Factors affecting the rate of an enzyme-catalysed reaction
These are four factors that can affect the rate of an enzyme reaction:-
- The pH
- The temperature
- The concentration of the enzyme solution
- The concentration of the substrate solution
The pH can affect the rate of an enzyme-catalysed reaction by affecting the bonds in the tertiary structure of the enzyme's active site. When bonds are broken, the shape of the active site is distorted, so the substrate molecule cannot bind to the enzyme causing no enzyme-substrate complex to be formed, therefore no products are made. The optimum pH for an enzyme is neutral or just slightly alkaline. If it is below or above the optimum pH, then the enzymes denature. Figure 5, below shows the effect of pH on the rate of an enzyme controlled reaction.
Figure 5: The effect of pH on the rate of
an enzyme controlled reaction
The temperature affects the rate of reaction also, an increase in temperature increases the amount of energy, and therefore more enzyme and substrate molecules will collide more often, increasing random movement. The more collisions, the higher the chance that a substrate molecule will fit into the active site. The optimum temperature (40°C to 50°C) is where the reaction is at its maximum temperature. If the temperature is above optimum, the rate of reaction starts to decrease as the active site starts to distort causing no enzyme-substrate complex and producing no products, as the substrate cannot bind. At a very high temperature the numbers of collisions are very high, but without the correct shape of the active site, no products can be formed. The enzyme can become denatured; which means that the enzyme's active site has become permanently damaged. Figure 6 below shows the effect of temperature on the rate of an enzyme-controlled reaction. As the temperature rises above its optimum, the rate of reaction falls rapidly as the enzymes denature.
Figure 6: The effect of temperature on
the rate of enzyme-controlled reaction
The concentration of the enzyme solution also affects the rate of an enzyme reaction. If there are more enzyme molecules than substrate molecules, there would not be enough substrate to continue forming products. When all the active sites are in use, the optimum rate will be achieved. The reaction will take place very quickly and then finish if no more substrate is added, because the substrate will run out.
Catalase
Catalase is a globular protein; it is present in the peroxisomes (micro body organelles that have various oxidation reactions in which toxic peroxides are generated as side products) of nearly all-aerobic cells. It catalyses the decomposition of hydrogen peroxide into water and oxygen. It also serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals an atom or a group of atoms with an unpaired electron. Radicals are usually reactive and are capable of causing a wide range of biological damage.
Aim
The aim of this experiment is to investigate how the concentration of the substrate hydrogen peroxide (H2O2) affects the rate of reaction of the enzyme catalase in yeast cells.
Prediction
I predict that the greater the concentration of hydrogen peroxide added on the faster the rate of reaction will be. This is because there are a lot of enzymes in the solution, and therefore a large number of active sites are available. Also, the number of collisions will increase, increasing the rate of reaction along with it.
The product produced from the formation of enzyme-substrate complex is oxygen. I therefore predict that the greater the volume of oxygen is, the faster the rate of reaction will be.
Preliminary work
To get an idea of what sort of method and apparatus should be used I consulted a number of biology books and found a very reliable method which measures the amount of oxygen produced while using a burette. I tried setting up the apparatus and had a go at the experiment by using water instead of hydrogen peroxide and water instead of hydrogen peroxide and yeast. Using this method would be very ideal for my experiment; this is because it is can be followed easily, it is reliable and it gives as accurate as possible.
Variables
The independent variable will be the substrate (hydrogen peroxide), as its volume will change throughout every test, because different concentration will be used.
I will use 10cm3 of hydrogen peroxide for every test, as I believe that it is the best measurement that could be used in this investigation. 5cm3 will not produce reliable results as it could be not good enough for producing enough amount of oxygen.
The dependent variable will be the total amount of oxygen produced, as it cannot be changed.
The concentrations to be used are: 5%, 7.5%, 10%, 12.5%, 15%, 17.5% and 20%.
The range will be 2.5 in between each concentration.
Each result will be repeated 3 times, this is to make sure that the results of each test is as accurate as possible and that the test was fair.
The variables which will have to be kept constant throughout the experiment are:
- Temperature
- pH
- The substrate concentration
- The volume of substrate added
The reason why I choose these variables to be controlled is because they are what will affect my experiment's results the most. In my method I will explain how I will be controlling them.
Fair testing
A good range (2.5) in between the hydrogen peroxide was used. The same volume of the yeast solutions will also be used. Equipments which need to be washed will be washed with distilled water between each test; this is to make sure that everything is decontaminated. The temperature (25°C) will be controlled throughout the experiment. Each experiment should be allowed to proceed its maximum time (5 minutes), and each concentration will be repeated three times (ensuring that valid and accurate data are collected).
Equipments
The equipments to be used during the experiment are:
Diagram of apparatus
The diagram below shows how the apparatus looks like when it is used during the experiment.
Safety
Coats and bags were put as far away as possible from where the experiment is taking place. Stools were pushed in, under the table. The experiment was carried out standing up to avoid any spills or danger.
Hydrogen peroxide can be very irritant to skin and eyes, therefore contact with eyes or skin should be avoided, if it does get in contact with the skin or eyes, then the skin should be washed as soon as possible. Any spills of hydrogen peroxide should be mopped as soon as possible.
Method
The temperature of the yeast cells will be set on room temperature, 25°C (a water bath will be used to keep it on that certain temperature). The volume of substrate added will be kept constant by using the same syringe throughout the experiment. The substrate concentration will be kept constant by using different concentrations for each test. All equipments will be washed with distilled water every time the experiments are repeated; so that the equipments are decontaminated.
The reason that these control variables will have to be controlled during the experiment is because it will give me accurate results and it will make each test fair.
To calculate the rate of reaction at given times, a tangent will be drawn on the graph on each of the concentrations, y over x (y/x), by doing this I will be able to draw up a graph to show the rate of reaction.
To calculate the mean (average result) all I have to do is add the results of the three repeats that I did and then divide the result by three. This is to be done for each three repeats and for each concentration. To draw a graph with the concentration of H2O2 against the total volume of O2 (cm3 collected), I will get the average for each concentration. For example for the 5% concentration, I will add up all the averages together and divide them by ten. I will do exactly the same for the rest of the concentrations. By doing this I will be able to plot a graph which will show the amount of O2 collected for each concentration.
The steps that I would have to follow throughout the experiment are:
- Wash all equipments with distilled water, so that everything is decontaminated.
- Care should be taken to avoid any gaps where oxygen could escape by making sure it is air tight.
-
Measure out 1 cm3 of hydrogen peroxide of the 5% concentration in a syringe and 10cm3 of the yeast solution in a measuring cylinder.
- Pour the yeast into the conical flask, place the bung and make sure that it is air tight, if it is not use Vaseline to make sure that it is.
- Connect the syringe to the conical flask.
- Gently push the syringe so that the contents are inserted into the conical flask, straight after you do that start the stop clock.
- Take readings for the first thirty seconds then for the next minute, then the next thirty seconds (a minute and thirty seconds) and so on.
- Record readings in a results table.
- Wash equipments after the first test has ended and then start with second test following the exact same rules.
- Each concentration is to be repeated three times (to get as accurate results as possible and to make it a fair test).
- Follow the same rules for each concentration.
The control for this reaction will be the enzyme catalase (which is in the yeast cells). Instead of using catalase I will use water. By taking the catalase away hydrogen peroxide will not be broken therefore it will not react with water.
Safety precautions have been taking by putting coats and bags away, and by wearing goggles to avoid any serious accidents. The apparatus was assembled correctly; minimum loss of gas was insured and readings were easily done. I have tried to control all the variables, but as the yeast was poured into measuring cylinder and into the conical flask, its temperature decreased therefore temperature was one variable which I was not able to control. The volume of hydrogen peroxide was controlled by trying to use the same gas syringe throughout the experiment. Hydrogen peroxide was pushed by the gas syringe into the conical flask, straight after doing that the stop clock was pressed to start, and readings were taking every thirty seconds.
Apparatus was cleaned with distilled water between each reading to make sure that everything was decontaminated. The same equipments were used for each reading. No anomalous results have been identified, but each test was carried out three times to make sure that results were as fair and as accurate as possible.
Results
The results table, on page 8, shows the results for each test, each result was recorded, and to get the mean I added the results for each test together and then divided it by three.
The formula for the mean is:
By getting the mean I was able to draw up a graph which shows the total of volume of oxygen produced for each concentration. The volume of oxygen kept on increasing when ever a higher concentration was used; we can see this by simply looking at the results of the 5% concentration of hydrogen peroxide and the results of the 20% concentration of hydrogen peroxide also. This increase is because particles are colliding successfully, making the rate of reaction goes successfully along with it. For the 5% concentration, the reaction stops at 3 minutes and thirty seconds producing 4.9cm3 of oxygen, and the 20%
started slowing down at 3 minutes and after, I gave 5 minutes for each experiment to take place, the oxygen collected for this concentration was 19.6 m3. This implies that the greater the substrate is added, the higher and faster the rate of reaction is.
A best fit curve was drawn on each result for each concentration. The reason that this was done was to draw out a tangent for the first thirty seconds, where the curve has touched the plots first, to find out the rate of reaction. The rate of reaction is calculated by adding the y- axis with the x-axis of the tangent. The total of my x-axis had to be timed by three, because each little square was 3 seconds. Below is the graph with the results.
The rate of reaction for each concentration was:
Conclusion
The results indicate that the higher the substrate concentration, the faster the rate of reaction,
This is because there is an increased chance of hydrogen peroxide molecule binding with a catalase molecule, producing an oxygen product. Hereby more enzyme-substrate complex can be formed. The increased rate of reaction will mean that more oxygen is formed. This increase in productivity of oxygen will form more oxygen filled bubbles. Each concentration rose on the first graph, but the second graph had three anomalous results.
Collision theory states that effective collisions between reactant molecules must occur in order for the reaction to occur. Particles must collide successfully in order for the rate of the reaction to increase. This proves that my prediction was right. The rate of 12.5% concentration was higher than that of 7.5 %. The same prediction went for all my results
for the first graph.
Evaluation
Possible errors that could have risen with my experiment is that the fact that the experiment was carried out over two lessons. We did not receive the actual set, but an identical one instead; therefore results could have been affected. The syringe in the second set did not insert all hydrogen peroxide into the conical flask, remains of hydrogen peroxide remained in the syringe thereby catalase did not break down as much hydrogen peroxide as it could have. This also means that not the same volume for each concentration was used throughout the experiment. The temperature of yeast also decreased during the reaction, it was a variable, which I could not control. The data obtained could have been affected by these possible errors.
If I were to do the experiment again, I would improve it by carrying out the experiment in one go and by using the same syringe throughout the experiment, and also by using a water bath (at 25°C) to put the conical flask in so that the temperature of the yeast cells and the hydrogen peroxide remains the same, in order that the reaction's results are not affected. I will also make sure that everything is airtight so that no oxygen bubbles are formed before the start of the reaction.
Bibliography
I have used the following books:
As Biology text book
A2 Biology text book
Revise Biology As with Letts
And these websites:
www.botany.uwc.ac.za
www.sonoma.edu