Name ______

Enzymes Lab

Before you begin, save this Lab Report Template on your computer as LastNameAPBIOEnzymes

Go to Biology Labs Online site (http://www.biologylabsonline.com) and log onto the Enzymes Lab. Read all the instructions below BEFORE you start!

Read the Background Information on enzymes so that you understand the terms used in this online lab as well as how enzymes function. Then you will be ready to experiment with the effects of temperature, pH, and substrate concentration on enzyme function. You will be completing Assignments 1, 2, and 3 for this lab.

To start the Enzyme Lab, scroll down on the left to find the START LAB link. Click and wait for the Enzyme Lab to open. You will see a lab bench. You can click on the various items to see their names and functions. You will also see links to the experiment area, a data plot area, a notebook, a calculator, a glossary, a help function, as well as links to the introduction and the assignments. Please spend time getting comfortable with the lab interface before you start!

Experiment 1: Temperature Optimum for Invertase Activity a. Set the water bath at 30oC, the substrate concentration at 90 mM sucrose, leave the pH at 7.0, and do not add any inhibitors. b. Click Add Enzyme & Go: A plot of product concentration vs time will appear. The enzyme kinetic data is plotted as data points in solid black circles. c. Move the red line to get a line-of-best-fit. Try to align the red line so that you have an equal number of data points above and below the line. Use your best judgment! Record the value of the slope in the data chart below. The record data button only functions when you have properly determined the slope. d. Click Clear Experiment, make sure the pH is 7.0 and the sucrose concentration is 90 mM. (These should not change, but it is a good habit to check!) e. Increase temperature to 35oC, Add Enzyme and Go, determine the slope of the line (rate), and record. f. Repeat at 5o increments until you reach 85o C. g. Click on Plot Data and you will see a data chart. The Assignments area of the Enzyme Lab provides details on how to use the data plotting function and what the abbreviations mean. Refer to this as necessary. You will not be able to cut and paste this data chart accurately into this lab report, so make sure that you have recorded the rate data (slope) in the data chart below. You will be able to copy, cut, and paste the graphs! h. Title your graph: Experiment 1—Temperature Optimum The default plot is Vo vs [S] so you need to change this to Vo vs Temp. i. Shift-Click to highlight the rows you want to include in the plot. Make sure you have highlighted all the temperature rows! j. Click Plot Selected Data and then move the arrow/ vertical line to find the temperature maximum. k. Click Export Graph and then copy and paste it below your data chart!

AP Biology Johns Hopkins Center for Talented Youth Fall 2006/2007 Temperature Rate (oC) (mM/min) 30 35 40 45 50 55 60 65 70 75 80 85

Paste your Vo vs Temperature Plot here:

Experiment 1 Analysis:

1. 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.

2. 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?

3. 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?

4. 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? How would you test this?

Experiment 1 Conclusion: The temperature optimum for invertase is ______.

AP Biology Johns Hopkins Center for Talented Youth Fall 2006/2007 Experiment 2: pH Optimum for Invertase

a. Set the water bath at the temperature optimum you determined in Experiment 1, the substrate concentration at 90 mM sucrose, leave the pH at 3.0, and do not add any inhibitors. b. Click Add Enzyme & Go: Find the line-of-best-fit as you did in Experiment 1. Record the value of the slope in the data chart below. c. Click Clear Experiment and repeat using the pH values in the data chart below. d. Click on Plot Data to see a data chart. e. Title your graph: Experiment 2—pH Optimum The default plot is Vo vs [S] so you need to change this to Vo vs pH f. Shift-Click to highlight all the rows for the pH experiment If you have run this experiment right after the temperature experiment, all the data is listed. You need to scroll down to where you set the optimum temperature and adjusted the pH values. If you get an error message, check to see that you have highlighted only the rows where you changed pH. g. Change the plot to # 2, click Plot Selected Data, and then move the arrow/ vertical line to find the pH maximum. h. Click Export Graph and then copy and paste it below your data chart!

Rate pH (mM/min) 3.0 3.4 4.0 4.4 5.0 5.4 6.0 6.4 7.0 7.4 8.0 8.4 9.0 9.4 10.0

Paste your Vo vs pH Plot here:

Experiment 2 Analysis

1. What is the optimal pH for invertase activity? Why and how do pH changes affect invertase activity?

2. 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?

3. 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? How would you test this?

Experiment 2 Conclusion: The pH optimum for invertase is ______.

AP Biology Johns Hopkins Center for Talented Youth Fall 2006/2007 Experiment 3: The Effect of Substrate Concentration on Invertase Activity

By now you are familiar with the operation of the Enzyme Lab, so the instructions will be simpler:

a. Set the water bath at the temperature optimum you determined in Experiment 1 and the pH at the optimum you determined in Experiment 2. b. Start with an initial substrate concentration [S] of 0.00 mM c. Click Add Enzyme & Go: Find the line-of-best-fit as you did in Experiments 1 and 2. Record the value of the slope in the data chart below. d. Click Clear Experiment and repeat using the[S] values in the data chart below. e. Click on Plot Data to see a data chart. f. Title your graph: Experiment 3—Substrate Concentration. And set plot type to Vo vs [S] g. Shift-Click to highlight all the rows for the [S] experiment If you have run this experiment right after the temperature and pH experiments, all the data is listed. You need to scroll down to where you set the optimum temperature and pH and adjusted [S] h. Change the plot to # 3, click Plot Selected Data. You will have to move the vertical and horizontal arrows/lines line to find Vmax and Km i. Click Export Graph and then copy and paste it below your data chart!

Note: You do NOT have to set yp Lineweaver-Burk or Eadie-Hofstee Plots, as suggested in the online instructions.

Sucrose Concentration [S] Rate mM (mM/min) 2.5 5.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 320.0 340.0 360.0 380.0 400.0

Paste your Vo vs pH Plot here:

Experiment 3 Analysis

AP Biology Johns Hopkins Center for Talented Youth Fall 2006/2007 1. What was the relationship between [S] and invertase activity? Was this relationship what you expected? Why or why not? Explain your answers.

2. What do the values for Vmax and Km indicate?

3. What do you think would happen if you carried out an experiment with concentrations of sucrose greater than 400 mM? How would you test this?

Experiment 3 Conclusion: The maximum velocity for Invertase activity at ____ o and pH ___ is ______

Assignments 4 and 5 are NOT required, but you may perform them if you want to!

AP Biology Johns Hopkins Center for Talented Youth Fall 2006/2007