322; d) the active site visualizing Glu 322, Asp 51, Asp 327, His 412, and Ser 102 residues.

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In Part I, students are guided to examine various views of alkaline phosphatase's structure, the active site of alkaline phosphatase, and the enzyme's secondary structural elements. Students are asked to answer the following questions.

  • How many subunits does alkaline phosphatase have?

  • How many disulfide bonds does alkaline phosphatase have?

  • How many disulfide bonds does each alkaline phosphatase subunit have?

  • What atom does the green sphere represent?

  • What molecule does the orange sphere with four red spheres attached to it represent?

  • What atom does the maroon sphere closest to the green sphere represent?

  • What atom does the other maroon sphere represent?

  • What secondary structural elements does the alkaline phosphatase contain?

  • Does one of these elements predominate in the structure?

After the students are familiar with Protein Explorer and Chime and have seen the overall structure of alkaline phosphatase, they start to examine the active site in extreme detail. Leaving the Mg2+ and Zn2+ cofactors in the structure along with the phosphate group, the rest of the enzyme structure is hidden. Students are then guided to add back a particular active site residue one by one in a ball and stick display. First, students add back residue Glu322. After manipulating the structure with Chime to examine various viewpoints of the structure, students are prompted to answer the following questions regarding Glu322. For this first amino acid, sample answers to the questions are given in bold.

  • What is the functional part of this amino acid (what part is in the active site)? The carboxyl group of the glutamate side chain.

  • What other active site atom(s) are in closest contact with this residue and what is this residue's possible role(s)? The Mg atom and thus the negatively charged side chain may be coordinated (bound) to the positively charged Mg atom thus holding the Mg atom in its proper conformation.

Students are then prompted to add Arg166, Asp51, Asp327, His412, and Ser102 one at a time into the active site and answer the same questions as shown previously for Glu322. It is important to convey to the students that their answers are just “potential” functions for the amino acids because at this point of the exercise they have only structural information in which to base their answers. Also, during this part of the exercise, students are asked to differentiate between the two zinc cofactors by referring to the zinc atom closest to the magnesium atom as Zn2 and the other zinc atom as Zn1. This distinction becomes very important in Part 2 of the exercise when the enzyme mechanism will be presented.

Part II: Correlation of Kinetic Data and Amino Acid Functions in Alkaline Phosphatase

All groups need to complete Part I before the class proceeds to Part II, in which the mechanism of alkaline phosphatase is studied, and several of the amino acids highlighted in Part I are further examined. As a class, the students are shown and guided through a published figure that illustrates the general mechanism of alkaline phosphatase[6]. To facilitate student learning, the published mechanism figure (Fig. 2) is slightly modified in that the amino acid that serves as the nucleophile is not revealed.

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Figure 2. Mechanism of alkaline phosphatase shown in the bioinformatics exercise. To maximize student learning during the exercise, the mechanism figure does not reveal serine 102 as the nucleophile.

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After the mechanism is reviewed, students are asked to focus on the two zinc atoms depicted in the mechanism and describe the role of Zn1 and Zn2 in the alkaline phosphatase mechanism. They are asked if each zinc atom is important for substrate binding and/or for the reaction in converting substrate to product. Through class discussion, it is discovered that the mechanism shows that both Zn1 and Zn2 are important in substrate binding and in converting substrate to product and thus if either zinc dissociates from the active site the enzyme's Km and Kcat will be affected.

In the last part of the exercise, students use information gained from the structure of alkaline phosphatase's active site, the general mechanism, and published kinetic data for various active site amino acid mutations (Table I) to propose specific roles for certain amino acids[6–9]. They are asked to answer the following questions for Ser102, Asp327, and His412.

  • 1
    From the kinetic data, determine whether Ser102 is largely involved in substrate binding or the reaction of converting substrate to product (or both).
  • 2
    What is the specific role of Ser102 in the mechanism of alkaline phosphatase?
  • 3
    What information supports this answer?
Table I. Alkaline phosphatase kinetic data incorporated into the bioinformatics exercise
MutationKcat (s−1)KmM)
Ser102 [RIGHTWARDS ARROW] Ala[8]0.0013444
Asp327 [RIGHTWARDS ARROW] Ala[9]0.0113,900
His412 [RIGHTWARDS ARROW] Asn[7]7210
His 412 [RIGHTWARDS ARROW] Asn (w/added Zn) [7]189
Wild type[7–9]358

Some students initially finish this part of the exercise; however, after reviewing the group's answers, it is found that the students focus too generally on the amino acid role and do not use the new mechanism information to formulate their answers. For example, students may initially answer that Ser 102 has the specific role of proton donor or that it stabilizes a transition state molecule, roles they have seen for other amino acids in enzyme mechanisms. However, when asked where the proton donor is in the alkaline phosphatase mechanism, they realize that the attention needs to be given to the role that Ser 102 plays specific to the alkaline phosphatase mechanism. After further thought, students may realize that Ser 102 actually acts as the nucleophile in the alkaline phosphatase mechanism. Students sometimes come to this realization quite quickly by using the structural information they gained in Part I and from their knowledge of serine's role in chymotrypsin, an enzyme they have studied in lecture. If not, then, several probing questions are needed to guide students to formulate that answer, such as “Where is Ser 102 positioned in the enzyme active site?” or “On the mechanism figure point to where Ser 102 would be found?” It is important that the students realize that multiple pieces of information were used to conclude that Ser 102 is the nucleophile, such as the structural position of Ser 102, that a serine has the capability of acting as a nucleophile and that when changed to alanine, an amino that cannot act as a nucleophile, the enzyme reaction virtually does not occur as indicated by the very low Kcat value as shown in Table I.

The specific roles for Asp 327 and His 412 are not as difficult for students to determine, because in Part I they have already proposed that both these amino acids are important in positioning/holding Zn1 in the active site. Some groups need the instructor's guidance to understand that the effects on the kinetic constants when Asp 327 or His 412 are changed results indirectly from the dissociation of Zn1 from the active site as supported by the “w/added Zn” His 412 [RIGHTWARDS ARROW] Asn kinetic data.

LEARNING ASSESSMENT

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Summative Assessment

The major purpose driving the development of the described alkaline phosphatase bioinformatics exercise was to increase the student's understanding of how kinetic data can be used to elucidate the function of an active site amino acid, a concept that was shown to be a challenge for many students. Summative assessment, using pre- and postexercise questions, was used to assess student improvement of this ability. Students answered a set of three questions before starting the bioinformatics exercise (pre) and after completion of the bioinformatics exercise (post) (Fig. 3). Each question showed mutational kinetic data in Michaelis–Menten graph form or in table form, and based on the kinetic data, students were asked to propose a specific function for a particular amino acid. Students had 10 minutes to complete each set of questions. Over a two-semester time frame, the assessment exercise was administered to 91 students.

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Figure 3. Sample of each of the pre/postexercise questions including sample answers ranked adequate and inadequate.

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Each student answer was rated by two different evaluators with the following rubric:

  • 1 = The answer shows adequate ability by the student to use kinetic data correctly to determine a probable catalytic function for an active site amino acid.

  • 0 = The answer shows inadequate ability by the student to use kinetic data correctly to determine a probable catalytic function for an active site amino acid.

The two evaluators initially reviewed 30 answers together to ensure that the criteria used to rate the answers was standardized. All student answers were then rated by each evaluator individually, a total of 546 pre- and postanswers. Student answers that did not receive the same rating by both evaluators were further evaluated. Figure 3 shows examples of answers rated adequate and inadequate for each question. Analysis resulted in each student receiving six ratings, three pre-exercise, and three postexercise. The preratings were summed together as were the postratings (maximum = 3, minimum = 0).

SPSS paired t-test analysis of pre- versus postexercise data showed a statistically significant (p < 0.05) improvement in student's ability (pre-exercise mean = 1.46 and postexercise mean = 2.65, n = 91) to use kinetic data correctly to determine a probable catalytic function for an active site amino acid (Fig. 4). Furthermore, the evaluators observed that the pre-exercise answers commonly contained vague language, which made it difficult to judge the level of student understanding. Whereas the postexercise answers overall showed the use of better kinetic terminology and contained more sophisticated justifications.

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Figure 4. Pre- versus postexercise assessment results.a) Comparison of ratings of student-assessment answers with error bars indicating 95.0% confidence intervals (n = 91). Paired t-test analysis of pre- versus postexercise data showed a statistically significant (p < 0.05) improvement in student's ability to use kinetic data correctly to determine a probable catalytic function for an active site amino acid. b) Comparison of means for reported student confidence levels with error bars indicating 95.0% confidence intervals (n = 95). Paired t-test analysis of pre- versus postexercise rankings showed a statistically significant (p < 0.05) increase in student confidence for all areas assessed.

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Student Perception of Learning Experience

Students were asked to complete a questionnaire in which they ranked “confidence in their ability to …” from high (score of 5) to low (score of 1) before and after the exercise for a variety of different skills relating to bioinformatics, kinetics, and protein structure (Fig. 4). Paired t-test analysis of pre- versus postexercise rankings showed a statistically significant (p < 0.05) increase in the student's confidence in all areas. Students reported the greatest confidence gain in their ability to 1) use Protein Explorer, 2) interpret the mechanism of alkaline phosphatase, 3) visualize an enzyme's active site, and 4) correlate the amino acid function with mutation effects on kinetic data. The responses to the Protein Explorer and the alkaline phosphatase mechanism items were not surprising because this was the first time many students were exposed to these items. However, students had been exposed prior to this bioinformatics exercise multiple times to the concepts of enzyme active site structures and the use of kinetic data in proposing amino acid functions. Therefore, it was encouraging to document that the completion of the bioinformatics exercise increased student's confidence gain for these two items, which were also two major goals for the exercise. The large confidence gain in student's ability to correlate the amino acid function with mutation effects on kinetic data was corroborated by the pre- and postexercise assessment data.

ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The development and incorporation of an alkaline phosphatase structure/kinetics bioinformatics exercise into the UW-L Survey of Biochemistry curriculum, as described above, can readily be integrated at other institutions in a similar fashion. However, because biochemistry curriculums vary between institutions in regard to the number of laboratory meetings per semester, the length of each laboratory session, and access to computers, several adaptations of this exercise are described below.

Focus on Zn and Mg Cofactors

In 2002, Rob Dean published a kinetics exercise[3] that could be incorporated into the undergraduate laboratory curriculum that focused on studying the magnesium and zinc cofactors of alkaline phosphatase. This exercise goes beyond the typical kinetics study in that not only are the Km and Vmax values for alkaline phosphatase determined in the presence and absence of inorganic phosphate, thus illustrating competitive inhibition, but the kinetic constants are also determined with varying concentrations of magnesium or zinc ions. This wet-laboratory kinetics study could be performed in conjunction with the bioinformatics exercise described in this article to provide students with both data and structural images to illustrate the importance of metal cofactors in the function of alkaline phosphatase.

Molecular Biology Expansion Activity

In the bioinformatics exercise, published kinetic data is utilized to illustrate the importance of various active site amino acid residues. This exercise could be modified to have students generate specific mutations in alkaline phosphatase, purify the enzymes, and then determine and compare the kinetic constants for both wild-type and mutant alkaline phosphatase enzymes. Students would use Part I of the presented bioinformatics exercise, which focuses on the active site structure, to determine what specific mutations they would like to study.

Because a plasmid-containing phoA gene is not readily available, the gene would need to be PCR amplified from E. coli and then cloned into a plasmid. Once cloned, the specific mutations could be introduced into the phoA gene by site-directed mutagenesis. The plasmids containing the phoA mutations would then be transformed into E. coli strain E15, which contains a deletion in the phoA gene. This strain is available through the Coli Genetic Stock Center at Yale University (CGSC# 4829) and should to be used as a host strain to minimize the background expression of wild-type alkaline phosphatase. The plasmid proteins, both wild-type and mutated alkaline phosphatase, could then be expressed and purified via ammonium sulfate precipitation and anion exchange chromatography[7].

This potential adaptation would require a significant molecular biology component being added to the exercise, which may fit nicely into some biochemistry laboratory curriculums. If molecular biology is not a key goal for the biochemistry course curriculum, the cloning- and site-directed mutagenesis could be carried out within a separate molecular biology course, while the enzyme purification and kinetic studies could be carried out in the biochemistry course, thus forming a connection between both biochemistry and molecular biology laboratory course curriculums. Furthermore, the cloning and site-directed mutagenesis could be carried out via an undergraduate research project with the constructs then being utilized by students in the biochemistry laboratory course.

Prelaboratory Active Site Visualization

Another adaptation of the presented bioinformatics exercise could be for the students to explore the structure of alkaline phosphatase before they performed the traditional wet-laboratory kinetics experiment. Students could perform just Part 1 of the exercise whereby they would examine, either individually or as a whole class, the overall and active site enzyme structures before they worked with the enzyme in laboratory. This would provide more meaning to the kinetics experiment they then perform. Furthermore, the bioinformatics exercise would provide students with a visual representation of the competitive inhibition by inorganic phosphate, which they will experimentally determine from the kinetic data they generate in the laboratory.

CONCLUSION

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The alkaline phosphatase structure/mechanism bioinformatics exercise described in this article has been successfully performed by a large number of upper-level undergraduates at UW-L over a 5-year period. The exercise provides the students with a hands-on experience of visualizing and manipulating a protein structure using the protein-modeling bioinformatics program Protein Explorer. Furthermore, assessment data show that having students utilize published kinetic data, structural information, and general mechanism information together to elucidate the specific catalytic function for several alkaline phosphatase active site residues improves their ability to use kinetic data correctly in determining a probable catalytic function for an active site amino acid. In addition, the exercise reinforces or improves student's understanding of basic enzyme concepts such as active site structures, catalytic mechanisms, and kinetic constants and can be adapted to fit various biochemistry curricula.

REFERENCES

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. ALKALINE PHOSPHATASE BIOINFORMATICS STRUCTURE/KINETICS EXERCISE
  4. LEARNING ASSESSMENT
  5. ADAPTATIONS OF EXERCISE INTO VARIOUS CURRICULA
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1470-8175/suppmat .

FilenameFormatSizeDescription
sm_file_BMB120_1.doc237KStudent Handout [bmb120-StudentHandout-AnInsideExplorationofAlkalinePhosphatase.doc renamed to sm_file_BMB120_1.doc]
sm_file_BMB120_2.doc63KInstructor Guidelines [bmb120-InstructorGuidelines-AnInsideExplorationofAlkalinePhosphatase.doc renamed to sm_file_BMB120_2.doc]

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.