Enzyme Lab
Name ___________________________
Assignment 1: Getting to Know Enzyme Lab: Setting Up an Experiment
The first screen that appears in Enzyme Lab 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 Enzyme Lab.
When you set up an experiment in Enzyme Lab, you will add a buffered solution, sucrose as the substrate,
invertase (the enzyme), 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 so you can observe the effect of changing these variables on invertase activity. A simulated visible
light spectrophotometer will measure product as it is created. Data are recorded and plotted as a function of
product concentration [P] in micromoles (m m) versus time (minutes). The data you collect can then be
analyzed by several different types of plots that are commonly used for analyzing data for enzyme-catalyzed
reactions.
Assignment #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 Enzyme Lab 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. Just as real experiments are conducted in a biochemistry lab, the enzyme should always be
added to the tube last to prevent the reaction from starting before everything has been added to your test tube.
Develop a hypothesis to predict the effect of an increase in temperature on invertase activity:
We believe that as temperature increases, invertase activity will _____________________
because _____________________________________________________________________
____________________________________________________________________________.
Experiment #1 Set – up:
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 the 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.
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?
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Is this what you expected? Explain your answer. _______________________________________________
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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) is represented by the slope of the linear portion of the
curve in this type of plot
To determine the slope of the line, a red line will appear on the plot. You can 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. Follow the directions below to determine the slope of the line for this first measurement.
Click in 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 the data for your experiment, 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.
Experiment #2
Create a second experiment keeping buffer pH constant and [S] at 90 mM, but this time increase
temperature to 35° C. Run the experiment, determine the slope of the line, then record your data.
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 give the data plot a title, select a plot to create,
change the symbols, and view raw data as a table 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 use Enzyme Lab to carry out a number of important calculations and present
these data as plots that are traditionally used for studying enzymes.
VO vs. [S]: analyzes the relationship between reaction velocity (Vo) and substrate concentration [S].
VO vs. Temperature: analyzes the effect of temperature on reaction velocity.
VO vs. pH: analyzes the effect of pH on reaction velocity.
Lineweaver—Burk: Produces a linear plot for the inverse of velocity (1/V) versus the inverse of substrate
concentration [1/S].
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? Explain your answer. ___________________________________________
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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.
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Temperatures above or below optimum cause a decrease in invertase activity. Explain why this happens.
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What is happening to the enzyme to produce these decreases in activity?
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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?_________________________________________
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Experiment #3:
If you were to carry out these temperature experiments at a lower [S], what effect would [S] have on the
temperature optimum for invertase? Formulate a hypothesis.
I believe that as [S] decreases, the optimum temperature for invertase will _____________________
because ________________________________________________________________________________
_______________________________________________________________________________________.
Test your hypothesis and record your results.
What did you discover? Explain your results.
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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 protein found in the cytoplasm in a skin cell is
surrounded by a different fluid environment at a different pH than a membrane-bound enzyme like invertase
which is found in the small intestine. The following assignment 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.
Create a hypothesis to predict the effect of pH on invertase activity.
I believe that invertase activity will _______________when pH changes (choose increase or decrease)
because ________________________________________________________________________________
_______________________________________________________________________________________.
Set up an experiment at the optimal temperature that you determined in assignment 1, with a substrate
concentration of 90 mM. Begin at the lowest pH value, 3.0, measure invertase activity, find the slope of
the line, record data. Run experiments 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). Repeat for a second buffer with a different pH
value.
Create a plot of VO vs. pH. Click on this plot and drag the gray dashed pH line until you find V max.
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?
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Explain your answers? Why and how do pH changes affect invertase activity?
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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.
I believe that the optimum pH for invertase will _______________when temperature changes (choose
increase or decrease) because ________________________________________________________________
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What did you discover? Explain your results.
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Assignment 3: 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. 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. 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 and record your data. Repeat this experiment, keeping [I] at 0.0 m M
while increasing [S]concentration in 10 mM increments until you have run experiments at 60 mM, 70
m.M, 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 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].
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Why did this occur?
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What happened to Vmax in the presence of the inhibitor?
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What happened to KM?
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If either Vmax or KM changed, explain why.
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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.
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