In Catalase, heme functions as a prosthetic group. A prosthetic group is a tightly bound, specific non-polypeptide unit required for the biological function of some proteins.
There are factors, which will control/affect the rate at which an enzyme-controlled experiment will take place. These include:
The Enzyme/Substrate concentration:
Providing conditions such as pH and temperature are at the normal for that specific enzyme, and there is an excess amount of substrate, then the arte of reaction will be directly proportional to the enzyme concentration. Increasing the amount of enzyme will increase the rate of reaction. If the amount of enzyme stays the same, the rate of reaction will increase with the increase in substrate concentration up to a point. When the enzymes active sites are all working as quickly as they can, adding more substrate would bring about no further increase in the rate of reaction.
PH:
Most enzymes have an optimum pH at which the rate of reaction is fastest. As we know the three-dimensional shape of an enzyme is vital for it to function properly. The most abundant chemical bonds found in the enzyme are the hydrogen bonds.
Small changes in the pH can affect the rate of reaction without denaturing the enzyme. At the extremes of its pH range, an enzyme can become unstable and denatured. Acidity and alkalinity can affect the active site of an enzyme. Free hydrogen or hydroxyl ions can affect the charges on the amino acid side chains of the enzymes active site. This will also affect the hydrogen bonds. If the bonds break due to a change in charge the three-dimensional shape will be lost, thus changing the shape of the active site. The substrate will no longer fit into the active site and will not be able to for an enzyme-substrate complex. Meaning that the enzyme looses its activity and the rate of reaction falls.
Low pH Correct pH High pH
Temperature:
Heating increases the rate of reaction in most chemical reactions. Heating a substance gives it greater kinetic energy, thus making the substance move around more. This means that there is a greater chance of molecules colliding, increasing the rate of reaction.
Increasing the temperature of an enzyme-controlled reaction will increase the rate of the reaction but only up to a point. For most enzymes in the human body the rate will increase up to 40oc, a little higher than body temperature, known as the optimum temperature. This rise will bring about a corresponding rise in the rate of reaction. The optimum temperature is specific to the enzyme and has a lot to do where the enzyme is found.
Any increase in temperature will increase the energy of a single atom. This means that in an enzyme, the bonds between the polypeptide chains would be also be affected. The rise would mean the bonds would begin to vibrate. Eventually the vibrating would get so fast, the bonds would break this will start to happen as the temperature increases past the optimum. The bonds that hold the precise three-dimensional shape will be broken, and so the precise shape will also be lost. This means that the active site will have changed, and the substrate can no longer bond to the enzyme. We say that the enzyme has become denatured.
If the enzyme becomes denatured, it will no longer work, even if you cool it. If you cool the enzyme below the optimum temperature, the enzyme becomes inactive. The enzyme will work again once heated.
In our experiment we are going to be using the enzyme Catalase, found naturally in potato. In our experiment we are going to be observing what happens when you vary the temperature at which the enzyme is working at. Before I decide on my exact method I will conduct a few preliminary test to ensure that my method will be as accurate as possible.
Preliminary Tests:
Before we started our actual experiment there were a few factors we had to set and keep the same all the way through. This is what we found:
Surface Area: We thought it would be appropriate to see whether surface area made a difference on the amount of oxygen produced. We had a fair idea from what we knew before that it would, it was jut a question of finding the surface area that gave the biggest yield of oxygen. After five minutes of reacting 75 ml of hydrogen peroxide and 2cm2 of Catalase in this condition we found that:
In the end, although puréed gave the best results, we decided to use tubes made with a borer. This was because it was easy to keep the amount the same, and the readings you got went so big. Smaller readings meant that as we got near the optimum the amount of gas produced wouldn’t exceed the boundary of the measuring cylinder.
PH: we looked at what pH the experiment should be conducted at to maximise the amount of oxygen produced. We found that the best pH to use was 7. After five minutes of reacting 75 ml of hydrogen peroxide and 2cm2 of Catalase in this condition we found that a yield of 10.10ml of oxygen was produced. This was the greatest amount. We also compared it to pHs at either end of the spectrum. At pH 4 we found that 7.10ml were produced and at pH 10 9.00ml was produced. This is what we expected to see, as when researching the enzyme Catalase we found it worked best under neutral conditions.
Temperature: we decided to look and see what the optimum temperature would be, just so we were aware when the maximum amount of oxygen should be produced. We thought it would be about 40oC. This is because in the body, the temperature is kept at about this, and as Catalase is found in there it must be about this point. After five minutes of reacting 75 ml of hydrogen peroxide and 2cm2 of Catalase in this condition we found that:
This confirmed what we thought and so know knew to look out for the maximum being at 40oC. This test also showed us that we needed a bigger range of temperatures and to have many repetitions to get the best results we can.
Concentration of Hydrogen Peroxide: We also looked in which concentration of Hydrogen Peroxide gave the best amount of oxygen produced. We found that after five minutes of reacting 75 ml of hydrogen peroxide and 2cm2 of Catalase in this condition:
From this we could see that the best concentration to use was 20 Vol. We decided in the end though to use 15 vol. This was because it was less harmful than the 20 Vol.
Prediction:
I predict that as you increase the temperature the amount of oxygen produced will increase, up to the optimum temperature. This is because heating a substance gives it greater kinetic energy, thus making the substance move around more. This means that there is a greater chance of molecules colliding, increasing the rate of reaction. The rate will increase in a steep manner up until about 40oC after which the rate will drop. This is because any increase in temperature will increase the energy of a single atom. This means that in an enzyme, the bonds between the polypeptide chains would be also be affected.
The rise would mean the bonds would begin to vibrate. Eventually the vibrating would get so fast, the bonds would break this will start to happen as the temperature increases past the optimum. The bonds that hold the precise three-dimensional shape will be broken, and so the precise shape will also be lost. This means that the active site will have changed, and the substrate can no longer bond to the enzyme, the enzyme has become denatured.
Plan:
At first, I will have to get the potato so I will use a borer to cut a cylinder of potato out of the whole one and from there I will cut up the potato cylinder into segments of 2cm using a knife.
While this is happening the flask with the hydrogen peroxide will be warming in the appropriate water baths. The water baths range from 0°C to 80°C, with intervals of 10°C.
While it is warming I will set up the equipment< see below. This is the most efficient way to do it.
Once the Hydrogen Peroxide is at the temperature I want them to be, I will add the potato, place the bung and then start the stopwatch and after 5 minutes stop the experiment and collect my data.
The procedure will be repeated for the other temperatures. I will then re-repeat for each temperature to ensure my results are accurate and reliable.
Equipment:
Ruler
Stopwatch
Tile
Marker pen
Measuring cylinder
Ice cream tub
Conical flask
Water baths at different temperatures
Beaker
Bung and gas delivery tube
Potato
Potato borer
Knife
Paper towel
Hydrogen peroxide (15 vol.)
Goggles
Thermometer
Method:
- Measure out precise amounts of 15vol. Hydrogen peroxide. The amount you need is 75.00ml. Add each flask to the appropriate water bath, and place a thermometer in each.
- Then cut the pieces of potato with a borer, ensuring it is held vertical. Once you have the tubes cut them to a precise length of 2.00cm. Making sure that the knife I not held at a slant.
- Set up the equipment as below next to the water bath of your choice.
- Add the potato and the start the stopwatch to time for five minuets.
- Once the five minuets is up quickly remove the delivery tube and then recorded the amount of oxygen produced and collected in the measuring cylinder.
- Repeat this for each temperature and then re-repeat 3-4 times for each temperature.
Results:
Analysis:
Form my results I can see that my prediction was sort of correct. The amount of oxygen slowly rises up to the point of 40oC, there after the amount slowly decreases. As we began to heat the hydrogen peroxide it was gaining more kinetic energy. When we added the potato, the added kinetic energy meant that there were more collisions between the enzyme and the substrate and so more enzyme-substrate complexes could be formed.
This increase continued up to the 40oC mark, which I predicted we would see the most amount of oxygen produced. This is known as the optimum temperature. The point at which the enzyme is working the quickest it can and the point before which it becomes denatured.
We can see on the graph the point at which the enzymes begin to become denatured, as the slope in the graph falls and heads back towards the zero mark, although these points did not fit precisely on to my line of prediction.
We can show that that my prediction was correct by taking the gradient of the slope. If the gradient increases up to 40oC and then decrease post-optimum temperature then we know that my prediction is correct. I wasn’t able to this from my points because it was hard to draw a line of best fit through them
Over this sort of range of temperatures, the effect of temperature on the rate of reaction can be expressed as the temperature coefficient Q10.
Q10 = Rate of reaction at T+10
Rate of reaction at T
If we add in the values from our graph we will be able to see how much the rate of reaction is changed for every 100C rise in temperature.
Q10 = 400C 16.13 = 1.37
30oC 11.75
This means that for each 100 rise in temperature the rate of reaction changes by 1.37. Had my results been more accurate and reliable then the number I got for Q10 would have been 2.
Evaluation:
From my results I can see that I got didn’t really get the desired effect. Although my results did show that the rate rose then fell I would have expected to see them much nearer to my line of best fit. The reasons why this may of happened are listed below.
2 http://crystal.uah.edu/~carter/enzyme/catalase.htm
3 www.schoolscience.co.uk/.../ catalysis/catsch8pg1.html