When enzymes are in the tertiary structure, they have the capability of having a specific substrate enter the active site. Once the substrate enters the active site, it forms a short transient stage known as the enzyme substrate complex. This is where, if the substrate(s) fit, the enzyme will produce specific product through the reaction process of catalysis. The enzyme being studied, catechol oxidase, is a common and well known enzyme that helps in the process of bruising and browning in fruits and vegetables (Danyk, 2013/2014). It is known as catechol oxidase because the phenolic compound, catechol, is being oxidized into quinones, in this case, benzoquinone.
Enzymes activity is able to be sped up in many ways, including pH, increase in temperature, and the amount of substrate concentration. As the temperature increases, the rate of collisions between reactants also increases. This increase in rate affects the enzymes because the substrates move in and out of the enzyme faster, until the point at which a temperature will be too hot and it will rupture the bonds in potato juice being used. The acidity or alkalinity of a solution works in the same way, as high acidity or basicity will cause degeneration of the proteins as well. As for substrate available, there will come a time where the enzyme cannot attach and release the substrates any faster, resulting in a plateau in the benzoquinone concentrations. (Danyk, 2013/2014)
In our experiment we used potato juice extract to determine the influence of pH, temperature, and substrate concentration on the rate of enzyme activity. This being said, the increased speed of catechol oxidase should help produce higher concentrations of benzoquinone in the solutions.
Methods and Materials
The enzyme catechol oxidase was placed into a high speed blender to form a “juice”. All tests were watched for 5 minutes with gentle shaking at 1 minute intervals, and then placed into the 3°C ice baths and arranged from palest to darkest. Tubes were placed into 3°C ice baths in order to stop reactions.
For the substrate concentration with 5mL of pH7 buffer was added to each of the six tubes. Respectively, 1, 2, 4, 8, 16, and 24 drops of catechol were added to each of the tubes. To equal out the amount of solution in each of the test tubes, respectively 23, 22, 20, 16, 8 and 0 drops of pH 7 buffer were added. 30 drops of potato juice containing the catechol oxidase enzyme were then added to the solution, and then timed for 5 minutes at room temperature. The pH treatment only required 5 test tubes, with 3mL of matching buffer (pH4, pH6, pH7, pH8, and pH10) in the appropriate tube.
Following this, 10 drops of both potato juice and catechol are added to the tubes, and then timed for 5 minutes at room temperature. For temperature, 6 test tubes were required, and placed in their appropriate temperature treatments all with 3mL of pH7 buffer in them. These tubes were place in either 3°C, 12°C, 20°C, 35°C, 50°C, and 70°C baths for 15 minutes to warm each tube up to the appropriate temperature. After the 15 minutes, 10 drops of both catechol and potato juice are added to each of the pH7 solutions.
After arranging all the solutions from palest to darkest in their respective treatments, the spectrometer would be needed. Starting with the palest and moving to the darkest, all absorbance was measured at 460 nm. Once all these readings are found, divide by 0.0078 to calculate the benzoquinone concentration (µm). Mean and standard deviation were both calculated using Microsoft Excel 2007.
Based off the class date and the means calculated, it was found that all treatments had a point in which there would be no more benzoquinone released due to bonds breaking. The trend found when working with the amount of substrate availability showed a gradual plateau at concentration of about 85-90 µm (Figure 1). But based off the data collected, until this point, amount of substrate available did have an impact on how the concentration of benzoquinone.
After the results were calculated, there was a peak at which it had the highest concentration of benzoquinone, pH7. As the pH got lower, so did the benzoquinone concentrations. As well, when the pH got higher, more bonds would degenerate, causing the benzoquinone concentrations do be lower (Figure 2). The results were seen and could be predicted as to almost be a bell curve trend.
Temperature had almost the same effect as pH did. When the temperature reached a certain point, bonds started to be broken throughout the reaction. This caused almost a bell curve in the results. The point at which the highest benzoquinone concentration was found was at about 15 – 20°C (Figure 3). Even though the class data pointed out there was a higher concentration at about 70°C, in general there was a bell curve to the results.
Figure 1: Mean amount of benzoquinone concentrations from potato juice at different amount of substrate available including standard deviation with n=6. The bars above and below represent the standard deviation for the class. (n=6)
Figure 2: Mean amount of benzoquinone concentration from potato juice when working with different pH including standard deviation with n=5. The bars above and below represent the standard deviation for the class. (n=5)
Figure 3: The mean amount of benzoquinone concentrations from potato juice at different temperatures including standard deviation with n=6. Bars above and below represent the standard deviation for the class. (n=6) Discussion
The results seen during this experiment supported the hypothesis that temperature, pH, and the amount of substrate available all increase the rate of enzyme activity. This is proven by the results discovered in the reactions done. During the reactions, after putting them through the spectrophotometer, the results matched that all treatments would increase the rate at which catechol oxidase works. Both temperature and pH showed a peak before dropping again after a certain point, and the amount of substrate availability showed a gradual plateau.
As temperature increases, so does the frequency of the at which substrates collides with active sites. This is due to the increased rapid moving of the substrates. Once the temperature reaches a certain point, in this case about 15-20°C, the temperature actually starts to rupture the weaker bonds in the active site. This is why there is the drop in the benzoquinone concentration (Reece, 2011). However, in the graph, there was an unexpected result found at the reaction at 70°C (Figure 3). This result was not seen to be possible, but it was evident that at the 2 tables, due to the cloudyness of the reaction, more light was absorbed in the spectrophotometer.
Both the pH reaction and the amount of substrate available both agreed with the hypothesis given, and followed the trend lines expected. No unexpected results came from these 2 reactions. Both these reactions followed different trend lines, due to the effects of the treatments. PH was able to degenerate the ative sites, where as the amount of substrate just made it too much to actually coninue the reation any faster. Thats why there is a plateau when working with the amount of substrate. PH also works the same as temperature, where all enzymes work best at an optimal pH. Most enzymes work best at an optimal pH of 6-8. Beyond these optimal pHs, the active site usually degenerates, and in this labs case, they did degenerate, leaving a peak and drop in the reations.
To conclude, with the exception of the skewed result during the temperature readings, all three followed the general and predicted trend lines of the reaction. And with the reactions following the proper trend lines, it is evident that there is an understanding of how temperature, pH, and amount of substrate available affect the rate at which enzymes work Literature Cited
Danyk, Helena. 2013. The Cellular Basis Of Life Laboratory Manual. Department Of Biological Sciences. University Of Lethbridge. Reece, J.B., L.A. Urry, M.L Cain, S.A. Wasserman, P.V. Minorsky, and R.B. Jackson. 2011. Campbell Biology, Ninth Edition. Pearson Benjamin Cummings, San Fransisco, CA.