Articles

On the Convenience of Using a Computer Simulation to TeachEnzyme Kinetics to Undergraduate Students with BiologicalChemistry-related Curricula

Received for publication, June 10, 2002, and in revised form, September 30, 2002

Javier Gonzalez-Cruz‡, Rogelio Rodrıguez-Sotres§, and Mireya Rodrıguez-Penagos§¶

From the ‡Departament of Special Programmes and the §Department of Biochemistry, Facultad de Quımica,Universidad Nacional Autonoma de Mexico, 04510 Mexico DF, Mexico

Enzyme kinetics is a difficult subject for students to learn and for tutors to teach. During the practicalsincluded in the biochemical courses at the Faculty of Chemistry of Universidad Nacional Autonoma deMexico, we found that the students acquire good training in the calculations to obtain kinetic parameterssuch as Km, Vmax, optimum pH, etc., but, when they are questioned about the significance of the values andtheir relationship to enzyme catalysis, many confusing ideas arise. To provide extended practice opportu-nities that could aid in the learning process we developed computer software that simulates an enzymeassay for lactate dehydrogenase, named enzsimil, that was used as well as the practical sessions. Wetested three different levels of guidance to work with enzsimil, scripts where the students had to followdetailed (guided), intermediate (semiguided), or minimal (unguided) instructions, and for comparison, onegroup had a session of solving problems in class extracted from the program (class), and one more grouphad no additional sessions (control). After their respective sessions, the students either wrote a report orcompleted their script and undertook a laboratory practical. At the end, an exam was applied to allstudents. The reports and exams were graded, and the performance of the experimental groups wassubjected to a statistical analysis. In addition, we carefully read the answers trying to identify the morecommon errors and misconceptions. The study revealed that there are statistically significant benefits inthe use of the program. The semiguided scheme was more convenient to help the student in the short termfor the preparation of better reports, while in the long term, both the semiguided and unguided groupsperformed equally well. These results are discussed in terms of the convenience of guided or unguidedteaching strategies in computer-assisted learning.

Keywords: Teaching enzyme kinetics, computer-assisted teaching.

Enzyme kinetics in the Biochemical course has provento be too complex for the 3rd-year students of differentchemistry areas. Their knowledge of basic algebra andhow they interpret graphs requires improvement. This sit-uation is a crucial obstacle to their understanding of sev-eral subjects and in particular to the specific processes ofenzyme kinetics [1]. When we searched for ways to solvethis problem, we found that extending the laboratory prac-tice was expensive and time-consuming.

As an alternative solution, we developed a computersoftware program (named enzsimil)1 that simulates an en-zyme assay for lactate dehydrogenase. With this programthe student carries out a large number of enzyme assays ina short space of time. In this way practice should reinforcethe basic understanding of enzyme kinetics. Nevertheless,during the development of the program a number of ques-tions arose:

a. Will the simulator be a teaching aid that improvesthe performance of the students in the learning andcomprehension of enzyme kinetics?

b. What should be the role of the tutor during the useof the program?

c. Should the program be used without guidance toproduce a cognitive conflict and the spontaneousprocess of equilibration as suggested by Piaget [2]or as a teaching medium specially aimed to helpstudents in learning concepts and strategies as pro-posed by Vygotsky [3] or somewhere in the middle[4]?

With these questions in mind, an experimental model wasdesigned with different teaching methodologies withwhich the students used enzsimil.

In the present article, we describe the results obtainedwith five distinct groups during five different terms. Thearticle is divided in three parts. Under “Part I: Use andApplication of the Experimental Model” the experimentalmodel used and how it was applied is described. Under“Part II: Comparison of the Students’ Performance withand without enzsimil Using Different Teaching Modalities”

¶ To whom correspondence should be addressed. E-mail:penagos@att.net.mx.

1 The computer program described in the text is in Spanish, isfree for non-commercial use, and can be requested by e-mailfrom sotres@servidor.unam.mx.

© 2003 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATIONPrinted in U.S.A. Vol. 31, No. 2, pp. 93–101, 2003

This paper is available on line at http://www.bambed.org 93

the students’ performance with or without the use of thesoftware under the teaching modalities employed is sub-jected to a statistical evaluation. Finally, under “Part III:Interpretation and Discussion of Results” an interpretationof our findings is advanced, and a discussion is presentedabout the relationships between enzsimil, the teachingmethodologies used, and the students’ performance.

PART I: USE AND APPLICATION OF THE EXPERIMENTAL MODEL

Description of the Simulator

Kinetic Model and Algorithm—The program was writtenin Pascal and compiled under MS-DOS 6.0, so it can berun on most PC computers; it can also be run underWindowsTM in an MS-DOS-emulated environment. In de-signing the program a bi-bi ordered fully reversible kineticmodel was employed (Scheme 1, steps 1–5), consideringthe formation of two dead end complexes (Scheme 1,steps 6 and 7) when the first substrate to enter and the firstproduct to leave were simultaneously bound to the en-zyme, in both directions [5]. At the beginning of eachcalculation the program corrects the rate constants for thevarious reaction steps for temperature using the Arheniusequation [6] and determines the amount of active enzymeremaining after the preincubation period, if this was re-quested, assuming five ionic forms for the free enzyme(Scheme 2, E�2, E�1, E0, E�1, and E�2), which differ sub-stantially in their thermal stability (Scheme 2, rates k11 tok15). Of these five forms, only three were able to bind thesubstrates (Scheme 2, E�1, E0, and E�2), and the sub-strate-bound enzyme forms had enhanced thermal stabil-ity (Scheme 2, rates kn2, kn3, and kn4 smaller than k11 tok15). The program introduces pH effects on catalysis bycorrecting the kinetic constants in the steady state rateequation for pH [7]. For these calculations the three activeionic enzyme forms were considered to have differentbinding and catalytic properties. Then, at 1 � 10�4 sintervals, the distribution of enzyme forms is calculatedfrom the steady state distribution equations deduced fromthe kinetic model by the King-Altman method, and theamount of each form surviving the total time elapsed iscalculated from its respective first order rate law. Thedifferential form of the steady state rate equation is used tocalculate the amount of products formed during the 1 �10�4 s elapsed, and the concentrations of each substrateand product are updated. These operations are repeatedas many times as necessary to complete the desired assaytime. Conditional sentences were introduced at somepoints in the algorithm to prevent numerical errors result-ing from calculations with extreme values of the variables.For instance, high temperatures result in “overflow” errorsduring the attempt to calculate the exponentials in the

Arhenius equation for denaturation rates, but since largedenaturation rates imply that the activity goes to zero aftera few milliseconds, product accumulation is negligibleanyway, so the error can be avoided by setting the enzymeactivity to zero.

User Interface—The screen observed by the user isrelatively simple, mostly self-explanatory, and is depictedin Fig. 1. After entering the conditions and pressing F9, fivevalues of the absorbance of NADH at regular time intervalsare given by the program. The absorbance is calculatedfrom the Lambert-Beer equation using a molar extinctionof 6.22 �M�1 cm�1. The experimental error is simulated byrandom changes of the substrate, product, and enzymeconcentrations (within 5% of the given value), and in ad-dition, a random noise within �0.0005 absorbance units isadded to the calculated value. Absorbance has an upperlimit of 3.154 absorbance units. These last two calcula-tions intend to simulate experimental and instrumentallimitations found in a real laboratory. The final result aschange of absorbance per minute is obtained by non-weighted linear regression of the sampled points.

Design of the Experimental Model

When enzsimil was initially used, the general commentsof the tutors from the biochemistry department were thatthe students did not understand what they were doing andneither did they improve their performance in the exami-nations. To test how enzsimil could be a more efficientteaching and learning aid, four different teaching modali-

SCHEME 1

SCHEME 2

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ties were designed [8–10] to be used during and after thework with enzsimil. These modalities were Guided (n � 32),Semiguided (n � 26), Unguided (n � 20), and Class (n �10); a Control group (n � 31) was also included. Thestudents were ranked according to their general markaverage in their career and sequentially sorted (with the aidof a computer spreadsheet) into the four teaching modalitygroups. In this way, each group had students with a similardistribution of general averages. The Control group con-sisted of all the students registered in one group of thebiochemistry course for one term; with that exception, theother four modalities were applied in each term. A briefdescription of each modality follows.

Guided—In this modality, the students were given aprinted document (script) containing full detailed instruc-tions about:

i. How to carry out an enzyme assay using the com-puter program (section 1).

ii. How to perform the experiments and the values ofthe parameters to be used in those experiments.These parameters are as follows: effect of pH (sec-tion 2), effect of temperature (section 3), effect ofenzyme concentration (section 4), and effect ofsubstrate concentration (section 5).

iii. Closed questions were included that required thestudents to analyze their results and asked them toreach conclusions, giving a set of conditions wherethe LDH2 activity should be maximal (section 6). Thequestions required an understanding of how thevariables act and an analysis of possible causes forthe phenomena observed. At the same time, thismodality did not give room for the student initiativeto explore other assay conditions or to discussaspects that were not included in it. This modalitywas worded so that most possible mistakes madeby the students while studying enzyme kinetics us-ing enzsimil were constrained.

Semiguided—This second script had minimal instruc-tions about how to make an enzyme assay in the computerprogram (only section 1 of the Guided script) and a shorttext to remind the students that the enzyme activity mustbe “linear in the time interval selected and reasonablyhigh.” To achieve this, they had to find the assay condi-tions through several changes in the variables that influ-ence the enzyme assay. Further, they had to choose therange of values for the same parameters studied in theGuided script. These sometimes gave them unusual re-sults, deviating from the classic behavior obtained with theGuided script or in an experimental session. The scriptalso included some general questions to encourage stu-dents to analyze their results. To do that successfully, itwas essential that they reviewed the subject in biochem-istry textbooks as their results could have been atypical.Finally, they had to deduce optimal conditions for an LDHassay. The Semiguided modality allowed the students tomake mistakes in the experiments they were performing andto discuss what they thought was relevant to the study.

Unguided—In this third modality, the script includedonly section 1 and a list of the parameters to be investi-gated. The students have to work out for themselveswhether the results they obtained were in accordance withwhat they had studied or whether the conditions theychose were suitable for the enzymatic assay. After a week,they have to write a typical laboratory report as is custom-ary in the faculty of chemistry. The report included anIntroduction (with a general hypothesis), Materials andMethods, Results, Discussion, and Conclusions (they didnot have to follow any particular script). In the Unguidedmodality, we found frequent cases of non-classic enzymekinetic behavior due to unsuitable choices for conditions,for instance the enzyme activity being too low to see anychange due to unsuitable pH, temperature, etc. or a verynarrow range of the values of the factor under study (e.g.pH � 7–8).

Class—This fourth modality has exactly the same scriptas the Guided modality, but data were included as ob-tained from the computer program. The students solvedthe Class script in a classroom with a tutor present toassist the students when they had difficulties with thequestions. However, they were not allowed access to thecomputer program.

Control—There was a fifth option, the Control group, inwhich the students did not have access to the computerprogram nor did they carry out any enzyme kinetics exer-cises in the classroom.

Other Sessions and Evaluation

Following the computer session, the students had aweek to fill in the script (except for the Control group) or towrite the typical laboratory report. In the 2nd week, theyhad a practical session where they perform experimentsregarding the same aspects they had studied with thecomputer program. In the 3rd week, the students had adiscussion session with the tutor where their results (ex-perimental and from the computer program) were analyzedand discussed with the whole classroom, and at the end ofthat session, they took an exam.2 The abbreviation used is: LDH, lactate dehydrogenase.

FIG. 1. User interface of enzsimil. Preincubation (Preincuba-cion) can be requested, but an evaluation of its use was beyondthe scope of the present study. The student can vary all theparameters shown, including pH, temperature (Temperatura), to-tal assay time (Duracion), and the total initial concentrations ofadded enzyme (Concentracion de enzima), substrates (SUSTRA-TOS: NAD� and Lactato), and products (PRODUCTOS: Piruvatoand NADH). The forward reaction was taken after the enzymename, but if pyruvate (Piruvato) and NADH are added at thebeginning a decrease in absorbance is observed (as actuallyshown).

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The students were presented with an exam of multiplechoice questions concerning:

i. Previous knowledge acquired in subjects other thanbiochemistry or in the theoretical biochemistrylectures,

ii. Specific knowledge that allegedly had been taughtduring the computer program and the practicalsession,

iii. The application of the knowledge recently obtained,and

iv. A last section consisting of the interpretation ofgraphs elaborated with data from experiments sim-ilar to those performed with the computer program.

In the last section (iv) they had to apply the abilities andknowledge supposedly acquired in the solution of twoproblems. The first one asked the student to tell the dif-ference between two isozymes based on the kinetic re-sponse of those enzymes, and the second one asked thestudent to choose the conditions where an enzyme activityshould be maximal. The data for both problems werepresented in plots similar to those expected from thestudents in their previous work. The results were subjectedto statistical analysis using the SigmaStat statistical pack-age (Jendel Corp., San Rafael, CA).

PART II: COMPARISON OF THE STUDENTS’ PERFORMANCEWITH AND WITHOUT ENZSIMIL USING DIFFERENT

TEACHING MODALITIES

Does the Computer Program Influence thePerformance of the Students in Scripts and Exams?

To answer this question we started looking at possibledifferences in the marks of the students in scripts of thosegroups that did not use the program compared with thosewho worked with the program in a fully Guided, Semigu-ided, or Unguided manner. The statistical analysis of thedata (Table I) revealed no significant differences betweenthe Guided and the Semiguided groups: both performedbetter than the Class group with the performance of thestudents in the Unguided group being the worst.

The above results indicate that the students who used

the program with Guided or Semiguided scripts performedwell. However, it is not so surprising that the use of theprogram was not essential for the average student to scorea mark above 60% (minimal mark to pass). At the sametime, it is noticeable that the worst group was the Un-guided group. A reasonable conclusion of these results isthat although the program seemed to be a good teachingaid, guidance is essential, especially if we expect the stu-dents to make an ordered analysis of those factors, whichenable them to reach some conclusions. Additional obser-vations on each group follow.

Guided and Semiguided Groups—The Guided groupwas given specific instructions regarding the magnitudeand ranges of the numerical variables to be tested,whereas the Semiguided script only suggested which pa-rameters should be tested and how to decide whether theresponse obtained is acceptable. Therefore, the fact thatthe Guided and the Semiguided groups performed equallywell indicates that the knowledge and experience gainedin the investigation of the adequate conditions for thebasic enzyme assay lacked relevance in relation to theability of the students to give an adequate description ofthe effects of the various parameters to be studied whenanswering the scripts.

Unguided Group—It is important to mention here that tomark the Unguided group report (as they did not have anyscript to answer) we followed the criterion of awardingpoints for each parameter affecting the enzyme that theytested successfully and additional points if the experiencewas ordered enough to allow them to describe the char-acteristics of the response even if the description was onlysuperficial.

Class Group—It is worth pointing out that the Classscript was identical to the Guided protocol except that theresults of the experiments were already in the text (ob-tained from the program). The data in Table I indicate thatalthough the difference between the Class and Guidedprotocols was not very large (7%), it was statistically sig-nificant (p � 0.05). This result reveals that there is somebenefit associated with the students’ involvement in get-ting the numeric values. On the other hand, students in theUnguided group, who were only told what variables theyshould change and what was the main goal of the experi-ence (to find the best set of conditions for an enzymeactivity assay), had even worse performances than thestudents in the Class group.

Are There Particular Aspects of the Subject Where thePerformance Differences Are More Evident?

Second, we analyzed each distinct section of the scriptsand found that the student groups performed differently inthese sections. The script started by asking the studentsfor a set of initial conditions where the enzyme assays givean activity that is linear in the time interval employed andwhere the activity is reasonably high, in other words thatgives them enough room for changes in enzyme activity.Afterward they were asked to analyze the effect of severalvariables that can be freely manipulated in the program,and finally they had to give a set of conditions where theenzyme activity should be maximal.

TABLE IStatistical comparison of the performance in scripts of the groups ofstudents in a self-teaching session of written exercises (Class) or in a

self-teaching session with the computer program

Those working with the program were given a brief outline offactors to investigate (Unguided), a list of experiences to perform withminimal details (Semiguided), or a detailed guide to the requiredexperiences (Guided). The mean represents the percentage of correctanswers. The statistical test applied was the Kruskal-Wallis analysisof variance in ranks and multiple comparisons by the Dunn’s test ofdifferences in ranks.

Group Groupmean

Mean differencea

Unguided Class Guided

%Semiguided 79.5 25.9 (551)b 13.3 (221)b 6.0 (77)Guided 73.5 20.0 (473)b 7.3 (144)b

Class 66.2 12.6 (329)b

Unguided 53.6a The number in parentheses indicates the rank difference from the

Dunn’s test comparison.b Statistically significant with p � 0.05.

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The complexity of these questions is low for the first task(Assay conditions, section 1), medium for the secondgroup of tasks (pH, section 2; T, section 3; [E] section 4;and [S], section 5), and high for the Optimal conditions(section 6). The results in Fig. 2 show that the studentsperformed well in finding an initial set of Assay conditions,but they had a slightly poorer performance in the secondgroup of tasks, particularly in regard to the effects ofenzyme and substrate concentration, probably becausethese last variables are less related to everyday life expe-rience in the school of chemistry than temperature or pH.Finally they had bad marks in the Optimal conditions. Inour point of view, the most interesting facts are (see Fig. 2)that the Class group performed better in the second groupof tasks, but both the Unguided and Class groups hadequally bad performances in the Optimal conditions. Onthe other hand, the Guided and Semiguided groups hadbetter marks in the first and second group of tasks andhigher marks in the Optimal conditions as compared withthe Unguided and Class groups.

This behavior is statistically corroborated in Table II asthe Unguided group has the lowest marks in all the scriptsections (Fig. 2); the only significant difference within thegroup performance in the different script sections is be-tween the Temperature and the Optimal conditions, thesections with the group’s largest and smallest percentageof correct answers. In the Class group the significant dif-ferences start with the enzyme concentration where thedecrease in correct answers occurs to a greater extentthan in previous sections (Table II and Fig. 2).

In the Guided group the percentage of correct answerscontinuously decreases, so there are significant differ-ences between each script section, although their marksare higher than the marks from the Unguided and Classgroups and similar to the marks from the Semiguidedgroup (Table II and Fig. 2). The Semiguided group has the

larger percentage of correct answers in all the script sec-tions (Fig. 2); nevertheless, they had difficulties with theeffect of substrate concentration and with the Optimalconditions, which proved to be difficult for all the groups,so in those sections the correct answers diminish in asignificant way with respect to the previous sections (TableII).

The data strongly indicate that a teaching guide is es-sential for the students’ understanding with regard to en-zyme kinetics. Some conventional guidance, in addition tothe use of the computer program, facilitates the compre-hension of the different subjects in the scripts and hencethe students’ capability of reaching conclusions. A com-pletely Unguided teaching strategy is not advisable at leastif the evaluation closely follows the students’ work in thesubject. In addition we found of interest the followingparticular observations on the performance of each group.

Guided Group—The Guided group performed statisti-cally significantly better than the Unguided group withrespect to the Assay conditions (section 1, Table III) andthe effect of pH and of the enzyme concentration (sections2 and 4, Table III). Teacher guidance during the elaborationof experiences in the computer program seems to be,once more, important for the comprehension and learningof how an enzyme is assayed, how the pH affects thereaction rate and the enzyme stability, and how the en-zyme concentration alters the reaction rate.

Semiguided Group—The Semiguided group was moresuccessful than the Guided group in sections 2 and 4(Table III). In fact, this is the group with the largest per-centage of correct answers in the questions of the sectionsregarding pH effects and enzyme concentration (sections2 and 4). In principle, the main difference between theGuided and the Semiguided group is that the students inthe Semiguided group had more freedom to work with thecomputer program. Therefore, it is reasonable to assumethat this was the one factor that contributes the most to abetter performance of the Semiguided group with respectto the other groups; however, it is important to mentionthat the students in the Semiguided group may have spentmore time than the Guided group in choosing the range ofvalues for the variables under study, thus, along with thefreedom of choice, they also were forced to collect a largerset of data regarding the enzyme responses to variousfactors.

Unguided Group—The Unguided group is the one thathad the largest number of differences with the Semiguidedgroup apart from the enzyme assay conditions (section 1,

FIG. 2. Percentage of correct answers in each section of thescripts from the four experimental groups. Sections are asfollows: 1, the Assay conditions; 2, the effect of pH; 3, the effectof Temperature (T); 4, the effect of Enzyme concentration ([E]); 5,the effect of Substrate concentration ([S]); 6, conclusions with aset of conditions for Optimal activity. The line segments representthe standard error. Hatched bars, Class group (n � 10); light graybars, Guided group (n � 32); gray bars, Unguided group (n � 20);black bars, Semiguided group (n � 26).

TABLE IISignificance of rank differences between each script section mark

within different teaching modalities

The letters of the teaching modalities where the respective com-parison gave a statistically significant difference (p � 0.05) are shown.G, Guided; S, Semiguided; U, Unguided; C, Class.

pH Temperature [Enzyme] [Substrate] Optimalconditions

Assay G G G G, S C, G, SpH C C, G, S C, G, STemperature G C, G, S, U[Enzyme] G, S G, S[Substrate] G

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Table III); as we have mentioned before, the pH is one ofthe parts with which the Unguided group seems to haveproblems as they had the worst results (section 2, Fig. 2).One of the probable causes for this is that students arereluctant to choose values of pH larger than 7; they thinkthat as enzymes are in the human body it is only naturalthat the enzymes work better at pH values around 7. Theysimply do not discuss their results even when they aredifferent from what they expect. The same happens withtemperature (section 3). For them, the optimum tempera-ture is around 30 °C, so they do not try temperatureshigher than that, or if their results show that the optimumtemperature is above 30 °C, they do not discuss why; infact, the Unguided group seems to be the only one thathad problems with the study of the effect of temperature.We have interpreted these findings as an indication thatthe lack of guidance makes them prone to be misguidedby their own preconceptions.

The most difficult subjects for all the students were theeffect of the enzyme and substrate concentrations uponthe reaction rate (sections 4 and 5): the Unguided groupwas the worst one in these matters; they made the samemistakes that we found in the other groups but morefrequently. The most common explanation for their resultsobtained in both subjects is that the curve obtained fromthe variation of the enzyme concentration is similar (mostof the time) to the one of the effect of the substrateconcentration; thus for them, the effect is the same: theenzyme is saturated in both curves by the substrate evenif in the first case the substrate was held constant. Since athigh enzyme concentrations the limitations in the assayquickly results in NADH exhaustion resulting in roughly thesame rate for every enzyme assay, they believed that theenzyme was saturated and the maximum velocity wasreached.

Regarding question 6 where the students have to con-clude with the Optimal conditions for measuring LDH ac-tivity, the Class and the Unguided groups were the oneswith major problems making correct conclusions; a largerfraction of the students in the Unguided group were unableto change their preconceptions of the best conditions foran enzyme, so they concluded that the optimum values forthe temperature and pH are 30 °C and pH 7. Anothercommon conclusion is that the optimum enzyme concen-tration is the one that corresponds to the start of theinflection of the curve or to the value obtained at theplateau. The plateau here is an unexpected result for most

students since textbooks indicate that the response toenzyme concentration should be linear. Most students failto interpret this plateau as a limitation of the assay andattribute the curve of the enzyme response to substrateconcentration. Many of them also tend to interpret satura-tion as a bad condition for the enzyme; thus, they choosethe inflection point of the curve as an optimum substrateconcentration. The same mistaken ideas are found in theother groups but in a smaller fraction of the students.

Class Group—Although the questions given to the Classgroup and the Guided group were identical, the perform-ance of the Class group was significantly inferior to that ofthe Guided group in section 1 (Assay conditions section,Table III). As the Class group did not obtain the resultsfrom the computer program, this suggests that the use ofthe computer program in finding a set of suitable condi-tions for the enzyme assay may help in the understandingof how some variables affect the reaction rate (Table III).

The Class group also has a lower percentage of correctanswers than the Semiguided group in sections 1 and 4(Assay conditions and enzyme concentrations, Table III).However, the effect of enzyme concentrations also provedto be difficult for the students from all the groups. Incomparison with the Unguided group, the performance ofthe Class group was significantly superior in sections 1and 2 (Table III). Section 2 is about the effect of the pHupon the reaction rate. As pH is a factor that all thestudents review in subjects other than biochemistry, famil-iarity with this factor could be a reason for the best per-formance of the groups in this section. The enzyme con-centration effect, however, is new to them.

Students’ Performance in Exams

Week 1 involved those students who worked with thecomputer program (Guided, Semiguided, and Unguidedgroups) and the Class group that only did enzyme kineticsexercises. Week 2 involved a traditional practical in thelaboratory of the four groups, and in the 3rd week they hadthe exam. The Control group just had a traditional practicalin the laboratory (Weeks 1 and 2) and the exam in Week 3.The Control group had no contact with the program andwas not given a script of any kind before the experimentalsession.

Table IV shows the comparison of the various groups’performance during the traditional written examination.The first observation is that all the groups have an averagemark above 60. Surprisingly, the Unguided, the Semigu-ided, and the Class groups obtained the best marks withno significant differences between them. The marks of theUnguided group were significantly higher than those of theGuided and Control groups, and this was statistically sig-nificant. This suggests that given time the average studentmanages to pass the exam; those who were given thefreedom to literally “play” with the program made the mostof their later work. In addition, the poor performance of theControl group must be interpreted with care because,although given the same time to prepare for the exam,these students were not required to answer any script andmay have dedicated considerably less time to the study ofthe subject. In any case, in our experience, teaching en-

TABLE IIIComparisons between the median values of the correct answers from

each section of the scripts

The table shows only the number of the sections where statisticallysignificant differences between groups, two at a time, were foundaccording to the Dunn’s test (p � 0.001). Section numbers are asfollows: 1, Assay conditions; 2, pH effects; 3, Temperature effects; 4,Enzyme concentration dependence; 5, Substrate concentration de-pendence; 6, a conclusion with a set of conditions for Optimal activity.

Group Class Unguided Semiguided Guided

Guided 1 1, 2, 4 2, 4Semiguided 1, 4 1, 2, 3 4, 5, 6 2, 4Class 1, 2

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zyme kinetics demands special requirements. One singlepractical session (4 h each) on enzyme responses to var-ious factors seems hardly enough for the average studentto get a basic understanding of this subject. Thus, workingwith the computer program or with exercises before thepractical session should have beneficial effects, whichmay help to explain the results in Table IV.

To identify specific learning problems within the differentteaching modalities studied, we decided to look for differ-ences in the students’ performance in each question of theexam. Those questions where the average marks of thestudents were at or below 65% were selected, and wesearched for any evident particular trend in the wronganswers given by the different teaching modality groups.We translated from Spanish the questions and theanswers.

• Question 1: How do you measure the rate of a chem-ical reaction?

a. Quantifying the amount of synthesized substrateb. Calculating the rate of absorbance/change over

timec. Measuring the changes in product concentration in

a given set of timed. Measuring the time that it takes for the substrate to

be exhaustede. Making a graph of ln[Product] versus time

• Percentage of correct choice: 57.1 (c)• Percentage of most frequent incorrect choice: 39.4 (b)

In this question, the most frequent mistake was theconception that the experience performed in the programand in the practical session can be applied to all enzymereactions. This mistake was, however, uncommon in theClass group. We believe that this is due to the participationof the tutor overseeing the group during the class sessionbecause the students could have made mistakes relatedto the calculation of enzyme activity while working on theexercises, and the tutor probably made the observation ofhow the exercises could be extrapolated to othersituations.

• Question 2: If the enzyme concentration is increasedin an enzyme assay what happens?

a. The reaction rate does not change.b. The product accumulation is faster.c. The reaction rate diminishes abruptly.d. All the above statements are incorrect.

• Percentage of correct choice: 31.6 (b)• Percentage of most frequent incorrect choice: 54.0 (d)

This was a difficult question, and we believe that thedifficulty lies in the way of stating the correct answerbecause it demands the combination of two apparentlyunrelated pieces of knowledge. First, it requires the defi-nition of reaction rate, and second, it requires the responseof the enzymatic reactions to increases in enzyme concen-tration. Here most students simply fail to identify a rightanswer, which may be in some way related to the problemfound in the first question.

• Question 8: If we add more reactant to a chemicalreaction mixture, whose reaction order is differentfrom zero, the most likely outcome will be:

a. The reactant consumption will be slower.b. The reaction rate will become smaller because of

substrate accumulation.c. The reaction activation energy diminishes.d. The reaction rate does not change.e. At the beginning of the reaction the reactant con-

sumption will be faster.

• Percentage of correct choice: 64.7 (e)• Percentage of most frequent incorrect choice: 23 (d)

In this question, the students had to use a piece ofknowledge that they could not acquire from other sub-jects’ experimental sessions because in those subjectsthey did not analyze the general effect of reactant varia-tions in chemical reactions. In the biochemistry experi-mental session they studied the specific effect of changesin substrate concentration in enzymatic reactions. It is notsurprising that many students answered that the additionof more reactant results in no change in the reaction ratejust as when the enzyme is saturated with substrate andthe addition of more substrate makes little difference.However, it is noteworthy that the general response of theUnguided group was different; many thought that the re-action was slowed down by the addition of more reactant.One of the interpretations of this misconception is that thestudents of the Unguided group rely upon the commonhuman experience of the reduced efficiency of individualswho are given an excessive amount of work, that is to saythey are replacing a scientific framework with a commonhuman experience. However, other explanations are fea-sible, and clarification of this point may well deserve fur-ther studies.

To elucidate whether the students that answered onequestion correctly were the same students that correctlyanswered other questions, we made an analysis by Pear-son Product Moment Correlation. In the Control groupquestions 1 and 8 showed significant positive correlation(p � 0.003); nevertheless, questions 1 and 8 had an aver-age of 58 and 38% of correct answers, respectively. Itlooks as if the students that had a correct idea of how therate of a chemical reaction is measured were more likely to

TABLE IVStatistical comparison of the mean percentage of correct answers tothe exam questions by the experimental groups (Guided, Semiguided,

Unguided, Class, and Control)

The Control group did not use the computer program nor did theyuse enzyme kinetics exercises in class. Since the normality assump-tion was fulfilled by these data (Kolmogorov-Smirnov with p � 0.05),the statistical test applied was the one-way analysis of variancefollowed by the Tukey test for all pairwise multiple comparison.

Group Groupmean

Mean differencea

Control Guided Class Semiguided

%Unguided 70.0 9.1b 7.2b 4.3 2.4Semiguided 67.6 6.7 4.8 1.9Class 65.7 4.8 2.9Guided 62.8 1.9Control 60.9

a From the Tukey test.b The difference was significant at 0.05 level of p.

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know how the concentration of the reactant affects thereaction rate. It is worth pointing out that the Control grouphad the lowest mean for question 2. The failure to give theright answer for question 2 could be the result of thestudents’ inability to identify the enzyme as the cause ofthe decrease in the absorbance as we observed in somelaboratory reports or exams of those students who did nothave contact with the program or with class exercises. Infact, when the students were asked to explain why thisdecrease is produced, they answered that it is caused bythe pH or the temperature, both of which are physicalphenomena common in their past experiences.

Another correlation exists between questions 2 and 8 inthe Unguided group (p � 0.03) as the means of questions2 and 8 are in the range of other groups; this could signifythat the students who understand how the reaction rate isaltered by the enzyme concentration also had a correctidea about how the increase in the concentration of thereactant affects this rate. As the students of the Unguidedgroup are forced to interpret the effects of various vari-ables over the reaction rate by themselves, it is possiblethat they paid more attention to this particular aspect thanthe other groups because under guidance they may nothave been so aware of their misconceptions.

As stated in the exam description, in the last section ofthe exam the interpretation of two graphs from the stu-dents is required. The first one obtained a low percentageof correct answers as described below.

• Question 17: the students were presented the graphin Fig. 3 together with the text included in the legendto Fig. 3.

• Percentage of satisfactory answers: 41.5• Percentage of unsatisfactory answers: 58.5

In this question the students were asked for knowledgeof various characteristics of Km: its definition, meaning,and understanding of how Km affects the shape of a sub-

strate saturation curve of an enzymatic reaction. Heremost students were only able to describe the shape of thecurves but could not identify that the different shapes wererelated to the enzyme affinity for the substrate. We believethat as simple as the Km concept seems, most studentslearn the mechanical procedures to derive Km values fromraw data, as deduced from the answers to other questions,but they hardly understand the meaning of the number thatthey have calculated and its relation to the enzyme behav-ior. In other words, most students focused on calculationprocedures and descriptive knowledge but put little em-phasis on analysis, abstraction, and judgement.

PART III: INTERPRETATION AND DISCUSSION OF RESULTS

Clearly, giving the students the opportunity to use theprogram brought benefits because the Control and Classgroups showed poorer performance in both the scriptsand the exam. The fact that the Class group showed asimilar behavior to the Unguided group strongly argues infavor of having a session of work with the computer sim-ulation because in the short term their results are equiva-lent to a traditional lecture, and in the long term, using theprogram benefits the learning process as shown by theUnguided group, which had a better performance than theClass group.

For those groups that had contact with the program, theanalysis of the scripts seems to support different conclu-sions than those supported by the analysis of the examperformances. The fully or partially guided schemes led tobetter performance in the script reports, but the full orpartial lack of guidance resulted in better exam marks.However, when considered in more depth, the Unguidedgroup was indeed guided a posteriori because their re-ports were marked and corrections to their mistakes wereannotated, and with these, their reports were given back tothem before they do the laboratory practical and study forthe exam. So a definite, closed answer to the value ofguidance cannot be drawn from our data. Nevertheless,our data do say that some level of guidance is required ifthe intention is to favor an ordered study of enzyme be-havior through enzsimil, especially if the students are re-quired to organize and interpret the data shortly afterward.

On the other hand, lack of guidance seems to makestudents prone to be misguided by their own preconcep-tions [11]. For instance, in the script where they have tofind the optimum conditions for the LDH activity assay, theClass and the Unguided groups were the ones with majorproblems making correct conclusions. Many concludedthat the optimal values for the temperature and pH are30 °C and pH 7. Also mistakes such as considering theoptimum enzyme concentration in an assay as the one thatcorresponds to the start of the inflection of the curve andthe interpretation of enzyme saturation as a condition ap-plicable to all curves showing a plateau or regarding sat-uration as a bad condition for the enzyme are more fre-quent in the Unguided group than in other groups.

We believe that if the goal is to encourage the student tomake the most of later work then it is better to give thestudents some freedom of choice during the computersimulation session, provided that the tutor reviews andcomments on their work later. In that sense, a Semiguided

FIG. 3. Graph presented to the students in question 17. Theheading stated: “Two enzymes catalyze the same reaction, andyou are required to identify their differential kinetic properties.Find those kinetic parameters that are similar and those that differbetween the two enzymes represented in the graph.” (Both thegraph titles and the statement are translations from the original inSpanish.)

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strategy partially covers both situations and may be rec-ommended if the tuition goals are not sharply defined. Inaddition, a more subtle adjustment of the level of guidanceis always possible and even advisable.

Perhaps the most significant conclusion of this study isthat the best teaching strategy depends on what is to beachieved. To that respect, training “report-makers” or “ex-am-answering technicians” is hardly ever the main goal ofeducation; and thus a study on how the use of computersimulations and similar tools affects meaningful, long termlearning is required. Unfortunately such study requirestracking the development of the students in their profes-sional life and is beyond our current capabilities. In addi-tion, differences in teaching goals may help to explain whysome studies found that traditional teaching is as effectiveas more active approaches like problem-based learning[12], while other studies found benefits in the latterapproach [13].

In addition to the aspects mentioned in the previousparagraph, we think that the tutor must be aware that hisrole should be in accordance with the different teachingmodalities. In the full guidance modality, scripts and ex-ams from the students will all be similar, while the partialguidance, or the lack of it, will require the tutor to make useof wider criteria and a deeper knowledge of the subject inmarking the students’ work because the students will usetheir own individual strategies to solve problems. There-fore, their results will be heterogeneous but not necessarywrong.

Finally, there is extremely valuable information in thestudents’ scripts, reports, and exams regarding concepts

of enzyme kinetics that are particularly difficult to teach.We are currently working on their analysis, which will bethe subject of a future article.

REFERENCES

[1] F. Ranaldi, P. Vanni, E. Giachetti (1999) What students must knowabout the determination of enzyme kinetic parameters, Biochem.Educ. 27, 87–91.

[2] B. Inhelder, J. Piaget (1955) De la Logique de l’Enfant a la Logique del’Adolescent, Presses Universitaires de France, Parıs.

[3] L. Vigotsky (1979) El Desarrollo de los Procesos Psicologicos Supe-riores, Grijalbo, Barcelona.

[4] J. Castorina, J. Castorina, in: E. Ferreiro, M. Kohl de Oliveira, D.Lerner, Eds. (1996) El Debate Piaget-Vigotsky, la Busqueda de unCriterio para su Evaluacion, Paidos, Mexico, pp. 9–44.

[5] H. I. Segel, in: H. I. Segel, Ed. (1975) Enzyme Kinetics, John Wiley &Sons, Inc., New York, pp. 561–590.

[6] H. I. Segel, in: H. I. Segel, Ed. (1975) Enzyme Kinetics, John Wiley &Sons, Inc., New York, pp. 819–826.

[7] M. Dixon, E. C. Webb (1964) Enzymes, 2nd ed., Longman, Ltd.,London, pp. 54–166.

[8] G. Perez-Serrano (1991) Elaboracion de Proyectos Sociales. CasosPracticos, Narcea, S. A. de Ediciones, Madrid, pp. 105–162.

[9] R. Bisquerra (1989) Metodos de Investigacion Educativa. Guıa Prac-tica, CEAC, Barcelona.

[10] P. Baeza, A. Cabrera, M. T. Castaneda, J. Garrido, A. Ortega (1999)Aprendizaje colaborativo asistido por computador: la esencia inter-activa, Contexto Educativo, Revista Digital de Educacion y NuevasTecnologıas 1, nota-8: contexto-educativo.com/ar/1999/12/nota-8.htm.

[11] J. I. Pozo, M. A. Gomez-Crespo (1998) Aprender y Ensenar Ciencia,Ediciones Morata S. L., Madrid, pp. 149–204.

[12] D. T. A. Vernon, R. L. Blake (1993) Does problem-based learningwork? A meta-analysis of evaluative research. Acad. Med. 68,550–563.

[13] C. L. Harris, G. Guner, J. Arbogast, L. Salati, J. M. Shumway, J.Connors, D. Beattie (1997) Integrated problem based learning for firstyear medical students: does it teach biochemical principles? Bio-chem. Educ. 25, 146–150.

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