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LAB 2: ENZYMES Virtually every chemical reaction in organisms involves enzymes. What are enzymes? How do they work? Under what conditions do they function best? Under what conditions do they not function at all? These are some of the questions you will address in this laboratory session. QUESTIONS/OBJECTIVES OF LABORATORY: · How do enzymes work? What factors influence the activity of enzymes? · How do enzyme/substrate concentrations affect enzymatic activity? · What effects do specificity, temperature, and pH have on enzymatic activity? · What role do cofactors play in enzymatic activity?
RELEVANT READING : Chapter 6 (pp. 106-124) in text. INTRODUCTION: · What is an enzyme? Enzymes are proteins that catalyze chemical reactions in living cells. A catalyst is simply a compound that speeds up a reaction without being permanently changed during the reaction. Enzymes can be reused. They are neither destroyed nor altered while catalyzing reactions.
Catalysts work by reducing the amount of activation energy that a reaction requires. Note that enzymes do not make anything happen that would not otherwise occur on its own—they just make it far more likely to happen (and hence happen faster), by reducing the activation energy.
With few exceptions, enzymes are complex, three-dimensional proteins (it is exceedingly rare for any other kind of molecule to possess catalytic abilities, but RNA is one). Each enzyme functions by forming a reactive region that binds to a specific compound. The reactive area on the enzyme is called the active site. The specific compound(s) it binds is the substrate, and the result of binding is a temporary enzyme-substrate complex. Although this was once envisioned in terms of a rigid “lock-and-key fit,” biologists now subscribe to the “induced fit” model, which holds that structural changes during binding allow a more close, precise fit as reactants are brought together to reach a transition state which allows the reaction to proceed.
Enzymes are extremely selective about the substrates that they will bind. In other words, they exhibit high specificity. Most enzymes recognize both substrates and products and so can catalyze reactions in both directions. In other words, enzymes are reversible.
Enzymes have limitations and are vulnerable to several factors. Not only are they (like all proteins) subject to mutation—many “inborn errors of metabolism” involve faulty enzymes—but they are also dependent on other conditions. For example, temperature and hydrogen ion concentration (i.e., pH) often play a major role in determining an enzyme’s effectiveness.
Enzyme activity is also affected by the presence of cofactors, either certain metallic ions or organic molecules called coenzymes. Some of these ions/molecules compete for the active site (these are competitive inhibitors), whereas others bind to regulatory sites that change the shape of the active site and hence prevent substrate binding (these are allosteric inhibitors).
In this laboratory session you will test the effects of all these factors on the enzyme catecholase (also known as tyrosinase). Notice that the suffix –ase denotes an enzyme, while the word it is attached to describes the substrate (what the enzyme binds). Lipase, carbohydrase, and protease enzymes catalyze reactions involving (respectively) lipids, carbohydrates, and proteins.
EXERCISES:
NOTE: Your instructor will determine the amount of time available for the following exercises. If there is insufficient time for everyone to do all seven exercises, the experiments can be divided among several small groups, and groups can combine their data at the end of the lab period. All groups should perform Exercise A, however, in order to understand the action of catecholase.
In this experiment, you will use the spectrophotometer to obtain data on enzyme function. Your instructor will provide details on its operation and on how to prepare appropriate blanks by which to “zero” your machine. For this lab you will read all samples at 360 nm wavelength.
CAUTION: Catechol, hydroxyquinone, and phenylthiourea (PTU) are hazardous if ingested or absorbed through the skin. The methods specified in these exercises are designed to prevent skin contact with these substances. Since screw-cap test tubes are not available for spectrophotometer use, wear plastic gloves when you handle the chemicals. If an accident occurs, immediately wash your hands thoroughly with warm water and soap.
A. What do enzymes do? [The effect of catecholase]Catechol is a derivative of benzene found in many fruits and other plant structures. Catecholase catalyzes the reaction of catechol and oxygen and is the enzyme that causes bruised or otherwise damaged fruit to turn brown. In the presence of catecholase, catechol is oxidized to form benzoquinone, which has a reddish brown color. Benzoquinone is thought to inhibit the growth of microorganisms that cause rot, an obvious adaptive advantage to plants.
MATERIALS: 3 spectrophotometer tubes; test tube rack; grease pencil; 40º C water bath (or hotplate); 1 percent catechol stock solution; catecholase stock solution in ice bath; distilled water
PROCEDURAL STEPS: 1. Label test tubes 1, 2, and 3, and place 3ml of each of the following solutions into the respective tubes (each test tube will contain a total volume of 6 ml): Test tube 1: catecholase (always keep on ice!) and catechol Test tube 2: catecholase and distilled water Test tube 3: catechol and distilled water Cover the tubes with Parafilm and gently invert them to mix contents.
Record the absorbency at 360 of each solution in item A of your worksheet.
2. Place the three test tubes in a 40º C water bath for 15 minutes.
Agitate the solutions and check the color intensity of each one when you begin and every 5 minutes thereafter. Read the absorbency in the spectrophotometer at 360. Record your data in your worksheet.
3. At the end of the 15 minutes, remove the test tubes from the water bath. Save the tubes for color comparisons in later exercises.
B. How does enzyme concentration affect enzymatic activity? Now that you have seen how catechol normally reacts to form benzoquinone in the presence of catecholase, you may wonder how much enzyme is required for this reaction. You can vary the enzyme’s concentration by diluting it with a buffer solution. A buffer solution resists any change in pH by releasing or taking up hydrogen ions (H+). Your buffer will have a pH of 7.
In this experiment, using the materials given, you will devise a way to test the effect of enzyme concentration. Remember to use the buffer to dilute the catecholase stock. Before you begin your experiment have your protocol approved by your instructor.
MATERIALS: 3-5 spectrophotometer tubes; test tube rack; 40º C water bath; 10 ml graduated cylinder; 1 % catechol stock solution; catecholase stock solution; distilled water; pH 7 buffer solution
PROCEDURAL STEPS: 1. Record your hypothesis and procedures (approved by your instructor!) in item B of your worksheet. 2. Once you have made your solutions and noted the initial color intensities, place the tubes in the water bath for 15 minutes. Always keep catecholase on ice!
Agitate the tubes every 5 minutes, and record the resulting color intensities.
3. Record your results and conclusions in your worksheet.
C. How does substrate concentration affect enzymatic activity? Once you have tested to determine the effect of enzyme concentration, a new question arises: What is the effect of substrate concentration on the reaction rate? If you keep the amount of enzyme constant, how will the enzymatic activity change as you add more and more substrate? Remember that the enzyme and substrate must combine to form an enzyme-substrate complex in order for the reaction to proceed.
MATERIALS: 3-5 spectrophotometer tubes; test tube rack; 40º C water bath; 10 ml graduated cylinder; 1 % catechol stock solution; catecholase stock solution; distilled water; pH 7 buffer solution
PROCEDURAL STEPS:
D. How specific are enzymes in terms of substrates? The specificity of an enzyme is that enzyme’s tendency to react only with a particular substrate. In this experiment, you will demonstrate that catecholase can distinguish between two quite similar compounds, catechol and hydroxyquinone.
MATERIALS: 2 spectrophotometer tubes; test tube rack; 40º C water bath; 10 ml graduated cylinder; 1 % catechol stock solution; catecholase stock solution; 1 % hydroxyquinone stock solution
PROCEDURAL STEPS: 1. Label test tubes 1 and 2.
2. Place 3 ml of catechol in test tube 1. Place 3 ml of hydroxyquinone in test tube 2.
3. Pour 3 ml of catecholase into each tube.
Place the caps on the tubes, and shake the tubes to mix the solutions. Note the color intensities, and place the tubes in the water bath.
Agitate the tubes every 5 minutes for 15 minutes, and once again note the resulting color intensities by taking absorbency readings on the spectrophotometer.
4. Record your results and conclusions in item D of your worksheet.
E. How does temperature affect the activity of enzymes? Enzymes, like all proteins, have specific three-dimensional shapes. The molecules are held in these shapes—which are critical to the enzyme’s proper functioning—by hydrogen bonds and other fairly weak bonds. Extreme heat can break the bonds and thereby change the enzyme’s shape. When a protein’s shape is changed, it is said to be denatured.
For this exercise, you will be provided water baths (or hot plates) at various temperatures.
MATERIALS: 6 spectrophotometer tubes & rack; 40ºC, 60ºC and 80ºC water baths or hot plates; thermometer; 1 % catechol stock solution; catecholase stock solution; 3 400-ml beakers; test tube clamps
PROCEDURAL STEPS: 1. Design an experiment to test the effect of temperature on catecholase. Remember to bring the substrate to the desired temperature before you add catecholase.
2. Record your hypothesis and methods (approved by your instructor) in item E of your worksheet prior to your tests.
3. Once started, your tests should run approximately 15 minutes each. Agitate the solutions every 5 minutes and note the resulting color intensities.
4. After running the experiment, record your data in a table in the worksheet.
5. Summarize your results and conclusions.
F. How does pH affect the activity of enzymes? In addition to heat, other factors can cause proteins to become denatured. Many of the chemical bonds that hold a protein in its three-dimensional shape are affected by the presence of hydrogen ions (H+). The buffer solutions in the series you will use in this exercise vary in pH. By exposing the enzyme-substrate complex to a range of pH values, you can test the effect of hydrogen ion concentration on enzymatic activity.
MATERIALS: 7-10 spectrophotometer tubes & rack; 40ºC water bath; 10-ml graduated cylinder; 1 % catechol stock solution; catecholase stock solution; pH buffer series (including pH levels of 2, 4, 6, 7, 8, 10 and 12)
PROCEDURAL STEPS: 1. Using the materials listed, design an experiment to test the effect of pH on enzymatic activity. Have it approved by your instructor.
2. Record your hypothesis and methods in item F of your worksheet. Once approved, perform your experiment and collect the data.
3. As in the previous exercises, agitate the solutions at the beginning of the test and every 5 minutes thereafter for 15 minutes.
4. Note the resulting color intensities on the spectrophotometer.
5. Briefly state your results and conclusions in the worksheet.
G. What role do cofactors play in enzymatic activity? Enzymes often (but not always) require “helper” substances to bind substrates and catalyze a reaction. These substances, called cofactors, generally occur in two forms: mineral elements and organic coenzymes. In this exercise you will explore a cofactor that catecholase needs to catalyze the reaction that turns catechol (and oxygen) to benzoquinone.
MATERIALS: 2 spectrophotometer tubes & rack; 40ºC water bath; 10-ml graduated cylinder; 1 % catechol stock solution; catecholase stock solution; phenylthiourea (PTU) crystals; chemical spatula or lifter (Note: Since PTU is a hazardous chemical, your instructor will dispense it.)
PROCEDURAL STEPS: 1. Label two test tubes 1 and 2. Pour 3 ml of catecholase into each tube.
To test tube 2, have your instructor add three or four PTU crystals. [Note: Too much PTU makes the solution opaque (cloudy), which can give a falsely high absorbance reading.]
Screw on the test-tube caps or cover with Parafilm and shake tubes for 4-5 minutes.
2. Add 3 ml of 1 percent catechol to each test tube. Shake the tubes briefly, and record the color intensities of the solutions. Place the tubes in the water bath for 15 minutes.
3. Record the resulting color intensities as in the previous experiments.
4. Record your data in item G of your worksheet.
CONCLUSIONS: Materials for Enzyme Function Lab
Enzymes:
300 mL catechol stock 1% **POISON** KEEP ON ICE 200 mL tyrosinase stock (kept in freezer) KEEP ON ICE 25 mL hydroquinone soln 1% (shelf)
Phenylthiourea crystals Waste container
Buffers at pH 2, 4, 6, 7, 8, 10, 12 (30mL each) More of pH 7 (150 mL)
Thermometers 10 mL grad cylinders spec 20 spec 20 tubes test tube racks test tube clamps 400 mL beakers spatulas sharpies parafilm scissors water baths at 40 degrees C, and 60 degrees C hot plate at 80 degrees C plastic pipets 5 ml pipets with pipet pumps
Ice
REFERENCES/SOURCES:
Helms, D.R., C.W. Helms, R.J. Kosinski, and J.R. Cummings. 1998. Biology in the Laboratory, 3e. New York : W.H. Freeman. Name:
A. What do enzymes do? [The effect of catecholase]
· Fill in your data from Exercise A on the effect of catecholase on enzymatic activity:
· Which of the test tubes represents the control?
· Describe your results…
B. How does enzyme concentration affect enzymatic activity?
· What was your hypothesis in the experiment in Exercise B on the effect of enzyme concentration on enzymatic activity?
· Outline the procedures you used to test your hypothesis:
· Fill in the data for Table B:
· How did enzyme concentration affect reaction rate?
· What do you conclude from your results?
· Why did you use buffer instead of distilled water to dilute the enzyme concentration?
· Must there be an equal number of enzyme molecules and substrate molecules in order for the reaction to take place? Explain…
C. How does substrate concentration affect enzymatic activity?
· What was your hypothesis for the experiment in Exercise C on the effect of substrate concentration on enzymatic activity?
· Outline the procedures you used to test your hypothesis:
· Fill in the data in the accompanying table for Exercise C:
· How did substrate concentration affect reaction rate? · What do you conclude from your results?
D. How specific are enzymes in terms of substrates?
· Fill in the data for Exercise D on the specificity of catecholase.
· What do you conclude from this test?
· How do you think catecholase distinguishes between substrates?
E. How does temperature affect the activity of enzymes? · What was your hypothesis in Exercise E on the effect of temperature on catecholase?
· Outline the procedures you used to test your hypothesis.
· Record the temperatures used and the resulting color intensities of the solutions at each of the times indicated in Table E:
· Briefly summarize your results in this experiment. Draw a simple graph of your results for the 10- or 15-minute interval. Plot temperature on one axis and color intensity on the other.
· What is the optimum temperature for catecholase? ____________
· Certain species of bacteria live in hot springs where the water temperature is above 70º C. How can these organisms survive? Relate your answer to enzyme function.
F. How does pH affect the activity of enzymes?
· What was your hypothesis in the experiment in Exercise F on effect of pH on catecholase?
· Outline the procedures you used to test your hypothesis:
· Record the data you developed for this experiment in Table F.
· Briefly state your results in this experiment. Make a simple graph of your data on the effects of pH on enzymatic activity after 15 minutes.
· What is the optimum pH for catecholase function? ______________
· The stomach enzyme pepsin is most effective in a very acidic environment (pH 2) and becomes inactive in an alkaline environment. Based on the results of your experiment with catecholase, explain how this is possible. G. What role do cofactors play in enzymatic activity? | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
How
is the secondary or tertiary structure of a protein responsible for an
enzyme’s activity? 