Enzyme Kinetics Experiment with the Multienzyme Complex

Laboratory Experiment pubs.acs.org/jchemeduc

Enzyme Kinetics Experiment with the Multienzyme Complex Viscozyme L and Two Substrates for the Accurate Determination of Michaelian Parameters Nelson Pérez Guerra* Department of Analytical and Food Chemistry, University of Vigo, As Lagoas, 32004 Ourense, Spain S Supporting Information *

ABSTRACT: A laboratory experiment in which students study the kinetics of the Viscozyme-L-catalyzed hydrolysis of cellulose and starch comparatively was designed for an upperdivision biochemistry laboratory. The main objective of this experiment was to provide an opportunity to perform enhanced enzyme kinetics data analysis using appropriate informatics and statistical tools. By fitting the Michaelis− Menten, Lineweaver−Burk, Eadie−Hofstee, and Hanes equations to the experimental data, students explore the strengths and weaknesses of each plot and determine whether the two Viscozyme-L-catalyzed reactions obey Michaelis− Menten kinetics. Comparing the values of the Michaelian constants (vmax and KM) and the efficiencies obtained for each enzyme-catalyzed reaction allows advanced undergraduates to determine if the differences between these parameters are statistically significant. KEYWORDS: Second-Year Undergraduate, Biochemistry, Interdisciplinary/Multidisciplinary, Collaborative/Cooperative Learning, Inquiry-Based/Discovery Learning, Enzymes, Kinetics, Laboratory Computing/Interfacing

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yields of cellulose.17 In addition, the use of the Viscozyme L complex in kinetics experiments has some additional advantages over the use of individual enzymes. First, because the kinetic behavior of this enzyme complex had not been described before, students can determine whether the Viscozyme-L-catalyzed hydrolysis of cellulose and starch follows Michaelis−Menten kinetics. Second, this enzyme complex is less costly than the single enzymes, thereby decreasing the overall cost of the kinetics experiment. Third, the use of Viscozyme L allows students to study different enzyme-catalyzed reactions with specific substrates (e.g., xylans or arabinans) or nonspecific substrates (e.g., starch). The results presented in this description of a biochemistry laboratory experiment are the mean values obtained from triplicate experiments performed by different second-year Food Science and Technology students at the University of Vigo (Spain) over a period of five years. In this experiment, students gain experience with the collection, analysis and statistical treatment of enzyme kinetics data using a reliable and well-defined procedure involving informatics and statistical tools. This experiment’s design develops students’ reasoning skills through the application of previously studied enzyme kinetics concepts. Using this

INTRODUCTION Understanding the Michaelis−Menten (MM) kinetics requires a strong background on the mechanism of single-substrate enzyme kinetics from which the MM equation arises. Knowledge of this mechanism allows the students to understand the meaning of the maximum velocity (vmax), the Michaelis constant (KM), and the catalytic efficiency of an enzyme.1 This topic is an important one in the study of biochemistry, which is taught for the Degree in Food Science and Technology at the University of Vigo. Although many undergraduate enzyme kinetics experiments for biochemistry laboratories have been published,2−15 in most, the rate data are only analyzed using one,2−7,13 two,8,9,11,12,14 or three10 types of plots and a single enzyme.2,3,6,9,10,13,15 This procedure does not provide advanced undergraduate students an opportunity to explore the strengths and weaknesses of the Michaelis−Menten, Lineweaver−Burk, Eadie−Hofstee, and Hanes plots, which are commonly used to describe enzyme kinetics.16 Herein, an enzyme experiment for determining the maximum velocity (vmax) and Michaelis constant (KM) of the hydrolysis of cellulose and starch is developed. In this experiment, both hydrolyses are catalyzed by the multienzyme complex Viscozyme L, which exhibits β-glucanase, arabinase, cellulase, hemicellulase, and xylanase activities.17 The use of this enzyme complex is supported by the fact that enzymatic cocktails containing cellulases and hemicellulases improve hydrolysis © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: May 13, 2016 Revised: March 10, 2017

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DOI: 10.1021/acs.jchemed.6b00351 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. Reaction progress curves for different enzyme concentrations at a fixed substrate concentration (A) and for different substrate concentrations at a fixed enzyme concentration (B) for both substrates. [RS]: concentration of reducing sugars.

The laboratory practice (15 h) was performed in 1 week divided into five 3 h laboratory sections: (session 1) determining the initial velocity (v0) conditions at six different enzyme concentrations ([E] = 0.06, 0.56, 1.70, 3.40, 4.53, and 5.66 g L−1) and one substrate concentration ([S] = 1 g L−1) and selecting the most appropriate [E] for further experiments; (session 2) determining the initial velocity (v0) conditions at three substrate concentrations (30, 40, and 50 g L−1) using the previously selected [E]; (session 3) determining the velocity dependence on substrate concentration and organizing the data according to the Michaelis−Menten, Lineweaver−Burk, Eadie− Hofstee, and Hanes equations; (session 4) fitting the four equations to the experimental kinetic data using SigmaPlot software to estimate vmax and KM and selecting the plot that achieves the best fit (group work); and (session 5) calculating the efficiencies of enzymatic catalysis (Ef = vmax/[E]/KM) for each substrate; comparing the values of vmax, KM, and Ef for each substrate using the SPSS Statistics software; and discussing (group work) the results. To promote pair and group interactions and cooperation, at the end of the fifth session, the students discussed and explained the differences observed by considering the composition of the multienzyme complex Viscozyme L. Instructions for each laboratory session are provided in the Supporting Information. During the following week, after the laboratory practice, the students checked their results, analysis, and conclusions with the instructor before creating the final version of their report.

procedure, students realize that accurate data analysis is as important as good data collection. The Supporting Information provides guidance for obtaining the experimental data; fitting the Michaelis−Menten, Lineweaver−Burk, Eadie-Hofstee, and Hanes equations to the experimental data using SigmaPlot software; and comparing the values of vmax and KM and the efficiencies (Ef’s) for each substrate using SPSS Statistics software.

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LEARNING OBJECTIVES

The main objective of this experiment was to provide students with an opportunity for enhanced enzyme kinetics data analysis using appropriate informatics and statistical tools. Via this approach, students learn and gain experience using accurate modeling procedures and performing statistical comparisons with an Excel spreadsheet and SigmaPlot and SPSS Statistics algorithms. By fitting the Michaelis−Menten, Lineweaver− Burk, Eadie−Hofstee, and Hanes equations to the data, the students comparatively analyze the strengths and weaknesses of the four plots and statistically compare the values of the kinetics constants (KM and vmax) and Ef for two substrates (cellulose and starch).

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EXPERIMENTAL PROCEDURE The laboratory practice has been performed in the first semester during five years since 2010, by 16 students per semester. The approximate cost of the experiment is 28 € per student per year. Approximately half of the students (group 1) are randomly assigned to perform the enzyme kinetics assay with cellulose, while the other half (group 2) perform the assay with starch. In each group, students working in pairs conducted triplicate experiments to obtain data for comparisons.

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HAZARDS The dinitrosalicylic acid (DNS) reagent employed in the spectrophotometric determination of reducing sugars contains 3,5-DNS, potassium sodium tartrate, and sodium hydroxide. According to the material safety data sheet (MSDS), 3,5-DNS B




(CAS number 609-99-4), potassium sodium tartrate (CAS number 6381-59-5), Viscozyme L (CAS number 62213-14-3), and citric acid (CAS number 77-92-9) may cause irritation of the skin, eyes, digestive tract, and respiratory tract. Sodium hydroxide (CAS number 1310-73-2) is corrosive by ingestion and a severe irritant to the upper respiratory tract, eyes, and skin. Disodium hydrogen phosphate heptahydrate (CAS number 7782-85-6) is slightly hazardous and may be an irritant in the case of skin or eye contact. The other chemicals used (glucose, cellulose, and potato starch) have minimal hazards. The experiment should be conducted using gloves, eyewear, and a lab coat.

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RESULTS AND DISCUSSION In this biochemistry laboratory, students collected and comparatively analyzed the kinetics data on the Viscozyme-Lcatalyzed hydrolysis of cellulose and starch and performed an accurate statistical analysis to support their results. This approach allowed the students to determine whether this enzyme complex obeys Michaelis−Menten kinetics and to calculate the kinetic parameters (vmax and KM) and the efficiency of enzymatic catalysis (Ef) for both substrates. This laboratory practice has been conducted for five years by 80 students working in pairs. Half of the students performed the enzyme kinetics assay with cellulose (group 1), and the other half performed the assay with starch (group 2). Because the Michaelis−Menten model requires steady-state initial velocity (v0) conditions, on the first day of the experiment, the students determined the linear dependence of v0 on the enzyme concentration. To this end, students working in pairs conducted triplicate experiments with six different enzyme levels and a substrate concentration of 1 g L−1. Then, they plotted the experimental product formation data against time (Figure 1A) to determine the v0 values using the Excel Trendline algorithm. The students found that the curves for 0.06, 0.56, and 1.70 g enzyme L−1 exhibited linear kinetics for periods of 6 min for cellulose and 5 min for starch (Figure 1A). On the second day, each pair of students corroborated these results in triplicate assays with three other substrate concentrations (30, 40, and 50 g L−1) and an enzyme concentration of 1.70 g L−1 (Figure 1B). On the third day, students obtained, in triplicate, the v0 dependence on the substrate concentration ([S] = 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, and 50 g L−1) and organized the data to generate the Michaelis−Menten, Lineweaver−Burk, Eadie− Hofstee, and Hanes plots16 (symbols in Figure 2) using an Excel spreadsheet. As a key novelty of this experiment, students found for the first time that the two Viscozyme-L-catalyzed reactions follow Michaelis−Menten kinetics (left upper part of Figure 2). On the fourth day, each pair of students fitted the four plots to their experimental data set to obtain triplicate significant (P < 0.05) estimates (±standard errors) of vmax and KM and the R2 values using the SigmaPlot Regression Wizard. Figure 2 and Table 1 show a typical student pair result in one of the three experiments. On the basis of the detailed discussion of the results obtained in their triplicate experiments, the majority of students concluded that the Hanes and Michaelis−Menten plots, in this order, achieved the best fits (the highest R2 values) for both substrates, probably because the errors in determining v0 at low substrate concentrations are greatly magnified in the Lineweaver−Burk and Eadie−Hofstee plots.10,14,16

Figure 2. Predictions of the equations () fitted to the first experimental data (symbols) of the Viscozyme-L-catalyzed hydrolysis of cellulose (●) and starch (○) obtained by student pairs. The estimated values of vmax, KM, and R2 for this data are presented in Table 1.

On the fifth day, the three values of vmax, KM, and Ef obtained from the Hanes plot for each substrate by each student pair were organized in a table. Then, each student compared the values of vmax, KM, and Ef obtained for cellulose with the corresponding values obtained for starch, using the paired samples t-test in SPSS Statistics software. With this procedure, they determined if the differences between these parameters were statistically significant at the 0.05 level. From the corresponding comparisons, the students concluded that (i) cellulose was more rapidly hydrolyzed than starch, (ii) the cellulose concentration at which v0 is one-half vmax was significantly lower than the starch concentration (KM‑starch > KM‑cellulose), and (iii) Viscozyme L was significantly more efficient at catalyzing the hydrolysis of cellulose than starch. One out of four students related these results to the composition of Viscozyme L, which contains cellulases but not amylases,17 and concluded that starch was a nonspecific substrate for Viscozyme L. Table 2 presents averaged results from 20 student pairs over a five-year period. The high reproducibility of student kinetic data (with relative low standard deviation values) indicated the ability of students to measure the enzymatic activities of Viscozyme L for both substrates and the high stability of the enzyme complex. Comparing the results obtained daily by the different groups of students reflected their ability to collect and analyze enzyme kinetics data using informatics and statistical tools. Pre- and postlaboratory skills assessment tests, daily comparison, and discussion of the results obtained in the laboratory practice were used to assess the achievement of the learning objectives proposed in this laboratory procedure and the skills acquired by the students. By evaluating these activities (on a scale from 0 to 10), we ensured that the students have gained experience in the collection, analysis, and statistical treatment of enzyme kinetics data. A week after the end of the activity, the students presented a written report, including tables and figures conveying their C




Table 1. Typical Student Pair Result for One Data Set Values ± Standard Errors, N = 1 vmax (mmol L−1 min−1)

Plot

a

Michaelis−Menten Lineweaver−Burk Eadie−Hofstee Hanesa

41.07 50.43 42.71 40.51

± ± ± ±

1.13 4.71 2.26 0.75

Michaelis−Menten Lineweaver−Burk Eadie−Hofstee Hanes

24.81 26.27 24.28 24.81

± ± ± ±

0.47 3.07 0.62 0.37

KM/(g L−1) Cellulose Substrate 4.01 ± 7.36 ± 4.99 ± 4.08 ± Starch Substrate 5.38 ± 5.32 ± 4.94 ± 5.35 ±

R2

Efa/(L g−1 min−1)

0.47 0.87 0.64 0.54

0.9835 0.9833 0.8593 0.9965

1.05

0.41 0.87 0.30 0.45

0.9937 0.9537 0.9649 0.9978

0.49

The efficiencies were calculated for the plot providing the best fit.

Table 2. Comparative Five-Year Kinetic Results Mean Values ± Standard Deviations, N = 20 Plot Michaelis−Menten Lineweaver−Burk Eadie−Hofstee Hanesb

vmax‑cellulose/ (mmol L−1 min−1) 42.47 47.71 42.88 42.27

± ± ± ±

1.13 5.65 1.36 1.30

KM‑cellulose/ (g L−1) 4.30 6.04 4.58 4.34

± ± ± ±

0.26 2.00 0.51 0.36

Ef-cellulose/ (L g−1 min−1)a

vmax-starch/ (mmol L−1 min−1) 26.39 24.28 25.60 26.03

1.04 ± 0.08

± ± ± ±

1.00 1.38 1.03 0.89

KM‑starch / (g L−1)

Ef‑starch/ (L g−1 min−1)a

± ± ± ±

0.50 ± 0.04

5.90 4.83 5.32 5.61

0.57 0.72 0.59 0.55

The efficiencies were calculated for the plot providing the best fit. bThe mean values of vmax, KM, and Ef for cellulose were, respectively, different (P < 0.05) from those for starch. a

results and a discussion of their results supported by the corresponding statistical analysis. The written report was used to assess the students’ ability to write a well-structured lab report to communicate the results and conclusions of their experiments using the appropriate technical language and enzyme kinetics concepts. A potential limitation of the methodology proposed in this work is that the statistical programs used might not be available in many colleges that could otherwise be potential users of this procedure. To address this limitation, a procedure using only an Excel spreadsheet is described in the Supporting Information.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Nelson Pérez Guerra: 0000-0002-8202-932X

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Notes

The author declares no competing financial interest.

CONCLUSION This laboratory experiment is a suitable activity that allows students to apply and corroborate learned theoretical concepts in the context of enzyme kinetics and analyze experimental results using appropriate modeling and statistical procedures to support their conclusions. Via this procedure, students learn to analyze the statistical significance of the Michaelian parameters and the models fitted for cellulose (group 1) and starch (group 2), to select the most appropriate equation to describe their kinetic results, and finally, working in groups, to determine if there are statistically significant differences between the mean values of vmax, KM, and Ef obtained for each substrate (all students). This laboratory experiment was designed to promote pair and group interactions and cooperation, favoring working in teams and developing critical thinking and reasoning skills.

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Instructor notes containing detailed information related to the procedure and data processing (PDF, DOCX)

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REFERENCES

(1) Sorensen, R.; Novak, N. The Use of Michaelis-Menten Kinetics in Cell Biology and Physiology Teaching Laboratories. Biochem. Educ. 1996, 24 (1), 26−28. (2) Moe, O.; Cornelius, R. Enzyme Kinetics. J. Chem. Educ. 1988, 65 (2), 137−141. (3) Rowe, H. A.; Brown, M. Practical Enzyme Kinetics: A Biochemical Laboratory Experiment. J. Chem. Educ. 1988, 65 (6), 548−549. (4) Bruist, M. F. Use of a Spreadsheet to Simulate Enzyme Kinetics. J. Chem. Educ. 1998, 75 (3), 372−375. (5) Hamilton, T. M.; Dobie-Galuska, A. A.; Wietstock, S. M. The oPhenylenediamine-Horseradish Peroxidase System: Enzyme Kinetics in the General Chemistry Laboratory. J. Chem. Educ. 1999, 76 (5), 642−644. (6) Bendinskas, K.; DiJiacomo, C.; Krill, A.; Vitz, E. Kinetics of Alcohol Dehydrogenase-Catalyzed Oxidation of Ethanol Followed by Visible Spectroscopy. J. Chem. Educ. 2005, 82 (7), 1068−1070. (7) Bozlee, B. J. Reformulation of the Michaelis−Menten Equation: How Enzyme-Catalyzed Reactions Depend on Gibbs Energy. J. Chem. Educ. 2007, 84 (1), 106−107. (8) Halkides, C. J.; Herman, R. Introducing Michaelis−Menten Kinetics through Simulation. J. Chem. Educ. 2007, 84 (3), 434−437.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00351. Student notes containing detailed information related to the procedure and data processing (PDF, DOCX) D




(9) Olsen, R. J.; Olsen, J. A.; Giles, G. A. An Enzyme Kinetics Experiment for the Undergraduate Organic Chemistry Laboratory. J. Chem. Educ. 2010, 87 (9), 956−957. (10) Barton, J. S. A Comprehensive Enzyme Kinetic Exercise for Biochemistry. J. Chem. Educ. 2011, 88 (9), 1336−1339. (11) Goličnik, M. Exact and Approximate Solutions for the DecadesOld Michaelis-Menten Equation: Progress-Curve Analysis through Integrated Rate Equations. Biochem. Mol. Biol. Educ. 2011, 39 (2), 117−125. (12) Antuch, M.; Ramos, Y.; Á lvarez, R. Simulated Analysis of Linear Reversible Enzyme Inhibition with SCILAB. J. Chem. Educ. 2014, 91 (8), 1203−1206. (13) Johnson, R. J.; Hoops, G. C.; Savas, C. J.; Kartje, Z.; Lavis, L. D. A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates. J. Chem. Educ. 2015, 92 (2), 385− 388. (14) Silverstein, T. P. The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed MiniProjects. J. Chem. Educ. 2016, 93 (5), 963−970. (15) Lin, Y.; Lloyd, P. M. An Enzyme Kinetics Experiment Using Laccase for General Chemistry. J. Chem. Educ. 2006, 83 (4), 638−640. (16) Rogers, A.; Gibon, Y. Enzyme Kinetics: Theory and Practice. In Plant Metabolic Networks; Schwender, J., Ed.; Springer Verlag Science +Business Media: New York, 2009; pp 71−102. (17) Rosset, M.; Prudencio, S. H.; Beléia, A. P. Viscozyme Action on Soy Slurry Affects Carbohydrates and Antioxidant Properties of Silken Tofu. Food Sci. Technol. Int. 2012, 18 (6), 531−538.

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Enzyme Kinetics Experiment with the Multienzyme Complex

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