Assignments for majors

The following assignment is designed to help you become familiar with the operation of EnzymeLab. For your ease in completing each assignment, the background text relevant to the experiment that you will perform is italicized, instructions for each assignment are indicated by plain text, and questions or activities that you will be asked to provide answers for are indicated by bold text.

 

Assignment 1: Getting to Know EnzymeLab: Setting Up an Experiment

The first screen that appears in EnzymeLab shows you a biochemistry lab containing all the reagents and equipment you will need to perform your experiments.

Click on each item in the lab to learn more about its purpose. Once you are familiar with the lab, click on the Experiment button to begin the first assignment. This assignment is designed to help you become familiar with the operation of EnzymeLab.

When you set up an experiment in EnzymeLab, you will add a buffered solution, sucrose as substrate, invertase, and, in some reactions, inhibitors to a test tube to measure the rate of invertase activity. You will have the choice of performing each reaction at different temperatures and under different buffer conditions in which you can examine the effect of these parameters on invertase activity. A visible light spectrophotometer will measure product formation by measuring the absorbance of glucose (released as sucrose is cleaved by invertase) at approximately 450 nm. Data are recorded and plotted as a function of product concentration [P] in micromoles (m m) versus time (minutes). Raw data you collect can then be analyzed by several different types of plots that are commonly used for analyzing kinetic data for enzyme-catalyzed reactions.

1. Effect of Temperature on Invertase Activity

Changes in temperature can dramatically influence the activity of most enzymes by affecting enzyme structure. This exercise is designed to help you learn how to set up an experiment in EnzymeLab and understand the effect of temperature on enzyme activity. You will also analyze data from this experiment to determine the ideal temperature optimum for invertase activity.

To begin any experiment, you first need to set the temperature of your water bath, then add buffer and substrate to the reaction tube. As is the case with real experiments in a biochemistry lab, enzyme should always be added to the tube last to prevent the reaction from starting before all necessary components have been added to your test tube.

Develop a hypothesis to predict the effect of an increase in temperature on invertase activity, then test your hypothesis as follows.

Notice that the default temperature for the water bath is 40° C. To change the temperature, you can either enter a temperature value in the text box or use the arrows. Change temperature to 30° C; this is the lowest temperature at which you can carry out an experiment. Notice that the default buffer pH is 7.0. Do not change the default pH for any measurements in this experiment.

Notice that the default value for substrate concentration [S] is indicated by the slider bar labeled [S] and the value of 25 mM appears in the text box to the far right of the [S] slider. Substrate concentration for sucrose is reported in millimolar (mM) units. You can change [S] by either moving the slider bar or typing a value in the [S] text box. For the first reaction in this experiment we will begin by carrying out a reaction with 90 mM sucrose. Change [S] to 90 mM. Do not select any inhibitors for this experiment.

Note: The [S] value that you use for any experiment will be reported in the table under the Plot Data view. However, two students running an experiment with the same [S] may see slightly different results because EnzymeLab is programmed to simulate differences that represent slight experimental variations in concentration measurements such as you would encounter if setting up this experiment in a wet lab.

To add enzyme to your reaction tube, click the Add Enzyme & Go! button. This will also activate the spectrophotometer to measure product concentration.

a. Determining Starting Velocity (VO)

After each time you add enzyme, a plot of product concentration versus time will appear with enzyme kinetic data plotted as data points in solid black circles.

What did you observe for the plot of product concentration versus time? Is this what you expected? Explain your answer.

To begin your analysis of this experiment, you first need to determine the starting velocity of the reaction (VO). This is easily accomplished because VO (the initial rate of the reaction showing first-order kinetics where the rate of the reaction depends on [S]) is represented by the slope of the linear portion of the curve in this type of plot. The plateau (asymptote) of the plot represents zero-order kinetics where the rate of the reaction does not depend on [S]. Note: For many of the reactions you will run, the reaction will not reach zero-order kinetics.

To determine the slope of the line, a red line will appear on the plot. You can then click on this red line and move it to find the best fit for the slope of the plotted points for invertase activity. Try to align the red line so that you have an equal number of data points bisected by the line and an equal number of points above and below the line. This is generally a good approach for finding the best-fit slope. The program will not tell you when you have found the best fit, so use your best judgment! Follow the directions below to determine the slope of the line for this first measurement.

Click within the plot and move the red line until you have found the slope of the plotted data points. Before we can analyze this information further, you must record your data by clicking the Record Data button. Note: The Record Data feature is not active until you have properly determined the slope of the plotted line.

After you have recorded data for a measurement, you must click the Clear Experiment button before you can take another measurement. Note: If you forget to record data and attempt to clear the experiment, a warning box will appear asking if you want to clear data without recording it.

Keeping buffer pH constant and [S] at 90 mM, create another experiment but this time increase temperature to 35° C. Run the experiment, determine the slope of the line, then record this measurement. Repeat this process to set up experiments increasing temperature in 5-degree increments (40° C, 45° C, 50° etc.) until you reach the maximum temperature of 85° C. Find the slope of the line for each experiment and record these data.

Click on the Plot Data button to prepare a plot of your data for this experiment.

b. Plotting Invertase Kinetic Data

In the first window that appears in the Plot Data view you can title the plots for the experiment you are working on, select a plot to create, change the symbols for this plot, and view raw data in tabular form showing each measurement for a particular experiment–[S], the presence or absence of an inhibitor, inhibitor concentration [I], temperature, pH, and VO.

From the Plot Data view we can now use EnzymeLab to carry out a number of important calculations and present these data as plots that are traditionally used for studying enzyme kinetics. Each of the plots available in EnzymeLab is briefly described below. It is very important that you are comfortable with the abbreviations referred to below and the purpose of plotting data with these different types of plots. These principles form the basis for the majority of experiments that you will be carrying out with EnzymeLab.

VO vs. [S]: convenient way to express the relationship between reaction velocity (Vo) and substrate concentration [S]. Used to determine Vmax, the maximum velocity of an enzyme, represented by the asymptote (plateau) of the line. We can also use this plot to measure the Michaelis constant (KM). To find the Michaelis constant, you need to locate -Vmax then find where this value would intersect the x-axis. The [S] represented by this intersect point is the KM. This type of plot is a simple way to represent and determine Vmax and KM; however, it is not the most accurate way to determine these values because we are extrapolating from the plot.

VO vs. Temperature: analyzes the effect of temperature on reaction velocity.

VO vs. pH: analyzes the effect of pH on reaction velocity.

Lineweaver—Burk: also called a double-reciprocal plot. Produces a linear plot for the inverse of velocity (1/V) versus the inverse of substrate concentration [1/S]. Used to determine two important characteristics of enzymes that follow Michaelis—Menton kinetics: KM (substrate concentration at which a reaction has reached half of its maximum velocity), and kcat (turnover number = number of substrate molecules undergoing a reaction per enzyme molecule per second). The Lineweaver—Burk plot is a more accurate plot for determining Vmax and KM than a plot of VO versus [S] because Lineweaver—Burk plots are based on algebraically arranged equations of the Michaelis—Menten equation.

Eadie—Hofstee: a plot of V versus V/[S]. Another way to determine parameters of Michaelis—Menton kinetics. The y-intercept indicates Vmax, the x-intercept determines Vmax/KM, while the slope of the line determines -KM.

Note: Any of the plots that you generate can be saved to disk or printed by clicking on the Export Graph button, which appears to the left of each plot. Clicking on this button will open a separate window with your plot. From this window you can then save your plot to your hard drive or a disk, and you can print your plot by using the print feature of your browser software.

Plotting VO vs. Temperature: Click in the Title box and type in the title "Experiment 1 — Temperature Optimum." VO vs. [S] should appear as the default plot in the Plot Type box. Click on the popup menu and select VO vs. Temperature. For any plot, you must first select the data that you want to plot. You can click on an individual row to select it and the row will be highlighted, or you can select several rows by holding down the Shift key and clicking on each row. Shift-click on each row of measurements that you recorded for this temperature experiment, then click the Plot Selected Data button to produce a plot of VO versus temperature.

Click anywhere on the VO vs. temperature plot and drag the vertical gray dashed line to locate the highest value for enzyme activity (Vmax). When you have correctly located this value, the gray line will become black and it will freeze in place. This value, indicated in the best-temperature text box, represents the optimal temperature for invertase activity under these conditions of pH and [S].

What is the optimal temperature for invertase activity? Is this what you expected? Would invertase isolated from any two organisms (for example, yeast invertase vs. invertase from the small intestine of humans) show the same temperature optimum? Why or why not? Explain your answers.

Explain why temperatures lower or higher than the optimum cause decreases in invertase activity. What is happening to the enzyme to produce these decreases in activity?

Carefully examine the curve for VO vs. temperature. Is the slope of the line on both sides of the curve the same or different? If the slope of the line to the left of maximum velocity is different from the slope of the line to the right of maximum velocity, explain why this is. What is responsible for these differences in enzyme kinetics?

If you were to carry out these temperature experiments at a higher or lower [S], what effect would [S] have on the temperature optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results.

If you were to carry out these temperature experiments at a higher or lower pH value, what effect would this have on the temperature optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results.

 

Assignment 2: pH Optimum for Invertase

Another factor that strongly influences enzyme activity in living cells is the pH of the environment in which the enzyme is designed to function. For example, in humans, a cytoplasmic protein in a skin cell is surrounded by a different fluid environment at a different pH than a membrane-bound enzyme like invertase with an active site that projects out into the lumen of the small intestine. The following exercise is designed to help you understand the effect of pH on enzyme activity by studying invertase activity over a range of different pH values from acidic to basic conditions.

Formulate a hypothesis to predict the effect of pH on invertase activity.

Set up an experiment at the optimal temperature that you determined in assignment 1, with a substrate concentration of 90 mM. Beginning at the lowest pH value, 3.0, measure invertase activity, find the slope of the line, record data, and repeat this process for other buffer solutions with different pH values. Run experiments for at least two different buffers for each whole number change in pH units until you reach the maximum pH value of 10.0 (e.g., 3.0, 3.4, 4.0, 4.4, 5.0, 5.4).

Create a plot of VO vs. pH. Click on this plot and drag the gray dashed pH line until you find Vmax. When this line is correctly aligned, you will have found the pH optimum for invertase under these reaction conditions. The optimal pH value will appear in the best pH text box.

What is the optimal pH for invertase activity? Do the results of this experiment support or refute your hypothesis? Why or why not? Explain your answers? Why and how do pH changes affect invertase activity?

If you were to carry out these pH experiments at a higher or lower temperature, what effect would this have on the pH optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results.

 

Assignment 3: The Effect of Substrate Concentration on Invertase Activity

This exercise is designed to help you interpret Michaelis—Menten parameters for invertase by learning how to create and interpret plots of enzyme kinetics.In this experiment, you will add different concentrations of sucrose to invertase and then study the effects of sucrose concentration on invertase activity.

Set up an experiment at 50° C with a buffer pH of 4.0 and a [S] of 0.0 mM. Add enzyme. Find the slope of the line and record data for this first measurement. Continue to take measurements by carrying out separate experiments with increasing concentrations of [S]. Take a measurement at 2.5 mM, 5 mM, and 10 mM, then continue in 10 mM increments (e.g., 20 mM, 30 mM, 40 mM, 50 mM, 60 mM) until you have reached a [S] of 400 mM. Find the slope and record data after each measurement. Once you have finished these measurements, click the Plot Data tab.

Follow the directions below to use these data to create some of the plots described in assignment 1. Each plot is selected from the popup menu for Plot Type. Refer to these directions if necessary when creating plots for other experiments that you are working on.

Plotting VO vs. [S]: Click in the Title box and type in the title "Experiment 1 —

Substrate Concentration." VO vs. [S] should appear as the default plot in the Plot Type box. Shift-click on each row of measurements from 0 to 100, then click the Plot Selected Data button to produce a plot of VO versus [S].

Before you can determine Vmax and KM from this plot, you must first find the best-fit line for these data points. Click on the black KM arrow at the x-axis. Slide this arrow until you have found the best-fit line for these points. Adjust this line as needed by clicking and sliding the black Vmax arrow at the y-axis. A numerical value for Vmax and KM will appear in the Vmax and KM text boxes.

Setting Up a Lineweaver—Burk Plot: Return to the Data view and plot your data as a Lineweaver—Burk plot. Find the best-fit line for these data points by clicking and sliding the arrows at the x-axis and y-axis. The arrow at the intersect of the black plotted line and the y-axis indicates 1/Vmax. Notice that a value for 1/Vmax will appear in the 1/Vmax text box beneath the curve box. The arrow at the intersect of the plotted line and the x-axis determines —1/[S].

For a Lineweaver—Burk plot, Vmax and KM will not be calculated for you; you must calculate these values yourself. Based on the values you determined for 1/Vmax and —1/[S] calculate Vmax and KM from your plot. For your convenience a calculator can be accessed by clicking the Calculator tab at the top of the screen.

Setting Up an Eadie—Hofstee Plot: Return to the Data view and plot your data as an Eadie—Hofstee plot. Find the best-fit line for these data points by clicking and sliding the arrows at the x-axis and y-axis. The black arrow at the intersect of the black plotted line and the y-axis indicates Vmax. Notice that a value for Vmax will appear in the Vmax text box. The arrow at the intersect of the plotted line and the x-axis indicates Vmax/KM.

For an Eadie—Hofstee plot, KM will not be calculated for you. Calculate KM from your plot. For your convenience, a calculator can be accessed by clicking the Calculator tab at the top of the screen.

What was the relationship between [S] and invertase activity? Was this relationship what you expected? Why or why not? Explain your answers. What was the Vmax and KM for invertase for this experiment? What do these values tell you about invertase and its affinity for sucrose as its substrate?

How did the values for Vmax and KM derived from each of the three plots compare? Were these values similar or different? If they were different, explain possible reasons for these differences. Which plot did you find easier to use for determining these values?

What do you think would happen if you carried out an experiment with concentrations of sucrose greater than 400 mM? Formulate a hypothesis, then carry out an experiment to test your hypothesis. What did you observe? Describe your results.

 

Assignment 4: Effect of Inhibitors on Invertase Activity

Enzyme inhibitors (I) have played an important role in helping biochemists understand how enzymes function. By inhibiting enzyme activity, it is possible to learn a great deal about the biochemical properties of a particular enzyme. For example, in the absence of substrate, competitive inhibitors can be studied according to the following reaction:

E + I -> EI ->E + P

The equilibrium or dissociation constant for inhibitor binding (KI) in enzyme-inhibitor complexes is written as

KI = [E][I]
 
  [EI]

Typically, when determining how an inhibitor functions, experiments are carried out by measuring enzyme activity in the presence of the inhibitor and different concentrations of substrate. By comparing kinetic data for the inhibition studies to data from uninhibited reactions, it is possible to distinguish competitive inhibitors from noncompetitive inhibitors. When data from such studies are plotted in a Lineweaver—Burk plot, the slope of the plotted line indicates the apparent KM in the presence of inhibitor (a KM). From this value, KI can be determined from the expression

a = 1 + [I]
 
  [KI]

Two of the invertase inhibitors included in this lab are acarbose and Discorea rotundata invertase inhibitor B (DRI inhibitor B). The following assignments are designed to help you distinguish between different types of inhibitors by analyzing the effects of each inhibitor on invertase activity. Your goal is to determine how each inhibitor affects invertase.

1. Role of Acarbose as an Invertase Inhibitor

This experiment is designed to help you determine whether acarbose functions as a competitive or noncompetitive inhibitor of invertase.

Set up an experiment at 50° C, buffer pH 4.0, at a [S] of 50 mM with no acarbose. Run the experiment, determine the slope of the line, record data, and then repeat this type of experiment increasing [S] concentration (keeping [I] at 0.0 m M) in 10 mM increments until you have run experiments at 60 mM, 70 mM, 80 mM, and 90 mM. These uninhibited measurements will be important for studying the activity of invertase when it is inhibited by acarbose.

Repeat this series of measurements in the presence of 0.2 m M acarbose. Keep all other conditions the same as you did for the uninhibited measurements.

Plot a Lineweaver—Burk plot and/or an Eadie—Hofstee plot with both the uninhibited and inhibited data on the same plot as follows. Shift-click to select the five uninhibited measurements, select the plot you want (either Lineweaver—Burk or Eadie—Hofstee), then click Plot Selected Data. Return to the Data view by clicking the Data tab at the top of the screen. Shift-click to select the five inhibited measurements, then change the Data for Curve value to 2 (this indicates that the inhibited data will be plotted as the second curve on plot 1). Change the shape of the symbol to be plotted for these data by clicking on the popup menu for Symbol and choosing a different symbol for the inhibited data. Change the color of the inhibited data from the default, black, to another color using the Color popup menu, then click Plot Selected Data. You will now see a plot with two plotted lines. Your uninhibited data will be plotted in black and your inhibited data will be plotted in the color that you chose. Print this plot.

Determine Vmax and KM for the uninhibited and inhibited studies, then answer the following questions.

Compare your data from the inhibited reactions to your data from the uninhibited experiments. What did you find? Explain what happened to invertase activity as you increased [S]. Why did this occur? What happened to Vmax in the presence of the inhibitor? What happened to KM? If either Vmax or KM changed, explain why.

Based on these results and what you already know about inhibitors of enzyme activity, is this inhibitor functioning as a competitive inhibitor or a noncompetitive inhibitor? How do you know? Explain your answers.

2.Role of DRI Inhibitor B as an Invertase Inhibitor

To investigate how DRI inhibitor B inhibits invertase, carry out the same sets of experiments, both uninhibited and inhibited, with DRI inhibitor B that you set up for acarbose. For inhibited measurements, set DRI inhibitor B to 25 mM. Note: If you did not delete the uninhibited measurements from your experiment with acarbose, you do not need to repeat the uninhibited experiments; you can use your data from the acarbose experiment.

Compare your data from the inhibited reactions to your data from the uninhibited experiments. What did you find? Explain what happened to invertase activity as you increased [S]. Why did this occur? What happened to Vmax in the presence of the inhibitor? What happened to KM? If either Vmax or KM changed, explain why.

Based on these results and what you already know about inhibitors of enzyme activity, is this inhibitor functioning as a competitive inhibitor or a noncompetitive inhibitor? How do you know? Explain your answers.

Compare and contrast what happened as you increased [S] with acarbose to what happened with DRI inhibitor B. Were the results the same or different? If the effect of [S] on invertase was different in the presence of each inhibitor, explain why.

 

Assignment 5: Group Assignment

In the previous assignment, you learned about the effect of acarbose and DRI inhibitor B on invertase activity. A third inhibitor, l-arabinose, also inhibits invertase. Your goal is to work together in a group to determine the mechanism by which l-arabinose inhibits invertase. Work together in a group of four students to complete these exercises.

Divide your group into pairs. For one pair of students, set up and design a set of controlled experiments to measure the KI for l-arabinose, then carry out these experiments. This experiment should be carried out with different concentrations of substrate and inhibitor. Plot data from each measurement as separate Lineweaver—Burk plots. Determine Vmax and KM for each measurement. The KM in the presence of each different concentration of l-arabinose is the apparent KM (a KM). On graph paper, set up a Lineweaver—Burk plot of a KM versus [I]. If l-arabinose is acting as a competitive inhibitor, then KI is represented by the value of [I] that results in a KM of twice the normal KM.

If KM is unaffected by l-arabinose, use graph paper to plot 1/Vmax versus [I]. Vmax in the presence of inhibitor is the apparent Vmax. Estimate the value of [I] that reduces Vmax by approximately one-half. This value represents KI.

For the second pair of students, experiment. Based on your previous assignments, carry out uninhibited and inhibited experiments at a range of different substrate concentrations under conditions of optimal pH and temperature. Choose a [S] that is approximately 2—5 times higher than KM and arbitrarily choose a concentration of l-arabinose for these studies. Run two additional sets of experiments with the same conditions, but only run one set of experiments with a higher [I] and another set with a lower [I]. Plot data as needed to evaluate your results. When possible, plot both inhibited and uninhibited data on the same plots.

Once each pair of students has evaluated their data, discuss and answer the following questions

What is the KI for l-arabinose? Was KM affected by l-arabinose? Was Vmax affected by l-arabinose? What do the plots of apparent KM and apparent Vmax tell you about what type of inhibitor l-arabinose is?

Compare the inhibition of invertase by l-arabinose with the results obtained from inhibiting invertase with acarbose and with DRI inhibitor. Were the results similar or different? How were these results similar? How did these results differ? Explain your answers by describing what happened to Vmax and KM in the presence of l-arabinose.

Based on your results and your knowledge of enzyme inhibitors, does l-arabinose appear to be acting as a competitive inhibitor, noncompetitive inhibitor, or by another mechanism? Consult a biochemistry text for more details if necessary. Once you have a theory on how l-arabinose is working to inhibit invertase, describe your theory and explain how your theory is supported by your data. Design and run additional experiments if necessary until you have generated sufficient data to propose a theory for how invertase is inhibited by l-arabinose. Discuss your experiments and results with your instructor to help you answer this question.