Have an account?
Skip to Content
Build your own quiz
Suggestions for you
3 questions
Show Answers
See Preview
- 1. Open EndedBackground One of the qualities that distinguishes living things from non-living things is that living things have the ability to carry out chemical reactions that are crucial for survival. Even single-celled organisms are able to perform hundreds of chemical reactions within their cell wall. Imagine the countless number of reactions that take place in a large organism such as a human! None of these reactions are possible without enzymes. Enzymes are biological catalysts or reaction assistants. Enzymes are made up of various types of proteins that work together to drive a chemical reaction. Enzymes can get a reaction underway or can speed it up. For example, in the presence of certain enzymes, substrates (the chemical reactants) can be transformed into usable products at the rate of millions of times per second! In the absence of the enzymes, however, the reactants could take years to be converted into usable products, if at all. The work of enzymes is crucial to the existence of life on Earth. Enzymes are globular proteins: they have a specific 3D shape that is determined by the electrostatic charges of their constituents attracting and repelling one another. The 3D shape of a protein is critical to its function. Enzyme activity is described by a lock and key model, also known as the induced fit model. Enzymes have active sites where they come into contact with specific substrates. Once a substrate has come into contact with the active site of an enzyme, it is manipulated by the enzyme into the final product. When the process is complete, the enzyme releases the product and is ready to begin the process with new substrates. Enzymes are reusable and therefore always recycled. enzyme_substrate The environment of an enzyme also has an effect on its 3D shape. For example, pH affects the shape of the enzyme's active site. In the presence of excess H+ or OH- ions, the active site on the enzyme becomes progressively distorted; this decreases the enzyme's activity until it can no longer function as a catalyst. This process is called denaturation of the enzyme. Temperature also affects the activity of enzymes. Chemical reactions accelerate as temperature increases, so, in general, catalysis will increase at higher temperatures. However, each enzyme has an optimum temperature point beyond which the enzyme's functional 3D shape is lost and catalysis decreases. Boiling temperatures will denature most enzymes by stretching the molecular bonds present in the enzyme beyond repair. Many enzymes that are present in our bodies help us break down our food. Digestive enzymes are secreted all along the digestive track including the mouth (saliva), stomach, small intestine, and large intestine. Each stop along the digestive track has its own physiological environment and the enzymes in each environment must be able to perform certain functions. The enzyme, amylase, breaks down starches into simpler sugars. Another enzyme, lipase, breaks down fats into simpler molecules. Proteases break down proteins into simpler molecules called peptides. Enzymes make use of the chemical process of hydrolysis, in which a larger molecule is cleaved into two parts by the addition of a molecule of water. Thus, if a compound is represented by the formula AB in which A and B are atoms or molecules, and water is represented by the formula HOH, the hydrolysis reaction may be represented by the reversible chemical equation: AB + HOH ﾠ ↔ﾠﾠﾠﾠﾠﾠ AH + BOH As mentioned above, amylase breaks down starches into simpler sugars. Amylase catalyzes the breakdown of starch by cutting off the disaccharide maltose (two glucose molecules linked together). As the reaction progresses, the starch disappears and the sugar content (maltose) increases. Experiments In the experiments that follow, you will perform some tests to learn about enzyme activity. These experiments examine the effects that environmental pH and temperature have on the rate of amylase digestion of starch. This will be observed by using iodine to test for the presence of starch. Recall that iodine interacts with starch to form a dark blue-black color. As amylase breaks down starch, less and less starch is present, so the color of the iodine test solution becomes lighter and lighter until only the amber color of iodine ions will be seen. In order to stop the reactions at a specific time, we take advantage of the fact that enzyme activity is affected strongly by pH. When a strong acid (HCl) is added to an amylase solution, the pH falls below the functional pH of the enzyme and the reaction stops. In the case of amylase, the optimum pH is approximately 7 (neutral pH).
- 2. Open Endedone way to test the activity of amylase is to find the rate at which it breaks down a known quantity of starch. You may remember testing for the presence of starch with a solution of iodine (I2KI). Starch chains wrap around the tri-iodide ion in a coil. It is this shape, shown below, that produces the deep purple color.
- 3. Open Endedne way to test the activity of amylase is to find the rate at which it breaks down a known quantity of starch. You may remember testing for the presence of starch with a solution of iodine (I2KI). Starch chains wrap around the tri-iodide ion in a coil. It is this shape, shown below, that produces the deep purple color. iodine_in_starch If we add amylase to the solution, it breaks down the starch until there are no chains long enough to wrap around the tri-iodide, and the color of the solution returns to the yellow tint of plain iodine ions. 1. Exercise 3.1: Enzyme Activity vs Substrate Concentration a. purpose In this exercise, the student will investigate the effect of substrate concentration on the rate of the enzymatic reaction. How do you think an increased substrate concentration versus a decreased substrate concentration will affect the rate (speed) of the reaction? Why? b. procedure 1) Formulate a hypothesis about the effect of changing the substrate concentration on the speed of the enzymatic reaction. Record your hypothesis on Worksheet 2.1. 2) Using the "if-then" format, make a prediction for the proposed effect of substrate concentration versus the time required for substrate depletion. Record your prediction on Worksheet 2.1. 3) Before proceeding with the enzyme assay, perform the positive and negative controls as described below. a) Take three test tubes from the shelf and place them on the counter. b) Positive Control: To the first tube add 5ml of 2% starch solution and then 1ml of the iodine solution. Observe the color change and record it on the worksheet. c) Negative Control: To the second tube add 5ml of water and then 1ml of the iodine solution. Observe the color and record it on the worksheet. d) Negative Control: To the third tube add 5ml of the amylase solution followed by 1ml of iodine. Observe the resulting color and record it on the worksheet. e) When finished place all test tubes in the recycle bin 4) In order to perform this experiment, you will need several substrate concentrations. In order to produce the different concentrations, perform a 2-fold serial dilution of the substrate stock as described below. a) Place six (6) clean test tubes and an Erlenmeyer flask on the work bench. b) Add 99ml of water to the flask followed by 1ml of the 2% starch solution. This will give you a stock solution of 0.02%. c) To test tubes 2-5 add 2ml of water. d) To the first test tube at 4ml of the .02% stock starch solution. e) Transfer 2ml of the solution in tube 1 to tube 2 by dragging tube 1 over to tube 2. f) Now transfer 2ml of the solution in tube 2 to tube 3. Continuously transfer 2ml from one test tube to the next until you have deposited 2ml of solution in test tube 6. Place tube 6 in the recycle bin. You now have five test tubes that contain starch solutions of various percents (0.02%, 0.01%, 0.005%, 0.0025%, 0.00125%). 5) Add 1ml of iodine to each of the test tubes (Tubes 1-5). Note the color change. 6) Add 0.5ml of amylase to test tube 1. Be sure to note the virtual labs clock at the bottom of the screen. Record the time of adding amylase on the worksheet 7) Observe the reactions. What happens to the color as time passes? Continue to time the reaction until the solution is yellow/brown. This signifies that the reaction has completed since no starch is present. Record your observations and time of completion on the worksheet. 8) Repeat steps 6 and 7 to test tubes 2-5. 9) When finished, all test tubes in the recycle bin but save your stock starch solution. 2. Exercise 3.2: Enzyme Activity vs Enzyme Concentration a. purpose In this exercise, the student will investigate the effect of enzyme concentration on the rate of the enzymatic reaction. How do you think an increased enzyme concentration versus a decreased enzyme concentration will affect the rate (speed) of the reaction? Why? b. procedure 1) Formulate a hypothesis about the effect of changing the enzyme concentration on the speed of the enzymatic reaction. Record your hypothesis on Worksheet 2.1. 2) Using the "if-then" format, make a prediction for the proposed effect of enzyme concentration versus the time required for substrate depletion. Record your prediction on Worksheet 2.1. 3) Before proceeding with the enzyme assay, perform the positive and negative controls as described below. a) Take three test tubes from the shelf and place them on the counter. b) Positive Control: To the first tube add 5 ml of starch solution and then 1 ml of the iodine solution. Observe the color change and record it on the worksheet. c) Negative Control: To the second tube add 5 ml of water and then 1 ml of the iodine solution. Observe the color and record it on the worksheet. d) Negative Control: To the third tube add 5 ml of the amylase solution followed by 1 ml of iodine. Observe the resulting color and record it on the worksheet. e) When finished place all test tubes in the recycle bin 4) In order to perform this experiment, you will need several enzyme concentrations. In order to produce the different concentrations, perform a 2-fold serial dilution of the substrate stock as described below. a) Place six (6) clean test tubes on the work bench. b) To test tubes 2-5 add 2 ml of water. c) To the first test tube at 4 ml of the amylase solution. d) Transfer 2 ml of the solution in tube 1 to tube 2 by dragging tube 1 over to tube 2. e) Now transfer 2 ml of the solution in tube 2 to tube 3. Continuously transfer 2 ml from one test tube to the next until you have deposited 2 ml of solution in test tube 6. Place tube 6 in the recycle bin. You now have five test tubes that contain amylase solutions of various concentrations (100%, 50%, 25%, 12.5%, 6.25%). 5) Add 1 ml of iodine to each of the test tubes (Tubes 1-5). Note the color change. 6) Add 0.5 ml of starch solution to tube 1 and start the timer. 7) Observe the reactions. What happens to the color as time passes? Continue to time the reaction until the solution is yellow/brown. This signifies that the reaction has completed since no starch is present. Record your observations and time of completion on the worksheet. 8) Repeat steps 6 and 7 for test tubes 2-5. 9) When finished, place all test tubes in the recycle bin. 3. Exercise 3.3: Enzyme Activity vs Temperature a. purpose In this second exercise, the student will investigate the effect of reaction temperature on the rate of the enzymatic reaction. How do you think a decreased temperature versus an increased temperature will affect the rate (speed) of the reaction? Why? b. procedure 1) Formulate a hypothesis about the effect of changing the reaction temperature on the speed of the enzymatic reaction. Record your hypothesis on the worksheet. 2) Using the "if-then" format, make a prediction for the proposed effect of reaction temperature versus the time required for substrate depletion. Record your prediction on the worksheet. 3) Before beginning, place the following items on your work bench: a) One fresh Erlenmeyer flask b) Four (5) test tubes c) Four (4) constant temperature baths i) Set the one bath to "Ice" and the others to 21.5˚C, 37˚C, 50˚C, and 80˚C by clicking on the arrow button located on the right side of each bath. 4) Create a 0.005% stock starch solution by adding 50ml of water to the fresh Erlenmeyer flask followed by 25ml from the 0.02% solution. Once you have made the 0.005% starch solution, you may place the 0,02% solution in the recycle bin. 5) Place one test tube in each of the temperature baths. To each tube add 1ml of Amylase followed by 1ml of iodine. 6) To the test tube incubated in ice, add 4ml of the 0.005% starch solution. Be sure to note the virtual lab clock at the bottom of the screen. Record the time of adding starch on the worksheet. 7) Observe the reactions. What happens to the color as time passes? Continue to time the reaction until the solution is yellow/brown. This signifies that the reaction has completed since no starch is present. Record your observations and time of completion on worksheet 2.1. If the solution has not turned yellow after 5 minutes, you can assume the enzyme has been denatured. 8) Repeat steps 6 and 7 to the remaining test tubes (make sure you go one at a time). 9) When finished, place all materials in the recycle bin.
- Answer choicesTagsAnswer choicesTags