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Jessica Lee AP Biology Mrs. Kingston 23 October 2013
Abstract:
The purpose of this lab is to investigate the impact of temperature, substrate concentration, enzyme concentration, and the presence of an inhibitor on the effectiveness and rate of an enzyme. If the concentration of the enzyme or substrate is increased, the rate of the reaction will be increased as well. If the temperature of the enzymes surrounding environment is increased, then the rate of the reaction will also be increased. If the substrate concentration is increased then the rate of the reaction will increase. If an inhibitor is added then the rate of the reaction will be decreased significantly. The dependent variable is the rate of the reaction. The independent variable is the concentration of enzyme, the concentration of the substrate, the temperature, and the addition of an inhibitor.
Introduction:
Enzymes are proteins that carry out chemical reactions. They are catalysts within living organisms and regulate the rate at which a chemical reaction is carried out (Koshland). Enzymes range in their functions throughout living organisms; however metabolic enzymes are found in all cells of the body (Boncompagni). Enzymes are essential to the breakdown of lipids, carbohydrates, proteins and other molecules in the cell (Koshland). There are three types of enzymes that effect the location and function of the enzyme. These three enzymes are metabolic enzymes, digestive enzymes, and food enzymes found in uncooked nuts, vegetables, and fruits (Boncompagni). Enzymes specifically act as a catalyst, which is a chemical agent that is used to speed up the reaction without being used up
(Campbell and Recce, page 152). There are hundreds of different enzymes and the function of an enzyme is based upon the amino acids that make up the protein. Thus enzymes present in all cells play a major role in metabolism because without these enzymes reaction would take far too long to be carried out. Enzymes have this ability to speed up reactions by lowering the activation energy, the energy needed to break the bonds of the reactants (Campbell and Reece, page 152). For each reaction the reactants must absorb energy from the surrounding to be able to break bonds. Bonds are recreated as energy is given off to the environment. When the molecules reach the peak of the activation energy, they are very unstable and in the transition state. When taking in energy, this normally means that the reaction is doing so by taking in heat energy and giving off heat energy as bonds are formed again. This means that as bonds are being broken the reaction is endothermic, meaning that it is taking in heat energy from the surroundings, and exothermic as the bonds form, meaning that it is giving heat energy off to the surroundings (Campbell and Reece, page 152). Exergonic is synonymous to the term exothermic in this situation. However, in many situations there is not enough energy in the cell to overcome this energy barrier, and if the temperature was to be increased either the proteins would be denatured, or all the reactions would take place in the cell. Enzymes can thus speed up these reactions and allow for them to take place (Campbell and Reece, page 153).
Enzymes are also very specific to the chemical process and will only match with certain reactions. These reactions will take place in the active site of the enzyme. The substrate, the reactants of the reaction, bind to the active site, the only place on the enzyme a substrate can bind, creating an enzyme-substrate complex. The reactants are then converted to the products like in a normal reaction, but with the enzyme, the activation energy is much lower and the rate of the reaction is much faster. The shape of the enzymes, and active site, are a result of the amino acid sequence. As the substrate enters the active site the chemical groups and the R groups of the enzyme interact and the enzyme changes shape slightly to fit around the substrate even tighter. This is called induced fit
35-40 °C. Cells also have optimal pH values from about 6-8. Some enzymes, like those in your stomach, function best at a much lower pH (Campbell and Reece, page 155). Along with these factors, there are nonproteins that help the enzyme carry out this catalysis. These adjuncts are called cofactors. When these cofactors are organic molecules they are referred to as coenzymes and vitamins are a great example of this. While these factors most likely will enhance the performance of the enzyme there are also enzyme inhibitors that inhibit the function of the enzymes. There are two types of inhibitors, competitive inhibitors and noncompetitive inhibitors. Competitive inhibitors will attach to the enzyme in the active site, which blocks the substrate from attaching to the enzyme. Noncompetitive inhibitors do not bind to the active site of the enzyme, but rather bind to another site of the enzyme, thus changing the shape of the enzyme and making the active site less effective in carrying out the reaction (Campbell and Reece, page 156). These inhibitors are either reversible or irreversible. If the enzyme bonds to the enzyme through covalent bonds, it is usually irreversible. The use of inhibitors can also be done intentionally by the cell to regulate enzyme activity. When molecules bond to the enzyme and change the shape of the active site this is normally allosteric regulation. This can result in either inhibition or stimulation of an enzyme. This is seen through allosteric regulation. These enzymes are normally made of two or more subunits with their own active sites. There are both active and inactive forms of allosteric enzymes. If the inhibitor binds to an activator the shape is stabilized. If the inhibitor joins to an inhibitor, it stabilizes the inactive form as well (Campbell and Reece, page 157). In another situation, the substrate molecule that binds to the active site can stimulate catalytic powers of a multisubunit enzyme (Campbell and Reece, page 158). When these inhibitors bind to the allosteric site, they are changing the shape of the active site and not allowing the substrate to bind to the enzyme in many situations (Kornberg). This can result in feedback inhibition, which occurs once so much of a certain product is produced. This product will then switch off a metabolic pathway by this end product binding to the enzyme that is used to receive the reactants previously in the reaction. This will bind to the allsoteric
site on the enzyme. This can be essential to cells because it stops the production of excess products that just require extra expended energy to be made.
In all enzymes are extremely important to the reactions that take place within our bodies and cells. Specific to this lab, catalase was the enzyme used and hydrogen peroxide was the substrate used. Hydroxylamine was the inhibitor used. Catalase comes in many forms. It can protect our red blood cells or also found in bacteria (Goodsell). Catalase is most important because of its function and ability to break down millions of hydrogen peroxide molecules. It has four subunits, each with its own active site (Goodsell). Hydrogen peroxide decomposes into oxygen and water in the presence of heat or other substances. It is a colorless liquid that is commonly used for bleaching cotton and can be corrosive to the skin if the concentration exceeds eight (Curley, Robert). Hydroxylamine is an inorganic compound that is hygroscopic and acts as an inhibitor by binding to the enzyme, thus slowing down the rate of the reaction significantly ("Hydroxylamine.").
Experimental Design:
This lab consisted of four different experiments that were carried out to determine the effects of substrate concentration, enzyme concentration, and inhibitor, and temperature on enzyme activity. For each experiment catalase from the potatoes is needed. This required the potatoes to be blended with cold water and ice and then stored in an ice bath so that the enzyme is not denatured. For all experiments, 1% hydrogen peroxide was needed, but there is only 6% hydrogen peroxide that was readily available. In this case the hydrogen peroxide must be diluted. To do so a ration was needed between the hydrogen peroxide and water. Ten mL of hydrogen peroxide must be mixed with 50 mL of water to make a 1% dilution of hydrogen peroxide. This is used for all four trials.
In the enzyme (catalase) concentration lab collect eight beakers to prepare with eight different concentrations of catalase (40 mL, 32mL, 30 mL, 24 mL, 20 mL, 10 mL, 4 mL, and 0mL).Add distilled
This data table can be found on page seven and the graph can be found on page nine. The graph demonstrates the relationship between the rate of the reaction and the enzyme concentration and there is a direct relationship between rate and enzyme concentration. In the concentration of substrate lab, as the concentration of the hydrogen peroxide increased, so did the rate of the reaction. The data table that demonstrates this conclusion can be found on page eight and the corresponding graph can be found on page nine. The graph shows the direct relationship between substrate concentration and the rate of the reaction. In the in temperature portion of the experiment it was determined that the rate relatively increased as the temperature increased until the enzyme was denatured. This data table can be found on page eight and the graph that represents this data in found on page ten. The graph shows the direct relationship between temperature and rate of the reaction. The data for the inhibition lab can be found on page eight and there is no graph. The data clearly presents the effect of an inhibitor and how it significantly slowed the rate of the reaction. All data tables and graphs are found on pages seven through ten and allow for further conclusions to be drawn. Data:
Table 1
Enzyme Concentration^ Effect of Enzyme Concentration of Rate of Activity
(units/mL)
Trial 1 (Seconds)
Trial 2 (Seconds)
Average (Seconds)
Rate (1/seconds) 100 2.5 3 2.75. 80 1 3 2. 75 3 4 3.5. 60 4.5 4.7 4.6. 50 4 6 5. 25 6 8 7. 10 6 7 6.5. 0 52 72 52.
Table 2
Effect of Substrate Concentration on Enzyme Activity
H 2 O 2 %
(Substrate)
Group 1 Time (s)
Group 2 Time (s)
Group 3 Time (s)
Average Time (s)
Rate (s-^1 ) 0 N/A N/A N/A N/A N/A 0.1 16.295 20.45 38.9 25.215 0. 0.2 18.19 12.85 15.45 15.663 0. 0.3 10.31 9.45 13.4 11.053 0. 0.5 7.97 8.05 9.2 8.407 0. 0.8 6.455 6.85 7.9 7.068 0. 1.0 5.5 3.85 6.55 5.3 0. 2.0 3.02 3.8 5.3 4.04 0. 3.0 1.96 1.65 2.9 2.17 0. Table 3
Effect of an Inhibitor on Enzyme Activity
Concentration^ Enzyme (units/mL)
Time to float disk (seconds) (1/seconds) Trial 1 Trial 2 Average Rate Control 2.5 2.3 2.4 0. Hydroxylamine 73.8 66 69.9 0.
Table 4
Effect of Temperature on Rate of Enzyme Activity
Time to Float Disc (in seconds) Temperature (Degrees C) Trial 1 Trial 2 Average Rate (1/seconds) 5 9 8 8.5 0. 10 19 11 15 0. 22 11 6 8.5 0. 40 7 4 5.5 0.
70 n/a n/a n/a n/a
Discussion:
Once all this data was calculated and, investigated, and analyzed, it was evident the objectives and purposes of the lab were attained. This objective was to determine the effects of substrate concentration, enzyme concentration, temperature, and an inhibitor on the rate of a reaction. The final conclusions made were that as enzyme concentration, substrate concentration, and temperature were increased, the rate of the reaction also increased. When the inhibitor was added the rate of the reaction decreased significantly. All four of these parts of the experiment used the reaction between catalase and hydrogen peroxide. This reaction can be modeled as
2H 2 O 2 + catalase 2H 2 O + oxygen + catalase
Catalase clearly accelerates the process of the breakdown of hydrogen peroxide into water and oxygen. As discussed in the introduction, the enzyme catalase also remains after the reaction occurs because the
0
0 10 20 30 40 50 60 70 80
Rate of reaction (s
-^1 )
Temperature (°C)
Effect of Temperature on Rate of Reaction
Rate
reactants do not make covalent bonds with the enzyme at the active site. This reaction is so important to the cell because it makes sure that there is not a buildup of excess hydrogen peroxide that can disrupt the function of cells. For each of the four sections of the lab this reaction must be taken into consideration in order to fully understand why the results were the way they were.
The first independent variable tested was the concentration of the enzyme. Eight beakers were used to hold different concentrations of enzymes that the filter paper was put into. Then the filter paper was put into the substrate solution. The substrate in this case was the 1.0% hydrogen peroxide. The disc rose much quicker as the concentration of the enzyme increased, thus the rate of the reaction was much quicker. This can be concluded because as the reaction takes place oxygen is produced as a product. The filter paper absorbed the oxygen and rose once this reaction took place. As a result of the higher concentration of enzymes, the substrates had more possible active sites to bind to, thus more reactions were able to take place at once. As the concentration increased there were more and more opportunities for the hydrogen peroxide to bind the active sites of the catalase and react with a lower activation energy. In the second section of the lab the concentration of the substrate was varied while the concentration of the enzyme was kept constant. Nine beakers were prepared with differing concentrations of hydrogen peroxide as explained in the experimental design. The filter paper was placed in the enzyme solution and then placed into the substrate solutions and timed for how long it took for the filter paper discs to rise to the surface. As the concentration of the substrate was increased, the rate of the reaction also increased for reasons similar to the concentration of the enzyme. As the concentration of substrate increased there were more substrate molecules to bond with the active site of the catalase enzyme. Because there was this increase in the substrate concentration each time, there was a greater likelihood that the substrate would bind with the active site and carry out the reaction. The third section of the lab required five beakers that varied in temperature of the water bath that the reaction took place in. The enzyme and substrate concentration were all kept constant and the filter
to be produced and make the disc rise. This corresponds directly with the how inhibitors work as explained earlier in the lab.
When looking at the graphs and data on pages seven through ten it was evident that there was room for error in many respects. First off the, the most apparent error seemed to be in the temperature lab. As the temperature was increased the rate of the reaction should have directly increased as well. This was not completely apparent, because as the temper rose from five degrees Celsius to ten degrees Celsius the rate of the reaction decreased from 0.118 (1/s) to 0.066 (1/s). This could be a result of many errors or the fact that potatoes do not normally grow in high temperatures. This is an apparent discrepancy because it would be expected for the rate to increase as the temperature increased. The second place for errors could be basic human errors, while diluting the hydrogen peroxide from 6% to 1%. However, all the results followed a trend that was expected and agreed with the hypothesis that was made. Due to this fact, none of the errors could have been that significant to completely skew the meaning and significance of the analysis of the data. From this we can conclude that the results are relatively valid.
Conclusion:
Overall this lab was relatively successful in determining the effects of enzyme concentration, substrate concentration, temperature, and an inhibitor on the rate of the reaction. The objective of the lab was met, which was the rate of the reaction for all four sections of the lab. In doing so the time for the disc to rise to the surface was measured and then calculated into a rate. All this data that was collected was used to agree with not only the objectives of the experiment but also the hypotheses made on each individual lab. The results were relatively valid and showed a correct trend in data. To make these results even more valid, it would be necessary to complete the experiment multiple more
times because some sections did not have multiple trial to verify the results completely. All in all the results were reliable and showed the basic properties of enzymes and their role in chemical reactions.
