Examining enzyme activity in differing environments

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Enzymes are protein molecules which make things happen to other molecules that would otherwise remain absolutely static. They make stable carbohydrates, fats, and proteins susceptible to digestion products as building blocks to make new cells(Komberg). A protein is classified as an enzyme if it is know to catalyze a reaction. Some enzymes are purely proteins. In other cases consist of two parts: the protein portion and a cofactor. The cofactor may be an ion of a metal or an organic molecule (Campbell 2008).

Some enzymes control single chemical reactions and sometimes they bind with similar molecules and perform a small group of associated reactions. The substance in which the enzyme works is called a substrate. Only part of the substrate binds to the enzyme and that area is called the Active site. Enzymes, as protein are large molecules of a chain of amino acids that have at least tertiary structure, and in some cases quaternary structures (Marieb 2009). In order to speed up those chemical reactions an enzyme requires certain amount of energy. This energy is called activation energy. Activation energy is the amount of energy needed to start a reaction. Therefore, enzymes lower the activation energy so the reaction can occur quicker, but can not force chemical reactions to occur between molecules (Rittner).

Enzymes as proteins depend on its specific three dimensional structures, and its intermolecular bond. However, hydrogen bonds are easily broken by many environmental factors such as excessive pH, and temperature levels, causing proteins to unfold and lose their specific structure. In this case the enzyme is said to be denatured (Gale). Temperature and pH are environmental factors essential in the action of an enzyme. Most enzymes are optimally active at very low hydrogen ion concentration (pH ranges 5 -9). However, changes in optimal pH will impede proper folding, and disrupt the 3D shape by interfering in the intermolecular structure of enzymes (Kornberg). In the other hand, most human enzymes have optimal temperatures at 35° - 40° C (Campbell). Every single enzyme has an optimal temperature at which its reaction rate is greatest. Enzymes are not affected in their structural level in cold environments. Nevertheless, the reaction rate increases with higher temperatures until it reaches its optimal temperature; however, above optimal temperature the speed of enzymatic reaction decreases. (Campbell). The increase of kinetic energy on the enzyme disorders the week bonds, that stabilize the active site, and the protein eventually denatures.

The effects of enzyme and substrate concentration are strictly related to the rate of reaction, but they do not affect the three dimensional structure of the enzyme. (Campbell). The more substrate molecules offered, the more reactions occur in the active site. However, there is a limit to how fast the reaction can occur in relation to substrate and fixed concentration of enzymes. The enzyme is said to be saturated when the rate of reaction is determined by the speed at which the active site convert the substrate to product( Cambell) On the other hand, if substrate levels remain constant and enzyme levels increase, the enzymatic rate of reaction increase. However, enzyme activity will reduce as one enzyme reacts with one substrate, and consequently it will not find substrate to react with an outnumber amount of enzymes (Campbell).

Catalase is a protein that catalyzes the disproportional of dihydrogen peroxide to O2 and water.. It is found in nearly all microorganisms exposed to oxygen and resides in the peroxisomes of mostly all aerobic cells. Catalase optimal pH for human is approximately 7, and optimal temperature ranges between body temperature (Worthington Biochemical corporation). My prediction for the catalase experiment is to work better in neutral ph environments, and close to body temperature. It is predicted that enzyme reaction rate will increase as more enzyme as are add to the experiment. In the same manner, the reaction rate will increase as more substrate is add to the experiment until it reaches saturation point.

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. They are widely distributed throughout the animal and plant kingdoms and are found in many cellular organelles, in cytoplasm, and its membrane components (Taylor). In humans aminopeptidase is produced by glands of the small intestine I predict aminopeptidase pH optimal condition will be around 7 because it resides in the small intestine( Kornberg). In regard, to the aminopeptidase optimal thermal conditions my prediction is in a range close to body temperature. Abandoning body temperature, it will cause these enzymes to denature. I expect that the rate of reaction increase as more enzyme and substrate are release in the reaction, until it reaches saturation.

Methods

         

In this experiment, it was examined the characteristics, structure and effects of pH, temperature, enzyme and substrate concentrations on the enzyme catalase. During the experiment, we tested the efficiency of the enzyme catalase by altering the temperature, pH, substrate concentration, and enzyme concentration. The enzyme catalase was extracted from a yeast concentration, and we measured the production of oxygen gas for each trial. The measure of oxygen was accomplished using the Logger pro computer program, and the Vernier Gas Pressure Sensor.

            The first part of the experiment was to set up the Vernier LabPro, the Gas pressure Sensor and the LoggerPro Software to correctly measure the amount of oxygen gas discharged for every trial and their relation with temperature. This process is described as this: we connected the Gas pressure sensor into channel one of the computer interface. We prepared the computer for data collection by opening the file "06B Enzyme" from the Biology with computer folder in Logger Pro. Once the system was connected and the parameters were set up, we were ready to start the temperature experiment. In the experiment, we added three mL of 3% hydrogen peroxide and three mL of water to a 50 mL beaker and drew the solution into a syringe. The syringe was placed in ice for three minutes. After the time passed we removed the syringe from ice and record the temperature using a thermometer. Next, we filled an Ehrlenmeyer flask half full with ice water; we then placed three drops of the enzyme solution into a test tube and sealed it with the rubber stopper. After the c period, we connected the syringe to the gas pressure apparatus. We opened the valve on the side of the syringe and injected the six mL solution into the test tube. Quickly we closed the valve and collected the data on the computer software. The data collection ended after three minutes. We kept an eye on the reaction ensuring not to exceed 130kPa of pressure. After this procedure, we removed the syringe and robber stopper assembly. In the Logger Pro software, we selected experiment and then Store Latest Run. We saved our data by double clicking on the column. The same procedure was repeated to achieve the effects of the enzyme at room temperature and using water baths set at 30°C, 40°C, 50°C, and 60°C. For every trial the syringe was placed in H20 baths or kept at room temperature. Therefore,we repeated the same procedure describe above. At the end of this experiment, we found the rates of enzyme activity for each temperature trial. These rates were calculated as the slopes of the curves generated during the experiment. In order to measure the slope, we moved the mouse pointer directly to the point where the line started to increase. We pointed this section and dragged the mouse pointer to the point where the line does not look linear. From there, we clicked in the linear fit button to calculate a linear regression. A box appeared with a formula for best- fit line for every single trial. We recorded the equation of the line, the slope, and the regression coefficient in Table 3 of our catalase experiment handout.

            The second part of our experiment consisted to test the effects of different pH environments to the catalase .In the same way, we used the Vernier LabPro, the Gas pressure Sensor and the LoggerPro Software previously assembled. We added 3mL of a pH3 solution and 3mL of 3% hydrogen peroxide to a 50 mL beaker. Next we drew the 6 mL solution into a gas syringe. At the same time, we placed three drops of the enzyme solution into a test tube; we made sure to place the enzyme solution at the bottom of the test tube to avoid erroneous collection of data. We secured the stopper to the test tube creating a tight seal; we made sure that the stopper valve is in the closed position before injecting the solution. Next, we injected the peroxide solution to the test tube; immediately, we closed the valve and clicked the collect button in the computer. The trial lasted three minutes; we captured our data and removed the syringe from the apparatus. We followed the same procedure using pH 5, 7, 9 and 11 solutions. At the end of this experiment, we found the rates of enzyme activity for each pH trial in the same way we measured the equation of the line, the slope, and the regression coefficient in the above described. Finally, all the data was recorded in Table 4 of our catalase experiment handout.

Third, we proceed to test the effects of enzyme concentration for catalase. In the same way than prior experiments, we assembled the Vernier gas pressure sensor. Once the apparatus was installed, we added 3 mL of H20 and 3mL of 3% H2O2 to a small beaker. After that, we placed one drop of the enzyme solution into the bottom of a test tube. We tightly inserted the stopper into the test tube, and then drew up the 6ml substrate solution into the syringe. We assembled the syringe to the rubber stopper and proceed to inject the solution into the test tube. Rapidly, we closed the valve and clicked the collect button to begin data collection. This trial lasted around three minutes, when the data collection finished; we removed the stopper and discarded the substance of the test tube in the sink. In the computer software, we saved our data by selecting the experiment, and then the store latest run buttons. In the same manner, we repeated the same process using 2, 3, 4 , and five drops of the enzyme solution . At the end, we found the reaction rate for each quantity of enzyme solution by calculating the slope and linear regression in the above described. We just finished recording our data in Table 1 of our enzyme experiment handout.

Last, we were ready to test the effect of substrate concentration for catalase, we assembled the Vernier gas pressure sensor in the same way as described in experiments above. As the apparatus was installed, we added 1 mL of H20 and 5mL of 3% H2O2 to a small beaker. Later than that, we placed three drops of the enzyme solution into the bottom of a test tube. We tightly inserted the stopper into the test tube, and then drew up the 6ml substrate solution into the syringe. We assembled the syringe to the rubber stopper and proceed to inject the solution into the test tube. Rapidly, we closed the valve and clicked the collect button to begin data collection. When the data collection finished; we removed the stopper and discarded the contents of the test tube in the sink. In order to save our data, we selected experiment , and then the store latest run options in the logger pro software. Right after this, we repeated the same procedures above described using 2ml of H20 and 4mL of 3% H2O2. Followed by 3ml of H20 and 3mL of 3% H2O2, then by 4ml of H20 and 2mL of 3% H2O2, and finally by 5ml of H20 and 1mL of 3% H2O2. At the end, we found the reaction rate for each quantity of substrate solution by calculating the slope and linear regression in the above described. We just finished recording our data in Table 2 of our enzyme experiment handout.

Result

Figure 1. Relationship between rate of reaction and temperature for the enzyme Aminopeptidase. Note that the data was collected from a simulated experiment using the computer software Enzyme Incestigation. In order to prepare this simulation pH, substrate and enzyme concentration remained constant.

Figure 2. Relationship between rate of reaction and temperature for the enzyme catalase, remind that the data was collected from an actual experiment using the computer software Logger Pro. In this experiment, pH substrate and enzyme concentration remained constant.

Figure 3. Relationship between the rate of reaction and potential hydrogen for the enzyme Aminopeptidase. The data was collected from a simulated experiment using the computer software Enzyme Investigation from EME Corporation. In this experiment the temperature, substrate, and enzyme concentration remain constant.

Figure 4. Relationship between the rate of reaction ,and potential hydrogen for the enzyme catalase. In this actual experiment, it is observed the reaction of the enzyme in different pH environments while temperature ,substrate and enzyme concentration remained constant . Data collected from an actual experiment using the computer software Logger pro.

Figure 5. Relationship between the rate of reaction and substrate concentration levels for the enzyme aminopeptidase. In this simulated experiment temperature, pH and enzyme concentration remained constant. Note that the data was collected using the computer software Enzyme investigation.

Figure 6. Relationship between the enzymatic rate of reaction and the substrate concentration levels for the enzyme catalase. In this actual experiment, data was collected using the computer software Logger Pro. Note that pH, temperature and enzyme concentration remained constant.

Figure 7. Result of the enzymatic reaction of the enzyme catalase in regard its enzyme concentration. In this actual experiment, the pH, temperature and substrate concentration remained constant. Note that the data was collected using the computer software Logger Pro.

Discussion

In this study, it is examined the characteristics, structure and effects of pH, temperature, enzyme and substrate concentration on two enzymes catalase and aminopeptidase. Figure 1 and 2 describe as temperature rises, reacting molecules have more and more kinetic energy. This increases the chances of a successful collision and so the rate increases (Hoehn 2009). There is an optimal temperature at which an enzyme is at its greatest production. In figure 2, I observe catalase greatest rate of reaction at 31° C. However, In figure 1, the enzyme aminopeptidase greatest rate of reaction occur at 39°C. Above this termperatures the enzyme structure begin to break down ( denature) since at higher temperatures intermolecular shape are alter as the enzyme molecules gain even more kinetic energy ( Campbell 2009). Consequently, the rate of reaction start to decline as it seems in the simulated experiment of Aminopeptidase. The result of catalase was surprising because the trend does not follow my stated predictions. The rate of reaction started increasing up reaching its optimal temperature at 30° C. It is expected that as the temperatures leave optimal conditions the rate of reaction decrease. Then the rate of reaction increases at temperatures that are not optimal for the enzyme catalase. This trend reveals an error at 50 ° C caused by inappropriate acclimation the enzyme to the temperature.

An optimal range of pH exists for every enzyme. The optimal condition for catalase in humans is approximately seven (Worthington Enzyme Manual). Additionally, the optimal pH condition for aminopteptidase ranges between the pH of 8 or 9. (Jencks)Abandoning these levels cause the enzyme to denature decreasing its catalyzing abilities (Jencks). The result I obtained supports my predictions, aminopeptidase finds its optimal pH concentration between 7.5 and 8(see figure 3). On the other hand, catalase finds its optimal pH value at 7 ( see figure 4). A potential error is seen (see Figure 4) in catalase data. According to the graph catalase finds its optimal pH around 7, and start declining when abandons optimal condition as it gets more basic. However, when the reaction reaches pH 9 suddenly increase the reaction rate. This mistake might occur because we use a different pH solution ,and that might alter our reaction.

The effects of enzyme and substrate concentration are strictly related to the rate of reaction, but they do not affect the three dimensional structure of the enzyme. (Campbell).The results for substrate concentration prove my predictions, as more substrate molecules are offer, the more reaction in the active site ;therefore, an increase in the rate of enzymatic reaction. My study reveals that catalase and aminopeptidase increase the reaction rate as more substrate is offer to convert into product (see Figures 5 and 6). However, the reaction never reaches saturation point. Saturation point is the point where the reaction is limit by certain amount of substrates working on the enzymes (Campbell). It never reaches saturation because the amount of substrate is very low (see figure 6). My enzyme concentration behaves in the same way, as substrate levels remain constant, and more enzyme molecules are added to the reaction, the reaction occurs quickly (see Figure 7). Therefore, the reaction rate increases.

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