Study of Catecholase Activity

Enzymes are proteins that act as catalyst for cellular processes that would otherwise be too slow to maintain life. The enzymes speed up these chemical reactions by lowering the activation energy required for the reaction to take place. (Urry et al. 2017) Enzymes lower the activation energy by stabilizing the transition state of the reaction. The enzymes lower the activation energy this by forming weak bonds that will become stronger bonds, during the transition-state configuration. (The substrate binds to the enzyme at the active site which then forms an enzyme substrate complex. (Robinson 2006) The substrate will then break off, now changed into a product, while the enzyme is not changed nor consumed. There are processes that can affect either the structure or the active site of the enzyme. In the lab, the reaction activity of catecholase was tested as certain things about the environment was changed. According to the lab manual, changes in temperature, pH, and concentration will affect the rate of reaction. (Helms et al.1998) The first environmental change was a change in the temperature. Each enzyme has an optimal temperature range that will produce the highest amount of product. If the temperature is too low, then there will the molecules won’t be moving and therefore no reaction will take place. On the other hand, if the temperature is higher than its optimal range, the enzyme will denature due to the high temperature breaking the bonds. (Urry et al. 2017) The next environmental change is changing the pH of the reaction. Enzymes are known to have a small range of effectiveness. (Urry et al. 2017) Changing the pH to extremes will cause the enzyme to denature and therefore be inactive. The last environmental change is a change of the concentrations of the enzyme or substrate. Increasing the concentration of the enzyme will increase the speed of the reaction to a certain point. That point called the “point of saturation” is when all the enzymes are bound to a substrate and adding more enzymes won’t affect it. (Urry et al. 2017) Once it gets to this point the rate of the reaction crashes because there are no more substrates for the enzyme to bind to. Increasing the concentration of substrates will produce a similar situation but will there will be more products at the end since the enzymes will continue to react with every substrate until they are all products. Each one of these were changed to test and figure out how the enzyme activities affected. Hypotheses: Experiment one: Changing Temperature H0: Changing the temperature will not affect the reaction rate. HA: Changing the temperature will affect the reaction rate with the optimal range being 24oC. Experiment two: Changing pH H0: Changing the pH will not affect the reaction rate. HA: Changing the pH will affect the reaction rate with the optimal range being between pH 6-8. Experiment three: Changing Enzyme Concentration H0: Changing the enzyme concentration will not affect the reaction rate. HA: Changing the enzyme concentration will affect the reaction rate by increasing the reaction rate. Experiment four: Changing the Substrate Concentration H0: Changing the substrate concentration will not affect the reaction rate. HA: Changing the substrate concentration will affect the reaction rate by increasing the reaction rate as well as how much product is formed. Materials: For the first experiment: 7 test tubes, a hot water bath (60oC), an ice bath (2oC), catechol, potato juice, and a phosphate buffer (pH 7) are needed. For the second experiment: 5 test tubes, phosphate buffers (pH 4, 6, 7, 8, and 10), potato juice, catechol, and parafilm are needed. For the third experiment: 4 test tubes, potato juice, phosphate buffer (pH 7), parafilm, and catechol are needed. For experiment four, 8 test tubes, potato juice, phosphate buffer (pH 7), parafilm, and catechol are needed. Methods: Reaction: catechol + O2 --> benzoquinone In this lab, the reaction of catechol and catecholase is being tested. Potato juice is used since it is a natural source of catecholase. For the first experiment, 3 mL of phosphate buffer (pH 7) were added to three test tubes. One was labeled 2, while another was labeled 24, and the last was labeled 60. Test tube 2 was placed in ice bath (2oC), while test tube 60 was placed in a hot water bath (60oC). Test tube was left at room temperature (24oC). While these tubes were reaching the temperature desired, two more tubes were prepared with 3mL potato juice. One was added to the ice bath and the other was added to hot water bath. A third set of tubes were prepared with 3mL of catechol, one added to the hot water bath and the other added to the ice bath. After ten minutes, 10 drops of catechol (from a tube of the matching temperature) was added to each respective phosphate buffer tube. Likewise, 10 drops of the potato juice were added to each phosphate buffer tube of the matching temperature it was kept at. The change in color was recorded for the results. For the second experiment, five tubes were labeled with the following pH values: 4, 6, 7, 8, 10. 3mL of the matching phosphate buffer was added to the matching tube value. Shortly after, 10 drops of catechol were added to each of the tubes. Afterwards, 10 drops of potato juice were added, then the tubes were covered with parafilm and inverted. After the tube was inverted the parafilm was removed and the tubes sat there for five minutes then the change in color was recorded. For the third experiment, four test tubes were labeled as A, B, C, D. Each test tube had 3 mL of the phosphate buffer. Test tube A was prepared with an extra 20 drops of phosphate buffer and no drops of the potato juice. Test tube B was prepared with an extra 15 drops of phosphate buffer and 5 drops of the potato juice. Test tube C was prepared with an extra 10 drops of the phosphate buffer and 10 drops the potato juice. Test tube D was prepared with 0 extra drops of the phosphate buffer and 20 drops of the potato juice. Each tube then had 10 drops of catechol added to each of them, then covered with parafilm and inverted. The parafilm was removed and the tubes sat at room temperature for 4 minutes. The change in color was recorded for the results. For experiment four, eight tubes were labeled as 1, 2, 4, 8, 16, 24, 32, 48 and had 5mL of the phosphate buffer added to them. Test tube 1 was prepared with an extra 47 drops of the phosphate buffer and 1 drop of catechol. Test tube 2 was prepared with an extra 46 drops of the phosphate buffer and 2 drops of catechol. Test tube 4 was prepared with an extra 44 drops of the phosphate buffer and 4 drops of catechol. Test tube 8 was prepared with an extra 40 drops of the phosphate buffer and 8 drops of catechol. Test tube 16 was prepared with an extra 32 drops of the phosphate buffer and 16 drops of catechol. Test tube 24 was prepared with an extra 24 drops of the phosphate buffer and 24 drops of catechol. Test tube 32 was prepared with an extra 16 drops of the phosphate buffer and 32 drops of catechol. Test tube 48 was prepared with 0 extra drops of the phosphate buffer and 48 drops of catechol. Afterwards each tube was covered with parafilm and inverted. Shortly after 30 drops of diluted potato juice was added to each of the tubes, they were covered with parafilm and inverted. The parafilm was removed the tubes sat at room temperature for 5 minutes. The change in color was recorded for the results. Results: Table 1: Effect of Temperature Temperature Color intensity 2oC No color 24oC Darkish blue 60oC Clear Blue Table 1 shows, at specific temperatures, how intensely the color of the reaction is. At 2oC there was no color in the test tube. At 24oC there was a very vibrant color change, while at 60oC there was some color change, but not as much as at 24oC. Table 2: Effect of pH pH of buffer Color intensity pH 4 No Color pH 6 Light Blue pH 7 Dark Blue pH 8 pH 10 Almost Black No Color Table 2 shows, in specific pH, the color is more present. At pH 4 and 10 there is no reaction what so ever, while at pH 6 there is a small reaction. At pH 7 there is more color apparent, but pH 8 shows the most intense color change. Table 3: Effect of Enzyme Concentration Test tube Color Intensity A No Color B Light Blue C Dark Blue D Dark Blue Table 3 shows, when the enzyme concentration increases, the color is more noticeable. In test tube A there is no present color, while in test tube B there is some color. In test tube, C and D, the color is more intense than in the first two tubes. Table 4: Effect of Substrate Concentration Number of Drops of catechol Color Intensit y 1 2 4 9 16 24 32 48 No Color Light Blue Light Blue Dark Blue Almost Black Almost Black Almost Black Black Table 4 shows, when the substrate concentration is increased, the color increases in intensity. In test tube 1 there is no color present. In test tube 2 and 4 there is some color, but in test tube 9 the color gets more present. In test tube 16, 24, 32, and 48 the color is even more prevalent and keeps getting more intense as the numbers increase. Discussion: In the first experiment, the temperature of the substrate, enzyme and buffer were all placed in different temperatures, then the reactions were recorded. Table 1 shows that there was a correlation between temperature and the rate of reaction. For instance, there was no color at 2oC, suggesting that there was no reaction happening at that temperature. This is further supported by the lab manual, because it suggested that there was an optimal temperature for the reaction to occur. (Helms et al.1998) The reason there was no reaction occurring at that temperature is because the molecules would have slowed down as the temperature decreased to the point that there was no movement, which would produce no reaction. Looking at the higher temperature reaction, the reaction that took place at 60oC did have some color. While there was some color it was not as colorful as the room temperature reaction. The reasoning for this is that once the temperature gets above the desired range the extreme heat will denature the enzyme, therefore reducing how much enzyme is in the reaction.(Urry et al. 2017) The control group of this experiment was the 24oC, since it was at room temperature. This reaction was also the most colorful one, suggesting that room temperature was the most desired temperature for catecholase. Enzymes prefer an optimal pH as well as temperature, as shown in the next experiment. In experiment 2 the pH of the reaction was changed to test if the reaction would go forward or not. In table 2, it is shown that there is a relationship between the pH and reaction rate. When the pH was extremely acidic (pH 4) there was no color in the tube. Likewise, there was no color when the pH was extremely basic (pH 10), this is because at extreme pH the intra- and intermolecular bonds are broken, changing the shape and losing the effectiveness like the book stated. (Urry et al. 2017) While at a normal pH of 7 there was a good bit of color. When the pH is lowered even by small amount, in the experiment pH 6 there was little color suggesting that even a slight change in pH will greatly change the rate of reaction. But when the is slightly increased (pH 8) the color is much more prevalent than in the other reactions. This suggest that the preferred pH of catecholase would be between 6-8. In the third experiment the concentration of catecholase was changed to see if it would change the rate of the reaction. In tube A there were no catecholase added therefore there is no color in the tube, but in tube B, C, and D more enzyme was added. In tube B there was little color but in C and D there was equal but more color than in the previous tubes. The reason that B was so different than C and D, is because there was a low concentration of enzyme in the tube, so the substrate had to wait to bind to the enzyme. In tube C and D there was similar color but different amounts of enzyme. The reasoning for this that the reaction had reached a point where all the substrates were bound to the enzymes, but they produced the same number of products. The reason there wasn’t much difference in color in tube C and D is because there were no more substrates for the enzyme to bind to. Therefore, the rate of the reaction was dead, but all the available substrates were bound. In the fourth experiment the concentration of the substrate was changed. In tube 1 only one drop of catechol was added to the tube resulting in no color. In tube 2 and 4 a similar amount of color was produced in the tubes suggesting there was some reaction happening. Tube 9 had more color than any of the previous tubes, suggesting that more catechol added would produce a more intense color. This was further illustrated by the following tubes 16, 24, 32, and 48. Since more catechol was added to those tubes, increasing with each number, each tube had a more vibrant color than the previous tube. Conclusion Increasing the temperature of the reaction yields some reaction while decreasing the temperature of the reaction slows all reaction. Extreme pH levels inhibit the enzymatic reaction. Increasing the concentration of enzymes will increase the rate of reaction until it gets to a point where there is no more substrate to bind, therefore stopping the reaction. Increasing the concentration of substrates will increase the rate of reaction as well as number of products.