Enzyme concentration – at low enzyme concentration there is great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. Eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor.
Substrate concentration – at a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases. Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed.
COFACTERS
Most enzymes require additional help from cofactors, of which there are 2 main types-
Coenzymes – these are organic compounds, often containing a vitamin molecule as part of their structure. Coenzymes are not permanently bound to the enzyme but may be temporarily and loosely bound for the duration of the reaction and then move away once it is completed. For example NAD, which transfers hydrogen away from one molecule in a dehydrogenase reaction and takes it to another molecule.
Metal ions – most speed up the formation of the enzyme-substrate complex by altering the charge in the active site e.g. amylase requires chloride ions, catalase requires iron ions.
Inhibitors-
Inhibitors slow down the rate of a reaction. Sometimes this is a necessary way of making sure that the reaction does not proceed too fast, at other times, it is undesirable.
Competitive reversible inhibitors – these molecules have a similar structure to the actual substrate and so will bind temporarily with the active site. The rate of reaction will be closer to the maximum when there is more ‘real’ substrate, (e.g. arabinose competes with glucose for the active sites on glucose oxidase enzyme).
Non-competitive reversible inhibitors – these molecules are not necessarily anything like the substrate in shape. They bind with the enzyme, but not at the active site. This binding does change the shape of the enzyme though, so the reaction rate decreases.
Irreversible Inhibitors- These molecules bind permanently with the enzyme molecule and so effectively reduce the enzyme concentration, thus limiting the rate of reaction, for example, cyanide irreversibly inhibits the enzyme cytochrome oxidase found in the electron transport chain used in respiration.
PREDICTION
I predict that as I increase the concentration of the substrate (hydrogen peroxide), the rate of reaction will increase at a directly proportionally rate, until the solutions become saturated with the hydrogen peroxide. At this point adding more substrate will have no effect on the rate of reaction. The rate increases with a higher substrate concentration because more of the enzymes active sites are being used and occupied by the substrate, this will produce a greater amount of oxygen gas released. Once the amount of substrates exceeds the number of active sites the
rate at which the oxygen is produced will not change as the substrates will be queuing up to get into an active site.
From this a can predict that when I double the concentration of the hydrogen peroxide the amount of oxygen released will roughly double. This is because there will roughly be double the number of substrate molecules reacting with the catalase active sites.
I also think that at the higher substrate concentrations the amount of bubbles produced will be more than at the lower concentrations. This is due to the increased rate at which the substrate and enzyme react.
The theoretical maximum rate of reaction is when all the sites are being used but in reality this theoretical maximum is never reached due to the fact that not all active sites are being used at the same time. The substrate molecules need time to join onto the enzyme and to leave it so the maximum rate achieved is always slightly below the theoretical maximum. The time taken to fit into and leave the active site is the limiting factor in the rate of reaction.
APPARATUS
Burette- measures the amount of oxygen gas given off during the reaction.
Catalase
Stop watch- used to record the duration of the experiment.
Measuring cylinder
Hydrogen peroxide- (different concentrations)
Test tubes and rack
Large beaker
Extra large beaker
Metal stand and clamp
Rubber bung and connecting tube
METHOD
I will first get all the equipment. Then I will get a large beaker and fill it catalase, I do not need to measure the amount but I will not need more than 30cm3. I will now fill the extra large beaker with water. then I will fill the burette will water and place it upside down in the extra large beaker, I will do this careful so I do not loose too much water. I can now clamp the burette tightly so it is carefully balanced. It does not matter if the water level is not exactly on 0cm3, as long as I record the level of water correctly before the start of the experiment. I will now use a syringe to measure 5cm3 of catalase into the test tube. I will then collect all the different concentrations of hydrogen peroxide (2.0, 1.5, 1.0, 0.5, 01 molar). I will then get a clean syringe and measure out 5cm3 of the 2.0 molar hydrogen peroxide and keep it ready in the syringe. I will then get the stop watch ready. I can now quickly transfer the hydrogen peroxide into the test tube containing the catalase and quickly place the rubber bung onto the test tube. This bung has a tube connected to it which is placed directly under the burette in the water. I will now start the stop watch as quickly as possible. I will then record the amount of gas given off by seeing how much the water level dropped. I will record the burette level after 3 minutes. From this I will be able to calculate the amount of oxygen given off. I will now do the same thing for all the other concentrations.
FAIR TEST
To make this experiment fair I will take many precautions-
-I will try to keep the surrounding temperature the same. For example I will keep all the solutions out of the sunlight. I will also try and avoid as much contact with the test tubes and all the solutions as my body temperature could effect the solution temperature This is because the change in temperature can effect the rate of reaction. For example an increase in temp will cause the rate of reaction to increase, or a decrease will slow the reaction down, it has a negative relationship.
-I will use 2 separate syringes, one each for the catalase and the hydrogen peroxide. This is because they will be contaminated. Also after doing each experiment because we will using different concentrations of hydrogen peroxide
-As a precaution, I will limit my contact with the boiling tubes, as my body heat will raise the temperature, increasing the rate of reaction.
-I will monitor the temperature using a thermometer to ensure that it remains constant and not disrupt the results of the experiment by affecting the activity of the Catalase.
-I will look at all the measuring equipment at eye level and make sure the meniscus is level. This is very important as an incorrect volume can effect the final results dramatically.
-I will also be careful when handling the hydrogen peroxide as it is corrosive.
-After each experiment I will carefully note down the finish volume so that I can carry on the next experiment without filling the pipette again.
EVALUATION
I believe that the experiment was satisfactory in finding out how substrate concentration affects the rate of the reaction. However because of the fact that I had an anomalous result means that there can be many improvements, with the experiment and the equipment. I believe the reason the results weren’t accurate as possible are-
First of all I only did the experiment for each concentration once. For this reason we don’t have any average results which means we don’t know whether each experiment was accurate. When placing the hydrogen peroxide into the test tube, and then closing the bung and starting the stopwatch, there was a slight delay, which meant that there was some certain gas lose. However this transaction was the same for each experiment therefore my overall results were not affected. During the experiment the temperature of the chemicals changed. On the day of the experiment it was sunny and therefore the temperature of the catalase and the hydrogen peroxide increased during the experiment. This increase meant the rate of the reaction increased as the molecules have more kinetic energy, therefore the rate at which the hydrogen peroxide fits into the active site of the catalase increases. This is what I think the main reason for the anomalous was, because I did the 0.5 molar test near the end of the experiments the temperature of the solutions increased and therefore the reaction was quicker than it should have been. To stop any gas from leaking through the rubber bung I used plasticine, however I did not believe that it was working as best as it could have, and I felt that some gas was escaping. I think something like a cork would have been more suitable.
There can be many ways to improve the experiment so that these anomalies do not appear. These improvements can be from the procedure or the equipment used. First of all I should have done the experiment more than once. Because I only did the experiment once I do not have an average set of results for this reason I had to get an average form the whole of the classes results so that I could compare my results with the others. This way I can see whether my results were accurate. I also think that we should of used more concentrations of hydrogen peroxide. I only used 5 concentrations. I think that could of used 8 instead. This way I get a better idea of the results, and its more accurate as there is a smaller range. Also I could have made the solutions myself. All the different concentrations were made for us, however if we made the solutions our self it be more accurate. The equipment we used could have also been improved. Firstly all the equipment has a percentage error. The burette has a % error of 0.5 cm3. The syringe had an error of 1.0 cm3. These errors have large effects on the final volume of gas given off. To improve I could of used equipment with smaller % errors. For example I could of used a micropipette, which is very accurate at measuring small volumes to measure out the catalase and the hydrogen peroxide. I could have also used a machine, which feeds the correct amount of hydrogen peroxide with the catalse, and then use an electronic oxygen monitor to measure the volume of gas given off. This would rule out any human errors. Also when handling the test tubes containing the solutions, I should of wore gloves this way I would not be transferring any heat from my body to the solutions. I could of also used a ph buffer to maintain the ph of the solutions, this way there would be no change in the ph which could of affected the rate of reaction. I could have also drawn a trend line which would help me analyse al the problems with my predictions. Using this trend line I could have also found the r-squared value which would have showed me how closely the estimated values for the trend line correspond to the actual data. The trend line is most accurate when is r-squared value is at or near 1.
Overall I am pleased with my results however I would have liked more time do it at least two more times.
CONCLUSION
From my results you can see that my prediction was correct, as the concentration of the hydrogen peroxide increases so does the rate of the reaction. This is because as the concentration increases the amount of substrate molecules increases, therefore there will be more substrates to fit inside the catalase active sites thereby increasing the rate of reaction which is shown by the amount of oxygen gas given off. I also predicted that there would be more bubbles produced in the higher concentrations than in the lower concentrations this observation also was correct, again the reason for this is due to the increased rate of reactions. I also predicted that if I doubled the concentration of the hydrogen peroxide the reaction would also roughly double. This also appeared to be correct as at 1 molar the volume of gas given off was 2.1 cm3, and at 2 molar the amount of gas was 4.5 cm3, this is not exactly double but it is very close. The reason for this is that when you double the concentration there are twice as many substrate molecules reacting with the enzyme therefore the rate of the reaction would also roughly double. However I did obtain an anomalous result at 0.5 molar concentration. Because of this my graph was not as straight as it should have been. For example the graph of the average results of the class was much straighter. I think that the heat from my hands gave the enzymes more kinetic energy and so more collisions and reactions. Otherwise that I did not obtain any anomalous results. At 0.1 molar only a small reaction took place, which is why only a small volume of gas was given off. The reason for this is because there would have been only a small number of substrates available to bind with the catalase. However at 2.0 there were many substrate molecules available to bind with the catalase that’s why 4.5 cm3 of oxygen was given off. I believe that if I was to keep increasing the concentration of the hydrogen peroxide after a certain concentration the rate of the reaction would stay the same as there would not be enough enzymes to cope with the excess substrate, therefore the substrate molecules will have to ‘queue up’ for an active site. So overall the graph shows what I predicted however because I did not keep increasing the concentration I would not be able to see whether the reaction would have eventually stopped.
You can see that the gradients of the two graphs are very similar which show that my experiment was correct. Form this you can also say that the concentration is not directly proportional to the rate of reaction because the gradient is not 1 or near 1.
