NCSU BIO 183 - Exploring Properties of Enzymes – In Person Lab

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Exploring Properties of Enzymes In Person Lab LEARNING OBJECTIVES 1 Define the properties and role of enzymes in biological systems 2 Evaluate data and construct an argument for why the activity of the enzyme changes under multiple experimental conditions 3 Design an experiment to determine the effects of various experimental conditions on the enzyme catalase 4 Use a spectrophotometer to collect and evaluate results from the catalase experiment 5 Construct proper graphs using Excel 6 Practice scientific writing through reporting of experimental design and results INTRODUCTION Enzymes are biological catalysts that increase the rate of chemical reactions by lowering the activation energy needed for these reactions to proceed They help carry out thousands of chemical reactions at a rate of up to 1 000 000 times that of the same reaction without the enzyme present As such they are vital to the complex metabolic processes of living cells Figure 1 Activation energy for uncatalyzed and catalyzed reactions https openstax org books biology 2e pages 6 2 potential kinetic free and activation energy Enzymes are generally large globular proteins made up of several hundred amino acids They are often associated with a non protein prosthetic group that is important in the catalytic function of the enzyme Enzymes act on substances called substrates to produce one or more products A substrate could be a small molecule such as a sugar or lipid or a large macromolecule such as a protein or nucleic acid Substrates will bind to an active site forming an enzyme substrate complex held together by non covalent bonds such as hydrogen bonds hydrophobic interactions or ionic bonds If there is a prosthetic group present it will form part of the active site The substrate may undergo one or several changes which may involve covalent bonds before it is converted to the final product and released into solution Figure 2 Enzyme structure showing active site substrate and products https openstax org books biology 2e pages 6 5 enzymes The enzyme itself is then free to engage with another substrate In this way enzymes are recycled and may undergo thousands of chemical conversions at relatively low concentrations within cells If a substance binds to and blocks the active site it will interfere with the activity and efficiency of the enzyme These substances can bind irreversibly shutting down the enzyme permanently or reversibly meaning they will dissociate under the appropriate environmental conditions These substances are called inhibitors Enzymes often bind only to certain molecules or regions on larger molecules This property specificity allows the thousands of different enzymes to perform differing functional roles Sucrase for instance binds only to the disaccharide sucrose while another elastase binds only to regions on a protein where certain non polar amino acids are located The concentration of the substrate will have an effect on the activity of the enzyme too The structure of an enzyme can be affected by environmental conditions pH temperature and the concentration of salt and other small molecules can affect the activity of an enzyme either slowing or speeding its rate Physical alteration can also denature a protein meaning conditions have sufficiently altered the structure of the protein such that it can no longer function as an enzyme In lab today we will explore the properties of enzymes First we will use a simple qualitative test to explore a variety of environmental conditions on enzymes Then we will apply what we learn to run a quantitative test for enzyme activity and design experiments to further study these environmental effects ACTIVITY 1 A QUALITATIVE STUDY OF CATALASE ACTIVITY In this exercise we will explore the activity of the enzyme catalase which occurs naturally in the cells of many plants animals fungi and bacteria Catalase accelerates the breakdown of hydrogen peroxide H2O2 a common by product of oxidative metabolism into water and oxygen This catalase mediated reaction is extremely important to cells because it prevents the accumulation of hydrogen peroxide a strong oxidizing agent that can be harmful as concentrations build up Your instructor prepared an enzyme solution by cutting a potato into 8 10 pieces and putting them in a blender with 600 mL of cold dH2O The potato was blended for about 30 seconds and then filtered through four layers of cheesecloth The filtered solution was then placed on ice This solution contains many cellular extracts from the potato catalase is just one of the many enzymes in this solution The basic catalase reaction You can tell if this reaction is occurring if you can see 02 gas small bubbles rise and collect as foam at the top of the solution in the test tube You may need to wait a few minutes to see this and hold the test tube against a dark background PROCEDURE A TESTING THE ACTIVITY OF CATALASE Please highlight all your answers with a yellow background or use a different color font that is easy to read 1 Before you begin this experiment look at the components for each test tube in Table 2 below and fill in columns 1 and 2 2 Label your test tubes 1 4 Add the first component to each of the tubes 3 Add 5 0 mL of H2O2 to tube 1 and observe carefully for 5 minutes Record your results in column 3 in Table 2 below before moving onto tubes 2 3 and 4 Be sure to swirl your tubes to mix all the components together Record your results for each tube initially and check them frequently over the next 5 minutes Record any differences that you can see and briefly explain why you are seeing these results in column 3 Keep these tubes as reference while you move onto Procedure B Table 1 General catalase experimental tests performed by all groups of students Tube Contents 1mL H2O 5 0mL 3 H2O2 Column 2 Column 3 What do you think What happened Column 1 What is being tested here Control group will happen Produce a little bubbles x potato cube 5 0mL 3 H2O2 1mL Enz 5 0mL 3 H2O2 How starch enzyme affects reaction rate Enzymes from starch will speed up reaction producing many bubbles How the addition of enzymes affects rate of reaction Enzymes will speed up reaction producing many bubbles Why No change because no enzymes are present Many little bubbles produced because enzymes from starch speed up reaction producing O2 Many little bubbles are produced because the addition of enzymes sped up the reaction producing O2 1mL boiled Enz 5 0mL 3 H2O2 How the addition of heat affects

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NCSU BIO 183 - Exploring Properties of Enzymes – In Person Lab

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