Title: AP Lab #2: Enzyme Catalysts

Introduction: Enzymes are catalytic proteins, meaning they speed up – but do not create – chemical

reactions, without being used up or altered permanently in the process. Although various enzymes use different methods, all accomplish catalysis by lowering the free energy of activation – activation energy – for the reaction, thus allowing it to occur more easily.

Enzymes employ a variety of methods for performing catalysis. Some provide a micro environment within the active site where some of the side chains are H+ or OH- donors or receivers. Other enzymes work by bringing together substrates that would not normally meet outside the enzyme, or orienting them in a manner in which they would otherwise not occur. Still other enzymes stress the bonds of substrate molecules in order to make them easier to break; some take this a step further by actually forming temporary covalent bonds with the substrate molecules. Regardless of how it is done, all enzyme catalyzed reactions are reversible and will turn around when necessary.

As a result of four levels of organization, an enzyme has a very specific shape, which is called its conformation. Even more specific is the active site of the enzyme, where the actual catalysis occurs. The specific molecule or closely related molecules on which an enzyme functions is known as its substrate. Shape plays such an important role in enzymatic catalysis, that often even isomers of the substrate will be rejected. Once the substrate enters the active site, it may begin a process known as induced fit in which the enzyme perfectly conforms to the molecule to allow for more efficient catalysis.

Enzymes have specific environmental conditions at which they will function best. As a result, changes in environment can severely impact enzyme catalysis in both negative and positive ways. Each enzyme has specific ranges at which it optimally functions; in general, increasing the temperature will help the reaction along, until the point at which the protein degrades and denatures – or falls apart into its lower level structures. Denatured proteins will often return to their original state, after the removal of the denaturing agent, except when they are degraded multiple levels. The rate of reaction through catalysis can also be increased by increasing the concentration of either the enzyme or the reactants; enzyme if all the active sites are full or the substrate if the active sites are not all full.

Hypothesis: I predict that the reaction will begin rather quickly with the addition of the enzyme, but will slow as the H2O2 is converted to H2O and O2. I also believe that, given the amount of time the reactions are to be run for, not all of the H2O2 will react, thus leaving some to react with the KMnO4 even after the full 360 seconds.

Materials: Beaker, H2O2, H2SO4, KMnO4, water (boiling and unboiled), potato or liver, paper, syringe or pipette, burette, mixing stick, and a stopwatch.

Procedure:1) Add 10 ml of H2O2 to each of the labeled beakers for the set time they will be measuring. 2) Add 1 ml of H20 to the first beaker.

3) Allow the reaction to occur for the set time limit labeled on the specified beaker.4) Once the time limit is fulfilled, stop the reaction by adding 10 ml of H2SO4 to the beaker. 5) Repeat this procedure for all beakers with their specified time limit and mix well. 6) Gather a 5 ml sample from on of the beakers and transfer it to a new glass beaker.7) Record initial burette reading containing KMnO4.8) Continue to add KMnO4 until a faint brown color can be seen. 9) Record final burette reading.10) Calculate the ml of KMnO4 used to reach the end of the titration. 11) Repeat this procedure for the rest of the other time-labeled beakers.

Data:

Exercise 2A:1. a) Potassium Iodide. 1. b) Hydrogen Peroxide1. c) Water and Oxygen1. d) The bubbles forming2. The temperature denatured the protein making the enzyme useless.3. The H2O2 reacts with the liver by fizzing up. If the liver were boiled, there would be minimal to no reactions occurring.

Exercise 2B:Base Line Calculation

Final Reading of Burette 1.2 mLInitial Reading of Burette 5.0 mLBase Line 3.8 mL

Exercise 2C:Uncatalyzed Hydrogen Peroxide DecompositionFinal Reading of Burette 3.2 mLInitial Reading of Burette 10.0 mLAmount of KMnO4 6.8 mL

Exercise 2D:

Table 2.1: KMnO4 (mL) Time (seconds)

10 s 30 s 60 s 90 s 120 s 180 sa) Base Line 4.3 ml 4.3 ml 4.3 ml 4.3 ml 4.3 ml 4.3 mlb) Final Reading 1.8 ml 1.8 ml 2.0 ml 2.0 ml 2.4 ml 3.0 mlc) Initial Reading 5.0 ml 5.0 ml 5.0 ml 5.0 ml 5.0 ml 5.0 mld) Amount of KMnO4 Consumed 3.2 ml 3.2 ml 3.0 ml 3.0 ml 2.6 ml 2.0 mle) Amount of H2O2 Used 1.1 ml 1.1 ml 1.3 ml 1.3 ml 1.7 ml 2.3 ml

Graph 2.1:

Analysis of Results:

1.

Time Intervals (seconds)0 to 10 10 to 30 30 to 60 60 to 90 90 to 120 120 to 180

Rates* 0.11 ml 0.00 ml 0.00667 ml 0.00 ml 0.01333 ml 0.01 ml

2. At the beginning. When the hydrogen peroxide and catalase are first mixed, the substrate concentration is high and the molecules meet active sites often and with consistency.

3. Near the end. As the hydrogen peroxide is decomposed into H2O and O2, there is less of the substrate to bind with the active site, thus slowing the reaction. Had there still been sufficient H2O2 for the active sites, the reaction would not have slowed.

4. Sulfuric acid acts to inhibit the function of catalase because it denatures the enzyme which removes its catalytic abilities. As we know, enzymes have very specific and their structures are specially developed (conformed) to fit the molecule with which it reacts (substrate). When the sulfuric acid is added, it changes the conformation of the protein enough that is can no longer catalyze the hydrogen peroxide – this process is called denaturing.

5. As with any reaction, the decomposition of hydrogen peroxide catalyzed by the catalase enzyme will slow down if the temperature was reduced. As the temperature decreases the average random kinetic energy of the molecules decreases and the hydrogen peroxide is less likely to bind with the active site of the enzyme and react. If the temperature gets low enough the reaction will appear to stop and at some point the solution will freeze. Eventually, the temperature may get so cold it will denature the enzyme, but that is not likely in a high school biology lab.

6. In order to test the effect of temperature on rate of reaction, one needs to set up a series of water baths at varying temperatures. For this hypothetical experiment, use 10°C, 20°C, 30°C, 37°C, 40°C, 50°, and 60°C; for purposes of this experiment, we will call 37°C the control because it is average body temperature and catalase naturally occurring in our bodies. The first step is to establish a baseline by mixing 10.0ml of H2O2 with 10.0ml of H2SO4 and 1.0ml H2O, swirl for five seconds, extract 5.0ml and assay with KMnO4. Second set up a water bath for each of the 7 temperatures and add 10.0ml of H2O2 to each of 7.50ml beakers; place one filled beaker in each water bath. Next measure out 7 syringes each with 1.0ml of catalase solution and 7 syringes of 10ml of sulfuric; make sure they are sealed (waterproof) and add one of these to each water bath. Allow all components 30 minutes to reach their appropriate temperatures before continuing. Remove the first (10°C) set from its water bath and place on an insulating surface such as a towel. Add the catalase from the same water bath and start the timer; swirl for five seconds and then allow to react for 55 additional seconds (total reaction time 60 seconds), after one minute, add the proper syringe of sulfuric acid and swirl for 5 seconds to completely denature the catalase. Repeat for the remaining six temperatures. Now, for each ending solution transfer 5.0ml to a separate container and use a burette to find out how much KMnO4 it takes to react with the H2O2 and subtract this from your baseline. This is how much H2O2 was used up. Record and analyze your data. The higher temperatures should catalyze more H2O2, and the lower temperatures less --- 37°C very may be the optimum temperature, as it is body temperature, or performance could continue to increase.

Conclusion: This lab was helpful in showing us how enzyme catalysis really happens in biology. I

believe we went wrong in establishing our baseline; we messed up quite a few times in everything. To get better results, we should have done this lab over again, but time did not allow us to do so. With that said, our results are not very correct for we did mess up and we also rushed at some parts where time was becoming a factor and we just had to get done, no matter how sloppy we did our work and procedure. We received corrupted results because we just rushed at the end and we had a huge rise in our graph where there should have been a slight rise if not a steady line.

My hypothesis was partially correct as the reaction most certainly did begin quickly and slow as the time moved on, but I was essentially incorrect in the idea that enough H2O2 would remain so that the KMnO4 would react.