Abstract Over a two week period of time in the laboratory, we experimented and tested the reaction rate of a peroxidase enzyme and the factors that affected it, both positively and negatively. The purpose of these experiments was to probe and manipulate the activity of the enzyme peroxidase by varying temperature, pH, the amount of enzyme compared to the substrate and the effect of hydroxylamine. Peroxidase activity is expressed when the potato extract is subjected to stresses such as low temperature (El-hilali et al. , 2012).
The most eye catching factors that we tested for their impact on enzyme activity involved change in pH, temperature, boiling extract, and the effects of probing the active site with hydroxylamine. In the first part of the experiment we needed to determine what the level of concentration was most effective and how it best fit to determine the effects of different factors on its reaction rate. We accomplished this by standardizing the enzyme, proteins that act as a catalyst in reactions, with various amounts of Peroxidase (Table 1 Graph 1). We found that tubes 4 and 5 containing a total of 1. 0 mL of Peroxidase proved to be the most effective.
It was then found that the optimal temperature for peroxidase enzyme activity would be around 4°C and that the reaction rate would increase or decrease from the optimum temperature (Table 2, Graph 2). We also found that enzymes have an optimum pH level at which they react most efficiently. Based on our results we found that the optimal pH level of peroxidase enzymatic activity is pH 7 (Table 3 Graph 3). This made it reasonable for us to use a buffer with the pH of 5 to allow us to examine different factors that affected the enzymes activity without activity level reaching V max (How fast an enzyme is working).
Denaturation of an enzyme showed to have a negative effect on the reaction rate (Table 4). The final substance we introduced to the enzyme was hydroxylamine, an oxygenated form of ammonia, which prevented the protein from binding with the substrate at the active site (Graph 5). As previously mentioned the experiments we performed have given us a solid infrastructure to determine what elements had the most impact under different conditions. Introduction The fundamental biochemical reactions in living systems are catalyzed by enzymes. Enzymes are protein catalysts that facilitate the conversion of substrates into products. Alexander, 2011)
Without enzymes, reactions just wouldn’t occur fast enough to sustain life. An enzyme removes the energy requirement that in the physical world is required to produce reactions. For example, as a spark would ignite a gasoline vapor (by providing the activation energy), an enzyme powers a chemical reaction by lowering the required activation energy required to trigger such a reaction. This is a fine example of the structure/function correlation that we see frequently in the study of Biology. The shape of the enzyme determines what chemicals can attach to it, and therefore undergo a change in composition.
This site of attachment is called the active site of the enzyme. The molecule that binds to the active site and gets modified is called the substrate. The most common representation of the enzyme substrate reaction is called the enzyme-substrate complex, and is given the formula: E + S = PE, which is translated as: An enzyme combines with the substrate which forms the product through the stresses of covalent bonds in the substrate or their orientation causing a break while the enzyme remains in its original shape ready for its next interaction (Berenbaum. Life, Science of Biology 10th edition. 151-155) In this experiment we set out to determine the effects of several different factors such as temperature, pH, boiling an enzyme, and the effect of inhibitor treated enzymes on the rate of an enzymatic reaction. An enzyme is typically a protein with a specific three-dimensional shape. As previously mentioned above a small part of this shape forms the active site, where the enzyme combines with the substrate.
The substrate actually fits into the active site, which is why enzymes are specific to the reaction they catalyze. Campbell, N, 2008) Before performing the experiment we were instructed per lab manual to make hypotheses for every procedure. In regards to peroxidase, several points can be made about the overall effects of these factors. Factors such as temperature and pH have been shown to have an effect on the performance of enzymes (Vishwanatha, K. S. , Food Chemistry. 2: 402-407; 2009). Adjusting the temperature will produce one of two results; if the enzymatic reaction occurs and the enzyme is colder than the optimum temperature, fewer collisions between enzyme and substrate will occur thus reducing the rate of the reaction.
On the other hand, if the enzyme has been heated to a higher temperature than the optimum but not high enough to denature the enzyme, more of these collisions will occur and the reaction rate should speed up (Table 2, Graph 2). The second of the common factors is pH. Everything in the world has extremes on each end of the spectrum where the body, cell, etc. stops reacting to its environment. This goes for enzymes as well. If a solution is too basic or too acidic in comparison to the enzymes optimum pH, the enzyme will begin to degrade until it eventually stops reacting all together.
It is important that students and biologists understand these and other optimal conditions for enzymes to react in. For the pH aspect of the experiment the group predicted, through reading of the laboratory manual and general knowledge of enzyme properties, that the rate would vary based on both ends of the PH spectrum due to the complex nature of the positive and negative charges in the enzyme molecules. There was to be expected an optimal pH for the enzyme to function as it evolved for the specific function of catalyzing the reaction in the cell.
For the boiling experiment, one would assume due to the nature of protein denaturation that unless the enzyme normally worked under boiling temperatures, such as an advanced Archea, that the enzyme would be denatured and not express any enzyme activity. As for the inhibitor experiment, it was surmised that the inhibitor would perform its role and inhibit the action of the enzyme by competing with the substrate for the active site of the enzyme.
