Determining the Properties of an Enzyme - Essay Example

Determining the Properties of an Enzyme (Peroxidase) Introduction Enzymes are an essential part of life. If these compounds werent present to contribute to the chemical reactions in living things by lowering the activation energy, life could not exist. Enzyme activity may be affected by many factors including concentration, temperature and pH. The enzyme examined in this study is turnip peroxidase TP 7 enzyme. It is composed of 296 amino acids, one hemin group and one neutral carbohydrate side chain attached through asparagine. The molecular weight of the polypeptide part is 31,060, and including hemin and carbohydrate the molecular weight of the native enzyme is close to 33,400 (Mazza and Welinder 1980). An enzyme is a catalyst for biological reactions, and it requires a specific structure to be active, therefore anything that affects the structure will influence the enzyme activity. The purpose of this study was to determine for turnip peroxidase: How the amount of enzyme influences the rate of a reaction. The hypothesis is that the greater the enzyme concentration the more activity there will be, because with many substances there is a link between the amount they are found in and their activity. How the temperature of the solution influences the enzyme activity. It is expected that the enzyme will be destroyed at high temperatures and will work slowly at low temperatures because the enzyme is found in a plant that lives approximately at 10C-30C. The influence of the pH on enzyme activity. Because the plant tissue that the enzyme is found in is not extremely acidic or basic, it is expected that extremes of pH will have a negative effect on enzyme activity. The influence of inhibitors on enzyme activity. It is assumed that inhibitors by definition slow or stop activity; addition of an inhibitor will negatively affect activity. Methods How the amount of enzyme influences the rate of a reaction Previously prepared enzyme extract at volumes of 0.5, 1.0 and 2.0 ml was added to tubes containing 1.0 ml guaicol dye (or 0 ml for control), 2.0 ml H2O2 (or 0 ml for control), and buffer (pH 5) at volumes of 3-5 ml to produce a total volume of 8.0 ml. Spectrophotometer readings at 500 nm were performed at 20 second intervals immediately after mixing the tubes for two minutes and the results were recorded. How the temperature of the solution influences the enzyme activity Mixtures of 4 ml buffer (pH 5) (or 0 ml for control), 2.0 ml H2O2 (or 0 ml for control), 1.0 ml of extract (or 0 ml for control) and 1.0 ml guaicol dye (or 0 ml for control) were combined at a total volume of 8 ml, incubated at 4C, 22C, 32C and 48C and 100C All the solutions were pre-incubated at the appropriate temperatures for 15 minutes to allow them to equilibrate before mixing. Spectrophotometer readings at 500 nm were performed at 20 second intervals immediately after mixing the tubes for two minutes and the results were recorded. The influence of the pH on enzyme activity To determine the effects of pH mixtures of 4 ml buffer at pH 3, 5, 7 or 9 (or 5 or 0 ml for control), 2.0 ml H2O2 (or 0 ml for control), 1.0 ml of extract (or 0 ml for control) and 1.0 ml guaicol dye (or 0 ml for control) were combined at a total volume of 8 ml. Spectrophotometer readings at 500 nm were performed at 20 second intervals immediately after mixing the tubes for two minutes and the results were recorded The effect of inhibitors on enzyme activity To see how inhibitors effect enzyme activity mixtures of 4 ml buffer (pH 5) (or 0 ml for control), 2.0 ml H2O2 (or 0 ml for control), 1.0 ml of either extract or extract treated with the inhibitor hydroxylamine and allowed to incubate for 10 minutes (or 0 ml for control) and 1.0 ml guaicol dye (or 0 ml for control) were combined at a total volume of 8 ml. Spectrophotometer readings at 500 nm were performed at 20 second intervals immediately after mixing the tubes for two minutes and the results were recorded Results How the amount of enzyme influences the rate of a reaction Figure 1 shows the results of different amounts of extract on enzyme activity. Figure 1 2.0 ml (the highest concentration) produced the highest absorbance at all time points. How the temperature of the solution influences the enzyme activity Figure 2 and Figure 3 show the results of the effects of temperature on enzyme activity. Figure 2 Figure 3 As can be seen the reaction rate is lowest at low (4C) while there is no activity at 100C. The highest activity is seen at 22C-48C The influence of the pH on enzyme activity Figure 4 and Figure 5 show the effects of pH on enzyme activity. Figure 4 Figure 5 As can be seen the rate of enzyme activity reaches its peak at between pH 5 – 7 The effect of an inhibitor on enzyme activity Figure 6 shows the effects of hydroxylamine extract on enzyme activity. Figure 6 The activity at all time periods was zero for the tubes containing hydroxylamine, while the tubes without the hydroxylamine showed a steady increase from 20 – 120 seconds. Discussion Increasing the amount of extract produced increased enzyme activity as we hypothesized. Enzymes are dependent on biding sites to bind the substrate (Bugg 2004). It might have been interesting to add increasing amounts of extract until we saw a leveling of activity. This would indicate that the available binding sites were filled. The optimum temperature was found to be between 22C-48C. This was the expected result because the enzyme must be most active in the temperature range that the plant that produces it in, is found in. We can assume that if the enzyme came from a plant that lived in the far North, it would probably be most active at a lower temperature. The fact that there is no activity at 100C is due to the fact that boiling destroys the structure of the enzyme (Berg et al 2002), thereby making it unable to function. A follow up experiment might be to see if freezing would also destroy the enzyme activity. Enzyme activity was found to be affected by pH. Between pH 5 and 7 were the optimum pH, while pH 3 and 9 had visibly negative effects on the enzyme activity. This finding is logical as these are generally the pH of living tissue which is where this enzyme is found. pH may change the bonds in the enzyme causing its structure to change (Berg et al 2002). As an additional experiment we could determine the pH of the plant tissue where the enzyme came from and then find the exact optimum pH in the laboratory and see if they are the same. It is not surprising that the inhibitor blocked the enzyme activity. Inhibitors block enzyme activity by binding to the enzyme (Berg et al 2002), so it may have been interesting to add the inhibitors at lower amounts to see if there is a point where there is not enough inhibitor to bind to all sites on the enzyme and there would therefore be only partial inhibition. Acknowledgements I wish to thank my lab partners Bora Lim, Yongwha Oh and James Tram for help in performing the experiments. References Berg J., Tymoczko J. and Stryer L. "Biochemistry". W. H. Freeman and Company. 2002 Bugg, Tim. "Introduction to Enzyme and Coenzyme Chemistry". Cambridge, MA: Blackwell Publishers. 2004 Mazza G and Welinder KG. 1980. Covalent structure of turnip peroxidase 7. Cyanogen bromide fragments, complete structure and comparison to horseradish peroxidase C. Eur. J. Biochem. 108:481-489. Read More