Enzyme Catalysis - Morgan Park High School



Enzyme Catalysis With Applesauce and Pectinase (adapted by A. Lyman-Buttler from Dr. J. Smith, Ball State Univ.)Name:______________________________PointsProduce an outline of your practical biological investigation, including your purpose and hypothesis, and all information relevant to the study design.Identify your dependent and independent variables, their range, and how you will measure them.Identify experimental constants (controlled variables) and their significance.Demonstrate an ability to systematically record data.Follow lab safety rules.Experimentally determine the effects of pH and temperature on the rate of enzyme-catalyzed reactions.Describe the benefits of graphing data and present different types of data appropriately in both graphs.Evaluate any sources of error.Present the findings of your investigation in a well organized scientific report.Identify and explain the important features of a scientific report includingdiscussion of the biological concepts involved (background)materials and methodsdataconclusions based on analysis of the data (results & discussion)evaluation of the investigation (discussion)Peer review a lab report.BackgroundIn this lab the activity of the enzyme pectinase will be studied. Pectinase breaks down the carbohydrate pectin in applesauce. Pectin is a common component of fruits; it helps the cells of the fruit stick to each other. When it is broken down, the applesauce turns into apple juice. Purified pectinase is often used industrially in the manufacture of fruit juices. The effects of heat, acid, base, and enzyme concentration will be determined by measuring the amount of juice produced as a function of time.Enzymes are proteins that act as catalysts in living cells and carry out many of a cell’s metabolic processes. The chemical that the enzyme acts upon is the substrate. In a typical enzyme-catalyzed reaction, the enzyme will bind briefly with the substrate, allowing the substrate to form its products. After the products are formed, the enzyme molecule remains unchanged and continues to react with more substrate molecules.Because enzymes are proteins, they possess highly specific three-dimensional structures. As a result, most enzymes only react with a specific substrate, and catalyze a single reaction, a property called specificity. An enzyme molecule has a region called the active site which is the binding site for the substrate. This matching of active site to substrate is sometimes compared to a lock and key, where the keyhole is the active site, and the key is the substrate. Due to the sensitivity of many proteins to changes in their chemical and/or physical environment, the three-dimensional structure of enzymes is sometimes easily altered. When such a change occurs, we say the enzyme has been denatured. Since denatured enzymes cease to undergo their normal chemical activities, rates of reaction will be affected. Acids and bases (which change the pH) and excessive heat are common causes of enzyme denaturation.Another factor that can change the activity of an enzyme would be the presence of chemicals called inhibitors that interfere with the enzyme’s normal reactivity. Inhibition may be reversible or irreversible, depending on the nature of the inhibitor. If the inhibitor acts by binding to the enzyme’s active site, it is called competitive inhibition. Purpose: This lab activity actually involves four different experiments, with four different purpose questions:How does an acid affect the rate of pectinase activity?How does a base affect the rate of pectinase activity?How does high temperature affect the rate of pectinase activity?How does enzyme concentration affect the rate of pectinase activity?Hypothesis & PredictionYou should be able to predict how each of the treatments will affect enzyme activity. You must give SPECIFIC reasons for your predictions! (Hint: refer to Background & enzyme notes.) Write down your predictions below BEFORE beginning your experiment.If the enzyme is treated with acid, then the reaction rate will be [ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative control, because __________________________________________________________________________________________________________________________________________If the enzyme is treated with a base, the reaction rate will be [ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative control, because __________________________________________________________________________________________________________________________________________If the enzyme is heated to 60°C, then the reaction rate will be [ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative control, because __________________________________________________________________________________________________________________________________________If the enzyme concentration is reduced by half, then the reaction rate will be [ faster than / slower than / the same as ] the positive control, and [ faster than / slower than / the same as ] the negative control, because ____________________________________________________________________________________________________________________________Experimental DesignIn this experiment, we will be performing various treatments on the enzyme, and recording the volume of juice produced. Which one is the independent variable?_____________________ Which one is the dependent variable?_______________________List at least three experimental constants: __________________________________________________________________________ ___________________________________________________________________________________________________________What control(s) is (are) used? Be specific:_____________________________________________________________________________________________________________________________________________________________________________________Methods and MaterialsSafety. WEAR GOGGLES. H2SO4 and KOH are corrosive substances that will cause serious chemical burns; if you get them on yourself, put it immediately under running water, and tell Ms. Powell. If you spill chemicals in the lab, or break glassware, notify Ms. Powell (do NOT attempt to clean it up yourself until given instructions). Any student seen working without goggles, or performing any other unsafe act, (including eating and drinking) will be removed from the lab and reported to the Dean.Because different lab groups are using similar methods to collect the same kind of data, and are then sharing data, it is very important that everyone pay careful attention to detail—don't screw up the procedure!Which treatment will your group be doing? WRITE IT DOWN! ________ Take a beaker and mix your ingredients (see below) using a glass stirring rod for 60 seconds. When you combine the ingredients, add the applesauce last. The different treatments are:TreatmentName of treatmentIngredients1positive control1 container applesauce + 20 mL pectinase + 20 drops water2negative control1 container applesauce + 20 mL water + 20 drops water3heated enzyme1 container applesauce + 20 mL heated pectinase (60°C) + 20 drops water4enzyme w/ base1 container applesauce + 20 drops KOH (strong base) + 20 mL pectinase5enzyme w/ acid1 container applesauce + 20 drops H2SO4 (strong acid) + 20 mL pectinase6lower enzyme concentration1 container applesauce + 5 mL pectinase + 15 mL water + 20 drops waterUse a piece of pH paper to determine the pH of your mixture. Write down the pH of your solution here: _________Set up your collection apparatus according to Ms. Powell’s demonstration. (You should have taken notes on this!)Carefully pour the contents of the beaker into the funnel, being careful not to get any of it past the filter paper.Start your stopwatch (or start watching the clock) and make your first measurement (should be 0.0 mL)For all of the treatments, measure every 2 minutes for 30 minutes. Record in the table on the other side. Only record data for the treatment YOUR group is doing; you will get data for the other treatments from the other lab groups.ResultsRecord the volume of liquid produced at each of the times listed on the data table. You need to get some data from other groups, since your group does only one of the treatments. All students are expected to submit a report with a completed data table.Volume (mL)Time (min)0:002:004:006:008:001012141618202224262830123456Graph Using the data from your group AND from the other groups, draw one graph showing the results of all treatments. In your graph, plot volume against time and use a separate color or symbol for each line. Include a key. In your key, do NOT use the treatment number—use the treatment name! This is because somebody who reads your graph without reading the rest of your lab report should still be able to understand your results. Make sure you include all the components of a good graph (you should already know these) and pick the correct variable to plot on the x- and y-axis. Ms. Powell has graph paper that you may use—ask if you need it. A spreadsheet (like Excel) would be another good choice.DiscussionAnalysis QuestionsAnswer the following questions. Many of the questions are based on the information in the “Background” section—read it!Treatments 1 and 2 are referred to as positive and negative controls. What does this mean? What was their purpose?What are experimental constants? Why is it important to identify them?Why was 15 ml water added to treatment 6? Why were 20 drops water added to treatments 1, 2, 3, and 6?Predict what may have happened in a treatment that was kept much colder than room temperature, e.g., at 5°C.What is the enzyme in this lab? What is the substrate?Why did the applesauce turn into apple juice when it was combined with the enzyme? (Hint: read the “Overview” section.)Suppose you were to repeat this experiment, but this time you add an inhibitor. Predict what would happen & why.Scientists have just discovered a new enzyme in the fruit fly Drosophila melanogaster. The function of the enzyme is to break down fructose. What name would they probably give the enzyme and why? (Hint: look in your lecture notes.)Discussion ParagraphsType a full 2-paragraph conclusion and attach it to this packet. Remember, you must describe what is shown by your graph in paragraph 1, and refer to specific evidence (numbers) to support your claim. Write your own—no group papers! ................
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