Introduction
Enzymes are proteins that act as catalysts which accelerate many biological chemical reactions (Funk and Wagnalls 2018), making them an important staple in linking metabolic networks (Chiu et al. 2006) as well as many biological processes. Thus, it is important to have a fundamental understanding of enzymes and their reactions with substrates, including measuring their activity based on the factors of inhibition and temperature. In this experiment, the enzyme catechol oxidase catalyzes the substrates of catechol and oxygen to produce a brown pigmented substance called benzoquinone. Since enzyme activity is a measure of how well a particular enzyme catalyzes its reaction (Shields 2016), the activity of catechol oxidase will be measured based on the extent of the color change that occurs when catechol oxidase catalyzes the reaction under normal conditions, with various inhibitors, as well as in differing temperatures in order to determine the effects these have on enzyme activity.
In order to determine the activity of the enzyme¬ catechol oxidase—based on both inhibition and temperature—an experiment was conducted in three parts, with the first determining the action of catechol oxidase. This initial component of the experiment was used to confirm the reaction that occurs when catechol oxidase is exposed to its substrate (catechol.) The second and third components used the fact that catechol oxidase reacts with its substrate to determine the extent of reaction based on the factors of inhibition and temperature. It is hypothesized that enzyme activity decreases with inhibition, but increases with temperature, and will result in the most enzyme activity occurring with no inhibitors present at an optimum temperature.
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Methods
In the initial stage of the experiment, three test tubes were gathered and labeled Tubes A, B, and C. The first of the three tubes (A) was filled with 15 drops of catechol oxidase and 15 drops of catechol solution, while the second tube (B) was filled with 15 drops of catechol oxidase and 15 drops of water. The final tube (C) was filled with 15 drops of catechol solution and 15 drops of water. After adding the reactants, each tube was observed, and their respective results were recorded.
Next, five tubes were gathered and labeled Tubes A, B, C, D, and E. Each of the tubes were filled with solutions added in a specific order of enzyme (potato extract) first, followed by the inhibitor (either Benzoic Acid or PTU), then by the distilled water, with the substrate (catechol) being added last. In the case that there was no solution in the order, the sequence was then continued with the next step. The first tube (A) was filled with 10 drops of potato extract, 20 drops of distilled water, and 10 drops of catechol. The second tube (B) was filled with 10 drops of potato extract, 10 drops of Benzoic Acid, 10 drops of distilled water, and 10 drops of catechol, while the third tube (C) was filled with 10 drops of potato extract, 10 drops of Benzoic Acid, and 20 drops of catechol. The fourth tube (D) was filled with 10 drops of potato extract, a few crystals of PTU, 20 drops of distilled water, and 10 drops of catechol, while the fifth, and final, tube (E) was filled with 10 drops of potato extract, a few crystals of PTU, 10 drops of distilled water, and 20 drops of catechol. After all the solutions were added in the correct order, each tube was observed, and their respective results were recorded.
Finally, four tubes were gathered and labeled Tubes A, B, C, and D. Each of the tubes were filled with 20 drops of catechol and incubated for five minutes at various temperatures: Tube A at 4 °C (ice), Tube B at 22 °C (room temperature), Tube C at 37 °C (incubator), and Tube D at 80 °C (water bath). After the five-minute incubation, the tubes were removed, and five drops of enzyme were added to each. After shaking each tube, they were returned to their respective temperatures for another ten minutes. After this time, they were removed and subsequently observed, and their results of extent of color formation were recorded.
Discussion
The initial segment of the experiment was done to confirm that the reaction that occurs when catechol oxidase is exposed to its substrate occurs due to those two factors and not to other solutions present. Tube A, which contained only catechol oxidase and catechol solution, was the only one to exhibit a color change. This proves that catechol oxidase reacts with the substrate catechol. Since no color change was observed when catechol oxidase was mixed with water, and no color change was observed when catechol was mixed with water, it can be concluded that catechol oxidase reacts with the substrate catechol. The second and third segments of the experiment used this fact of catechol oxidase and its substrate to determine the extent of their reaction based on inhibition and temperature.
The second segment examined the effects of inhibitors, specifically Benzoic acid and 1-phenyl-2-thiourea (PTU), on the reaction between catechol oxidase and catechol. It was hypothesized that, based on the structural formulas of catechol and the idea that the structural formula of many enzyme inhibitors share similar structures to substrates (Shields 2016), Benzoic acid, and PTU, that Benzoic acid would be a competitive inhibitor while PTU would act as a noncompetitive inhibitor. This hypothesis arose from the fact that competitive inhibitors are able to fit into the active site, preventing the substrate from interacting with the enzyme, as well as the fact that noncompetitive inhibitors denature the enzyme and change its shape, also preventing the substrate from interacting with the enzyme. These two facts were used to form the logical assumption that similarity between the structure of inhibitors and substrates relate to types of inhibition. Thus, it was hypothesized that, since the structural formula of Benzoic acid was more similar to substrate catechol than PTU, Benzoic acid would be the competitive inhibitor, while PTU would be the noncompetitive inhibitor. This hypothesis was supported by the experiment, since Benzoic acid resulted in a slight reaction compared to the no reaction observed when PTU was added. A control was also used to show that both are inhibitors, since the highest degree of reaction occurred when neither were present (a fact obtained from the initial segment of the experiment).
The third, and final, portion of the experiment examined the effect of temperature on enzyme activity, and the results appeared to follow a linear positive trend that suggested that the higher the temperature, the higher the extent of color change. The trend reached an inflection point after 37°C, and sloped downward to no reaction occurring at 80°C. Since the optimum temperatures for enzymes of many living organisms is 38°C, it was hypothesized that the highest extent of the reaction would occur at 37°C. This hypothesis was supported by the results, but it did not consider the idea that no reaction would occur at 80°C. Although it appears that increases in temperature accelerate the reaction, enzymes are unstable when heated (Funk and Wagnalls 2018) which appears to provide an explanation for the shift in slope after 37°C. Yet, other studies suggest that the idea of “optimum temperature” may be misleading in regard to characterizing enzymes (Almeida and Marana 2019).
Therefore, based on the data available from each section, it can be safely concluded that the most activity between enzyme catechol oxidase and substrate catechol occurs when there are no inhibitors present and at the optimum temperature of 37°C.
References
- Almeida, Vitor M., and Sandro R. Marana. 2019. “Optimum Temperature May Be a Misleading Parameter in Enzyme Characterization and Application.” PLoS ONE 14 (2): 1–8. doi:10.1371/journal.pone.0212977.
- Chiu, Shih-Hau, Chien-Chi Chen, Gwo-Fang Yuan, and Thy-Hou Lin. 2016 “Association Algorithm to Mine the Rules That Govern Enzyme Definition and to Classify Protein Sequences.” BMC BIOINFORMATICS 7. Accessed October 29, 2019. doi:10.1186/1471-2105-7-304.
- “Enzyme.” Funk & Wagnalls New World Encyclopedia, January 1, 2018, 1; http://search.ebscohost.com/login.aspx?direct=true&AuthType=shib&db=funk&AN=en047300&site=ehost-live&scope=site.
- Shields, George C., PhD. 2016. “Enzymes.” Salem Press Encyclopedia of Health. http://search.ebscohost.com/login.aspx?direct=true&AuthType=shib&db=ers&AN=87321995&site=eds-live&scope=site.
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