Chemical reaction (slow): Enzyme-catalyzed reaction (much faster):

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1 1 Enzymes Introduction Enzymes are Biological Catalysts Recall that a catalyst is an agent which speeds up a chemical reaction without actually being consumed or changed by the reaction. Enzymes are proteins that act as biological catalysts to speed up/catalyze biochemical reactions that occur in living organisms. Without the aid of enzymes, most biological reactions would be too slow to be useful, and life would not be possible. Since enzymes are proteins, they are folded chains of amino acids, similar to all other proteins. The 3-dimensional shape of the enzyme is essential to its function as a catalyst, just as the shape of any protein is essential to its function. An enzyme catalyzes a biochemical reaction by binding the reactant, which is referred to as the substrate, in an indentation or pocket on the surface of the enzyme s structure. This indentation or pocket is called the active site. The active site is where the reactant/substrate undergoes a reaction in which it is converted to the product. The enzyme helps out during the process of the reaction, thus enabling it to occur faster than it would otherwise. When the reaction is complete, the product leaves the enzyme active site. The enzyme is unchanged after the reaction, so it is then able to bind another molecule of substrate in its empty active site and catalyze another round of the reaction. A simple diagram of an enzyme-catalyzed reaction is shown below. Chemical reaction (slow): substrate product Enzyme-catalyzed reaction (much faster): enzyme enzyme with enzyme enzyme with product is released from active substrate bound helps out product bound site and enzyme is ready to to active site with reaction to active site catalyze another reaction Inhibitors of Enzyme-Catalyzed Reactions An enzyme inhibitor is a compound that slows or stops an enzyme-catalyzed reaction. Inhibitors can act in a number of different ways, but they often bind to the active site. When the active site is occupied in this manner, the substrate cannot bind, so the enzyme cannot help convert the substrate to product. Hence, the reaction occurs very slowly or not at all.

2 2 Enzymes Prelab Name 1. Most enzymes are proteins. What do all enzymes do in living organisms? 2. What is the term for the reactant (starting material) in an enzyme-catalyzed reaction? 3. If the product of an enzyme-catalyzed reaction happens to be a colored compound, how can you tell that the reaction is actually occurring and determine the rate at which it is occurring? (The answer to this question is not found in the introductory material on the first page. However, you should be able to answer the question by just thinking logically.) 4. What is the term for a compound that slows down the rate of an enzyme-catalyzed reaction? 5. Page through the experimental procedure on the following pages. What are the names of the three enzymes you will study in this experiment?

3 3 Enzymes Procedure and Report Sheet Name Studying the Activity of the Enzyme Catalase 1. Catalase is an enzyme that increases the rate of the conversion of hydrogen peroxide (H 2 O 2 ) into water and molecular oxygen. This reaction is shown below. 2 H 2 O 2 O H 2 O Catalase is found within the cells of many organisms and is involved in the protection of cells from the harmful effects of peroxides which are sometimes products of cellular respiration. One can observe the activity of catalase by monitoring the amount of oxygen gas produced by the enzyme. What is the substrate of catalase? What are the products of the reaction catalyzed by catalase? 2. Cut a thin slice of potato and place it in a large beaker. Add enough hydrogen peroxide (H 2 O 2 ) to cover the potato slice. Look for the formation of bubbles on the surface of the potato. Are bubbles originating from the entire surface, or from a particular region of the surface? If a particular region, which region? By cutting the potato, you damaged some of its cells, allowing the contents of the cells (including enzymes) to spill out. The bubbles you observed are bubbles of gas produced by the enzyme catalase from the potato cells as it catalyzes a reaction with H 2 O 2. What compound do these gas bubbles consist of? Discard the potato (in the trash) and hydrogen peroxide (down the drain). Studying the Activity of the Enzyme Peroxidase 1. The enzyme peroxidase is found within the cells of specific organs or tissues in many plants. It catalyzes the reaction shown below. OH catalyzed by peroxidase + H 2 O 2 colored products 4-chloronaphthol Cl or other similar aromatic compounds What are the names of the substrates of peroxidase? Based on the reaction shown for peroxidase, how could you tell that the enzyme-catalyzed reaction was occurring what would you observe as the reaction progressed?

4 4 Peroxidase in CELERY 1. Obtain two pieces of the special tissue-printing paper. Handle them by the edges so that you don t contaminate the middle section. Get two plastic boats; put the 4-chloronaphthol + H 2 O 2 solution in one (just enough to cover the bottom of the boat) and the 4-chloronaphthol + H 2 O 2 + ascorbic acid solution in the other. Label them in some way so you don t mix them up. 2. Obtain a clean razor blade. It is important that the razor blade be clean and free of any residue from other vegetables, because enzymes in the residue could contaminate the next vegetable you cut. Cut two cross-section slices (~3/8 inch thick) of celery. Don t touch the cut surfaces of the slices handle them by the dry edges. Carefully and neatly blot one celery slice on one of the pieces of tissue-printing paper by pressing it very firmly down on the lab bench for about 15 seconds, so that you have a celery print on the paper (the print may not be visible). Try not to move or slide the celery slice while pressing it down it s important to get a neat print. Then press the other celery slice on the second piece of paper in the same manner. By slicing the celery, you damaged some of its cells, causing the contents of the cells (including enzymes) to spill out. By pressing the celery slice on paper, these cellular contents have been transferred to the paper. 3. Use a pencil to label one of the paper prints 4CN by writing lightly on the edge of the paper. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 solution. Remove the paper quickly and place it on some paper towels. Blot the surface lightly with another paper towel to remove excess liquid. Do not get the solution on your fingers, but continue to handle the paper with forceps! Wash your hands if you get any of the solution on yourself! Use a pencil to label the other celery print 4CN + asc acid. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 + ascorbic acid solution. Quickly remove the paper, place it on some paper towels, and blot the surface lightly with another paper towel. The 4-chloronaphthol + H 2 O 2 solution contains the two substrates of the enzyme peroxidase. By dipping the paper print into this solution, you have exposed any celery peroxidase on the paper to its substrates. The peroxidase should then begin to catalyze the reaction, producing the colored product which will be visible on the paper. The 4-chloronaphthol + H 2 O 2 + ascorbic acid solution contains the two substrates of the enzyme peroxidase, along with ascorbic acid which prevents the reaction by acting as an antioxidant. By dipping the paper print into this solution, you have exposed any celery

5 5 peroxidase on the paper to its substrates. However, the presence of the ascorbic acid will either prevent the peroxidase-catalyzed reaction from occurring, or will drastically slow it down. Allow the two celery prints to sit undisturbed for minutes. During this time, watch for the development of any color and take note of how quickly it develops. Note whether or not the color develops much more slowly (or not at all) for one of the prints. Note whether the color is much darker on one of the prints. While the color is developing, you can continue working on the next few steps using other vegetables. Record your observations for CELERY: What color appeared on the paper? Did color appear on both prints, or only one (which one)? If color appeared on both prints, is it significantly darker on one? Which one? Does it seem that ascorbic acid slowed down/stopped the reaction catalyzed by peroxidase? How can you tell that it slowed/stopped the reaction? Describe the pattern of the color on the print that does not contain ascorbic acid. For example, is it darker in some areas than others, can you clearly see the celery structure, etc.? Based on the appearance of this print, does it appear that peroxidase is found in all of the celery cells, or only in certain cells? For example, is it only found in the cells of the outer skin, the cells of the interior, the cells of the celery veins, etc.?

6 6 Peroxidase in RADISH 1. Obtain two new pieces of the special tissue-printing paper. Handle them by the edges so that you don t contaminate the middle section. 2. Rinse and dry the razor blade you used previously. Cut two cross-section slices (~1/4 inch thick) of radish. Don t touch the cut surfaces of the slices handle them by the dry edges. Carefully and neatly blot one radish slice on one of the pieces of tissue-printing paper by pressing it very firmly down on the lab bench for about 15 seconds. Then press the other radish slice on the second piece of paper. 3. Use a pencil to label one of the paper prints 4CN by writing lightly on the edge of the paper. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 solution. Remove the paper quickly and place it on some paper towels. Blot the surface lightly with another paper towel to remove excess liquid. Use a pencil to label the other radish print 4CN + asc acid. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 + ascorbic acid solution. Quickly remove the paper, place it on some paper towels, and blot the surface lightly with another paper towel. Allow the two radish prints to sit undisturbed for minutes. During this time, watch for the development of any color and take note of how quickly it develops. Note whether or not the color develops much more slowly (or not at all) for one of the prints. Note whether the color is much darker on one of the prints. While the color is developing, continue on with the next few steps. Record your observations for RADISH: Did color appear on both prints, or only one (which one)? Does it seem that ascorbic acid slowed down/stopped the reaction catalyzed by peroxidase? Describe the pattern of the color on the print that does not contain ascorbic acid. For example, is it darker in some areas than others, can you clearly see the radish structure, etc.?

7 7 Based on the appearance of this print, does it appear that peroxidase is found in all of the radish cells, or only in certain cells? For example, is it only found in the cells of the outer skin, the cells of the interior, etc.? Peroxidase in PARSNIP 1. Obtain two new pieces of the special tissue-printing paper. Handle them by the edges so that you don t contaminate the middle section. 2. Rinse and dry the razor blade you used previously. Cut two cross-section slices (~1/4 inch thick) of parsnip. Don t touch the cut surfaces of the slices handle them by the dry edges. Carefully and neatly blot one parsnip slice on one of the pieces of tissue-printing paper by pressing it very firmly down on the lab bench for about 15 seconds. Then press the other parsnip slice on the second piece of paper. 3. Use a pencil to label one of the paper prints 4CN by writing lightly on the edge of the paper. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 solution. Remove the paper quickly and place it on some paper towels. Blot the surface lightly with another paper towel to remove excess liquid. Use a pencil to label the other parsnip print 4CN + asc acid. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 + ascorbic acid solution. Quickly remove the paper, place it on some paper towels, and blot the surface lightly with another paper towel. Allow the two parsnip prints to sit undisturbed for minutes. During this time, watch for the development of any color and take note of how quickly it develops. Note whether or not the color develops much more slowly (or not at all) for one of the prints. Note whether the color is much darker on one of the prints. While the color is developing, continue on with the next few steps. Record your observations for PARSNIP: Did color appear on both prints, or only one (which one)?

8 8 Does it seem that ascorbic acid slowed down/stopped the reaction catalyzed by peroxidase? Describe the pattern of the color on the print that does not contain ascorbic acid. For example, is it darker in some areas than others, can you clearly see the parsnip structure, etc.? Based on the appearance of this print, does it appear that peroxidase is found in all of the parsnip cells, or only in certain cells? For example, is it only found in the cells of the outer skin, the cells of the interior, etc.? Peroxidase in MUSHROOM 1. Obtain two new pieces of the special tissue-printing paper. Handle them by the edges so that you don t contaminate the middle section. 2. Rinse and dry a sharp knife. (A razor blade will probably be too small to properly cut the mushroom.) Cut two cross-section slices (~1/4 inch thick) of mushroom. Don t touch the cut surfaces of the slices handle them by the dry edges. Carefully and neatly blot one mushroom slice on one of the pieces of tissue-printing paper by pressing it down gently on the lab bench for about 15 seconds (don t smash the mushroom by pressing too firmly). Then press the other mushroom slice on the second piece of paper. 3. Use a pencil to label one of the paper prints 4CN by writing lightly on the edge of the paper. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 solution. Remove the paper quickly and place it on some paper towels. Blot the surface lightly with another paper towel to remove excess liquid. Use a pencil to label the other mushroom print 4CN + asc acid. Using forceps, quickly dip this paper print into the boat of 4-chloronaphthol + H 2 O 2 + ascorbic acid solution. Quickly remove the paper, place it on some paper towels, and blot the surface lightly with another paper towel. Allow the two mushroom prints to sit undisturbed for minutes. During this time, watch for the development of any color and take note of how quickly it develops. Note whether or not the color develops much more slowly (or not at all) for one of the prints. Note whether the color is much darker on one of the prints.

9 9 Record your observations for MUSHROOM: Did color appear on both prints, only one, or neither? Examine the print that does not contain ascorbic acid. Based on the appearance of this print, does it appear that peroxidase is found in any of the mushroom cells? Explain. Compare the peroxidase activity of the vegetables: View the prints of the four vegetables by placing them side by side. Compare the darkness of the color from one vegetable print to another. List the vegetables that appear to contain peroxidase: List the vegetables that do not appear to contain peroxidase: Of the vegetables that do contain peroxidase, which one seems to have the most peroxidase? Explain how you determined that this vegetable has the most peroxiase. Discard the vegetables slices and paper prints in the trash; dispose of the two solutions in the boats by pouring them down the drain.

10 10 Studying the Activity of the Enzyme Polyphenol Oxidase 1. The enzyme polyphenol oxidase is found within the cells of specific organs or tissues in many plants and mushrooms. It catalyzes the reaction shown below. OH OH catechol or other similar aromatic compounds catalyzed by polyphenol oxidase O further reactions colored + O 2 and H 2 O products O What are the names of the substrates of polyphenol oxidase? The products of the polyphenol oxidase-catalyzed reaction are colored compounds, so you will be able to observe the progress of the reaction by the development of a dark color. Since polyphenol oxidase is found in most fruits and vegetables, you have seen it in action before. When cut apples turn brown, when the edges of lettuce turn brown, or when white mushrooms develop brown spots, the brown color is due to polyphenol oxidase in the cells of the fruit/vegetable catalyzing a reaction between O 2 in the air and phenolic compounds within the cells of the fruit/vegetable. Polyphenol oxidase seems to become more active as fruits and vegetables age, which is why fruits and vegetables begin to turn brown as they age and spoil. 2. Obtain four pieces of the tissue-printing paper. Get a plastic boat; put the catechol solution in it (just enough to cover the bottom of the boat). Label the boat in some way so you don t mix it up with others later. 3. Rinse and dry the razor blade you used previously. Cut a cross-section slice (~3/8 inch thick) of celery. Carefully blot the celery slice onto a piece of the tissue-printing paper as you did previously (press very firmly for ~15 seconds). Rinse the razor blade clean and cut a cross-section slice (~1/4 inch thick) of radish. Carefully blot the radish slice onto another piece of the paper. Rinse the razor blade clean and cut a cross-section slice (~1/4 inch thick) of parsnip. Blot the parsnip slice onto another piece of the paper. Rinse the knife you used previously and use it to cut a cross-section slice (~1/4 inch thick) of mushroom. Blot the mushroom slice onto another piece of the paper. 4. Use a pencil to label each of the paper prints catechol. Using forceps, quickly dip one of the paper prints into the boat of catechol solution. Quickly remove the paper, place it on some paper towels, and blot the excess liquid with another paper towel. Do not get the catechol solution on your fingers; wash your hands if you do!

11 11 Dip the remaining three prints into the solution, one at a time, in the same manner. Allow the four prints to sit undisturbed for minutes. During this time, watch for the development of any color. Note how quickly the color appears and how dark it becomes. While the color is developing, you can do steps 5-7 below. Record your observations for polyphenol oxidase: What color appeared on the papers? Did color appear on all four prints, or only some of them (which ones)? Describe the pattern of the color on each print. For example, is it darker in some areas than others, can you clearly see the vegetable structure, etc.? Celery: Radish: Parsnip: Mushroom: 5. Obtain two more pieces of tissue-printing paper. You should still have the boat containing the catechol solution. Get another plastic boat. Put catechol + ascorbic acid solution in the boat (just enough to cover the bottom) and label it. 6. Choose one of the four vegetables used above (celery, radish, parsnip, or mushroom) for which a dark color DID develop on the paper print. Rinse the razor blade or knife and cut two cross-section slices of this vegetable. Carefully and neatly blot each slice on separate pieces of paper.

12 12 7. Use a pencil to label one of the paper prints catechol. Using forceps, dip this paper print into the boat of catechol solution. Remove the paper quickly, place it on some paper towels, and blot excess liquid from the surface using another paper towel. Use a pencil to label the other paper print catechol + asc acid. Using forceps, dip this paper print into the boat of catechol + ascorbic acid solution. Remove the paper, place it on some paper towels, and blot excess liquid from the surface. Allow the two prints to sit undisturbed for minutes. During this time, watch for the development of any color and take note of whether or not the color develops much more slowly (or not at all) for one of the prints. Note whether the color is much darker on one of the prints. Then record your observations below. Record your observations: Which vegetable did you use? Did color appear on both prints, or only one (which one)? If color appeared on both prints, is it significantly darker on one? Which one? Does it seem that ascorbic acid slowed down/stopped the reaction catalyzed by polyphenol oxidase? How can you tell that it slowed/stopped the reaction? Before answering the following two questions, carefully read the information about the enzyme polyphenol oxidase explained in #1 on page 10. Whether or not ascorbic acid appeared to slow/stop the reaction, ascorbic acid IS known to prevent the formation of colored reaction products by acting as an antioxidant. Ascorbic acid is actually vitamin C (this is the chemical name for vitamin C). Can you think of a possible explanation for why many fruits and vegetables contain vitamin C? How might the presence of vitamin C benefit the plant? Lemon juice contains vitamin C. Dipping apple slices in lemon juice will prevent them from turning brown. Explain why this prevents the browning of the apple slices.

13 13 You learned previously about denaturation of proteins. Enzymes are proteins. How will cooking a vegetable affect the enzymes within its cells? Will the enzymes in a cooked vegetable be able to catalyze a reaction? Why or why not? Discard all vegetable slices and paper prints in the trash; dispose of the two solutions in the plastic boats by pouring them down the drain.

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