Concentration of substrate molecule
Concentration of enzyme molecule
Temperature
PH
The effect of enzyme concentration
- The proportion of enzyme to substrate molecules is important when considering the rate of an enzyme catalysed reaction. Enzymes work efficiently at very low concentrations, the number of substrate molecules that an enzyme can act upon in a given time is called its turnover number. Substrate molecules fit into the active site and if there are more substrate molecules than enzyme molecules, the number of active sites available will be a limiting factor. The optimum rate of reaction is achieved when all of the active sites are in use and the addition of more substrate would bear no effect to the rate of product formation. If there were fewer substrate molecules than enzyme molecules a reaction would subsequently take place quickly.
Temperature
- An increase in temperature effects the rate of a reaction in two different ways:
As the temperature increases the substrate and enzyme molecules gain more kinetic energy, so they move faster. As these molecules move faster they collide more frequently and therefore the rate of the reaction increases. Secondly, as the temperature increases the enzymes begin to vibrate more and the hydrogen bonds that hold the molecules in the precise shape break. As the shape has changed, the active sites no longer fit the substrate and consequently the enzyme loses all of its catalytic properties and is said to have become, denatured.
The effect of temperature on the rate of reaction is the combined influence of the two factors shown above.
- pH
The pH can have a direct response on the bonding of secondary and tertiary structures of enzymes. Each enzyme has an optimum Ph and many enzymes work best at neutral or even slightly alkaline conditions. Enzymes are made up of hydrogen bonds which, may be broken by the concentration of hydrogen ions present. Ph is a measure of hydrogen ion concentration and is measured on a scale of 1-14, with pH 7 being neutral, less than pH 7 acidic and more than pH 7 alkaline. Ph can effectively denature enzymes by breaking there hydrogen bonds.
- Substrate Concentration
At a low substrate concentration not all of the active sites are in use because there are not enough substrate molecules to use all of the active sites. If the concentration of the substrate were to be increased then more of the active sites would be filled and used. Once all of the active sites are in use an increase in the substrate concentration would not increase the rate of the reaction, as the amount of enzyme now plays a part as a limiting factor
Enzyme inhibition
Cells posses chemical regulators that reduce the speed or even slow down a reaction, these chemicals are known as inhibitors. There are two types of inhibitors: competitive and non-competitive. Competitive inhibitors are able to bind to the active site because they are of similar shape to the normal substrate. The reaction is reduced because the substrate cannot fit into the active site, as a result the substrate molecules compete for the active sites and the rate of the reaction slows down.
Non-competitive inhibitors bind instead, to another part of the enzyme. They have a different shape to the substrate and can bind with the active site and change its shape, so that the substrate can no longer use that active site. This therefore reduces the rate of the reaction.
Catalyse
The enzyme catalyses speed up the breakdown of hydrogen peroxide to water and oxygen. It is able to do so because the shape of its active site matches the shape of the hydrogen peroxide molecule. This type of reaction is called an anabolic reaction.
Catalyse is widely distributed in living tissue and is found in different foods for example potato. Catalyse causes the breakdown of toxic hydrogen peroxide, which is a by-product of some metabolic reactions by removing it from cells.
The rate of an enzyme catalysed reaction is the amount of substrate that has disappeared or the amount of product that has accumulated in a given time. So when using a catalysed reaction it is convenient to measure the rate of oxygen production.
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Plan
For my investigation I will be looking into the effect that the substrate concentration (hydrogen peroxide) has upon the enzyme (catalyse found in the potato) and the rate of reaction. In order for me to investigate this I will need to find out the maximum rate of reaction that can be reached when looking into the effect that the substrate concentration has on the rate of an enzyme controlled reaction. If I were to start to increase the substrate concentration the rate of the reaction would also increase as the enzyme molecules would still have active sites available to occupy the substrate. However, there would come a point when there are no longer any active sites available, it is at this point that the reaction has reached its maximum rate. For my experiment I need primarily to find out the maximum rate of reaction for my particular investigation which, is to use potato and hydrogen peroxide. To enable me to do this I will set up a preliminary experiment to find the maximum rate
For my investigation
VARAIBLE
- Concentration of the substrate (H202)
KEEP THE SAME
- The enzyme catalyse(potato)
- Mass of potato
- Amount of water in the box and measuring cylinder
- Same equipment
- Same temperature
Prediction
I predict that the hydrogen peroxide will break down to the products of oxygen and water in the presence of catalyse. I think that an increase in substrate concentration will speed up the reaction. This will occur up until the substrate as taken up all the remaining active sites. It is then that the reaction will stop increasing.
Equipment List
- hydrogen peroxide
- test tube rack
- plastic container
- a potato
- a cork borer
- water
- measuring cylinder
- clamp/clamp stand
- stopwatch
- 2x 10 mml syringe
- scales
- a ruler
- 5 boiling tubes
- bung/rubber tubing
- tile
- scalpel
- 3x beakers
- safety goggles
Method
To investigate my task I set up the following experimentation:
- Fill up a plastic container to half its volume with water (make sure that this volume of water remains constant throughout the investigation). Fill a measuring cylinder to the brim with water. Turn the cylinder upside down so that it stands upright inside the plastic container. Use the clamp and clamp stand to support the free standing measuring cylinder.
- Set up the boiling tube inside the test tube rack, with the bung and rubber tubing connected to the mouth of the boiling tube. Whilst one end of the rubber tubing is attached to the boiling tube, the other end should be joined to the inside of the measuring cylinder, within the plastic container.
- Using the cork borer make small tubes of potato a section the tubes with a scalpel. Measure the mass of the potato with the scales and ensure that this remains constant throughout the experiment. With the correctly measured out potato place it inside the boiling tube (this potato will act as the substrate).
- Set up the test tube rack with four separate test tubes. Use two beakers, one to contain the hydrogen peroxide and the second to hold the water. Use two separate syringes to extract the correct measurement of both the water and hydrogen peroxide for the four different experiments.
- Measure out the correct amounts of water and hydrogen peroxide and place the two measurements in a beaker. Remove the bung from the boiling tube and add the water and hydrogen peroxide (begin the stopwatch) and place the bung back again. Continue timing and take a reading every 20 seconds for 3 minutes. Record the results.
The above method refers to my preliminary experiment. Although I will use the same method for the main part of my research the preliminary experiment will be slightly different. For this I will only investigate one measurement. I have chosen to use 10 cm3 of hydrogen peroxide/0 cm3 of H20. I hope that this will be sufficient enough to determine the maximum rate of reaction.
Results for preliminary
These results allow me to determine how long I shall record my results for until I reach the maximum rate of reaction
See graph
- For my investigation I will develop this experiment by researching five different measurements of water and hydrogen peroxide. I will follow the same method as above. This are the measurements that I will be recording:
- (10 grams of H202 in 100 cm3 of H20)
Results
To enable my results to be as accurate as possible I repeated each experiment 3 times and took an average.
The results for the 0 % of hydrogen peroxide came out as zero as there was no reaction
See Graphs
Results/ observations
From my results table above you can clearly see that the greater the concentration of the hydrogen peroxide, the faster the rate of the reaction. In this experiment I measured the rate of oxygen production, as this is most convenient when using the enzyme catalyse. As the concentration of the hydrogen peroxide is increased the rate of the reaction increases as a proportional rate until the reaction reaches greater than 200 seconds, It is here that the rate begins to level off.
At 0 there was no reaction recorded. However, with all of the other measurements it was clear to see a reaction from the levels of oxygen being produced from within the boiling tubes.
Limitations
- I tried to keep the timing as accurate as possible by recording my results at 20 second intervals but there may have been slight miscalculations.
- Although I measured out the same mass of the potato discs (4.8g) each time the level of enzyme in each was unpredictable.
- The amounts of H202 and H20 were measured as accurately as possible, but I could only read off from the measuring cylinder to the nearest mm3. Again this was the same limitation ofr the level of oxygen being produced.
Hazards
- When conducting an experiment caution has to be taken. For this investigation I was using hydrogen peroxide which, is corrosive and is damaging when it comes into contact with skin. In order to ensure that I was safe I wore safety goggles throughout the experiment and I made sire that the hydrogen peroxide did not come into contact with my skin or the workbench that I was using.
- I was also cautious of those working around me and cleared a large space for me to work in.
- Hydrogen peroxide is colourless and could easily be mistaken for water. To ensure that all accidents are avoided I labelled the container holding the H202 and the H20 and made them clearly identifiable.
- I had to use a scalpel to cut up the potato and I was careful to replace the knife back in the box after I had used it.
Conclusion
From my experiment I can now conclude that the rate of a reaction steadily increases when more substrate is added. This occurs because an increased amount of the active sites within the enzyme are being used, and this allows more reactions to take place. Consequently, more oxygen is produced. There is a maximum level of reaction because there comes, a point when the level of substrate is so great that there are not enough active sites available for them.
Hypothesis – Hydrogen peroxide will breakdown to oxygen and water in the presence of Catalase. The reaction will increase with increasing enzyme concentration when molecules of hydrogen peroxide are freely available. However, when molecules of the substrate are in short supply, the increase in rate of reaction is limited and will have little effect.
Variables – In this investigation, the variables that affect the activity of the enzyme, Catalase, were considered and controlled so that they would not disrupt the success of the experiment.
i) Temperature –To control this variable, the temperature was maintained at a fairly constant level that allowed the enzyme to work effectively (room temperature, approximately 23ºC). This was achieved by using a test tube rack and tongs to handle the apparatus so that the heat from my hands did not affect the Catalase.
ii) pH –In this experiment, the pH was kept constant using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (potato tissue).
iii) Substrate Concentration To control the substrate concentration, identical quantities of the substrate were used for each reading. To ensure that this was measured precisely, 5ml syringes were used to accurately gauge to exact quantities.
iv) Inhibition – Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow the rate of reaction. They should not have affected this investigation, however, as none were added) Enzyme cofactors – cofactors are none protein substances which influence the functioning of enzymes. They include activators that are essential for the activation of some enzymes. Coenzymes also influence the functioning of enzymes although are not bonded to the enzyme. Unless enzyme cofactors were present in the potato tissue containing the Catalyse, they were not included in this investigation and therefore would not have affected the rate of reaction and the results of this experiment.
vi) Enzyme Concentration –I varied the enzyme concentration by altering the number of equal sized discs of potato that contain the Catalyse, in the reaction. The greater the number of discs, the greater the enzyme concentration.