Non competitive is where the inhibitor has no resemblance to the substrate molecule and binds to the enzyme at a site away from the active site. The rate of reaction is therefore unaffected by the substrate concentration.
Non reversible inhibition is where it leaves the enzyme permanently damaged, e.g. mercury breaks the disulphide bonds which maintain the shape of the enzyme molecule.
•Substrate concentration – The michealis constant is the concentration of substrate needed to make the reaction proceed at half its maximum rate. A low michealis constant means that the reaction proceeds rapidly because the enzyme and substrate have a high affinity for each for each other.
•Enzyme cofactors – A cofactor is a non-protein substance that is essential for some enzymes to function efficiently. Cofactors may be metallic ions, or low molecular weight organic molecules that are either temporarily or permanently attached to the enzyme molecule. . They are of three types: activators, coenzymes and prosthetic groups.
What affect does catalase have?
Catalase is a very fast reacting enzyme, it is found in many living cells, and it breaks down hydrogen peroxide to water and oxygen. In fact one molecule of it can deal with six million molecules of hydrogen peroxide in 1 minute. Hydrogen peroxide is toxic so needs to be changed into harmless substances.
In order to observe the effect of temperature on catalase the amount of oxygen released will be looked at. The oxygen produces a froth which we will then measure in mm and the volume of oxygen given off which will be measured in cm³
Hypothesis
From my research I think that the enzymes will denature after 37ºC and any other temperature above that. Reason being those enzymes are proteins and their structure is three-dimensional. Increasing the temperature disturbs the intra molecular bonds that hold the 3D shape. Because of this the shape is altered. Enzymes have an active site. This fits into the substrate molecular (see diagram1-lock and key). If the active site is altered the substrate will no longer fit in and so the enzyme doesn’t work properly.
The rise of reaction rate is also due to the increase in temperature, relating to the kinetic theory. The higher the temperature, the faster they move. This happens but only to an optimum of 37ºC. The curve leading up to the optimum point is gradual but as it is reached it falls dramatically. The reason being that the active site is destroyed therefore no reaction can take place as there is only one specific active site per substrate.
Method- measuring the height of froth and volume of oxygen
1. Put work shirt on and goggles on. Carry out the rest of safety precautions.
2. Gather equipment as shown on diagram.
3. Mix 10cm³ solution of distilled water with 5g of yeast.
4. Measure out 10cm³ of hydrogen peroxide
10ºC- iced water
20ºC-no extra equipment
30ºC-water bath required
40ºC-water bath required
50ºC-water bath required 60˚c- water bath required
5. Put both hydrogen peroxide and yeast mixture in separate boiling tubes and place in water baths if necessary.
6. Once temperature is correct take them out and put one of them in to flask.
7. Once you put the other into the flask put the cork over the flask.
9. Start stop watch and record volume of gas collected every 5 seconds.
Apparatus
· Yeast for the catalase
· Measuring syringe
· Hydrogen peroxide
· Goggles
· Stopwatch
· Measuring cylinder
Safety precautions
· Wear goggles
· Tuck tie in skirt
· Wear work shirt
· Handle the hydrogen peroxide with care as it is corrosive and an irritant
Fair test
A fair test is where all of tests in the experiment have the same circumstances put upon them. For example as temperature is being tested then all other variables must be kept the same. If one was tested with 50% of yeast with 50% water and one with 40% of yeast with 60% water then this would NOT be a fair test. The concentration may affect the results so the results could show that the 30˚c temperature test had a peculiar reading.
As I am testing for the differences in temperature this will be the only variable that will differ. Other possible variables that could have an affect are: concentration, mass of yeast, volume of hydrogen peroxide, use of inhibitors etc.
Mass of yeast could cause a discrepancy in the results because when there is more yeast, there is more catalase. When there is more catalase there is more chance of hydrogen peroxide molecules bumping into catalase molecules. More collisions will take place increasing the speed of reactions. To stop this I will measure out the same mass of yeast each time. This will also be the same with hydrogen peroxide so I will also measure out the same volume of hydrogen peroxide each time.
Another consideration that needs to be taken into account is that the experiment may produce a couple of anomalous results. To combat this I will take 3 Measurements from each concentration, meaning that any anomalous results can be easily spotted as they will be different from the other two. This result can then be scrapped and that particular result redone.
Accuracy
In order to make my investigation go to plan I will be as accurate as I can be so I will measure to the correct measuring size.
• Measure the volume in cm³ and amount of potato in grams to make sure that they are exactly the same mass before using them in the experiment.
• Do the experiment three times to ensure that there isn’t an odd result. Three is a good number to use as you can see if there is one odd one where if you just done the experiment twice then you would not know which one odd and which isn’t.
• Also to average out the results.
• the potato will be weighed with an electric scale so it will be measured to the nearest hundredth of a gram.
Preliminary results
10˚c
40˚c
60˚c
Conclusion: The preliminary results show that I should defiantly carry out the experiments with a minimum range of 10˚c. I possibly should go lower to freezing however I believe I have a large enough range already. At 60˚c they are not reacting so there is no need to go any higher than that. 60˚c will be my maximum and 10˚c will be my minimum.
Conclusion:
From my results I can conclude that my hypothesis was correct. From the intervals I have used the highest rate of reaction was at 40˚c. This does not mean that it optimum rate of reaction is exactly 40˚c. It only means that it is the best to the nearest 10˚c. Therefore from my results alone I can say that the optimum temperature for catalase is between 35-45˚c. To work out a more exact optimum temperature I could draw a graph showing the rate of reaction for all temperatures. Drawing a curved line of best fit across this line would mean I can find an exact estimate for the optimum temperature for catalase, which I predicted was 37˚c.
Graph on next page
The graph shows that the optimum rate of reaction from my results is 37˚c. I know this because the curved line of best fit peaks at 37˚c.
From my first graphs I can see that there was a high reaction rate at first which is plausible from the research I did. While there are a lot of free enzymes more will react than later on when they have already reacted. So this is also why they slowed down after 5 seconds. This was a very steep rate of reaction at first and slowed down extremely quickly though. This also fits in with my research, which says catalase is one of the fastest enzymes in terms of rate of reaction. Many of the catalase enzymes react almost immediately, leaving very few to react later on.
My graph of averages is also very informative. It shows that at first the rate of reaction is the same at all temperatures. I think this is because catalase reacts so fast temperature does not make a difference at first. The rate of reaction is so fast at first even at 10˚c that it cannot get any faster; no matter how fast they enzymes molecules move. However once the enzymes which are still to react become more sparse, temperature begins to show its effect. Faster moving molecules at higher temperatures are able to find a hydrogen peroxide molecule more quickly than slower moving ones.
My results have also shown the denaturing process as well. 40˚c was fastest temperature I had included. However by then already some of the enzymes were denaturing. We can tell this from the scatter graph showing rates of reaction of different temperatures. The highest point was below 40˚c. At 50˚c you could see a significant fall in the rate of reaction, and by 60˚c there was no reaction taking place, the small amount of gas was from the air pressure increasing as I put the cork over the flask.
Evaluation
I thought my experiment showed all the predictions I had made and so I was able to link the research together with the results. Even though my results showed me what I had hoped for I do not believe they were as good as they could have been. A perfect set of data would have shown all the experiments at each temperature to be the same; this was defiantly not the case. Most results were acceptable though and I did not think any were too out of place not to be included in the averages. All at 10˚c were close to each other. At 20˚c there was one which was higher than the others but it was not anomalous as it still showed a decent curve and was not a major problem. As this was at room temperature it is possible that the room was hotter than before. However I think that it was the other two that could have been out of place. This was because when I poured the yeast and distilled water mixture in with the hydrogen peroxide I often noticed that there was still some of the liquid left in there. Maybe the first two at 20˚c had not been fully poured into the flask. As the temperatures increased I expected the results to become worse as they would be faster so would be more volatile and slight mistakes would be more noticeable. Also it was harder to read off the syringe as it was filling up quicker.
Other reasons which could explain the worse results would be the period between pouring the yeast into the flask and getting the cork onto the flask so the gas could be measured and I could start timing. Some gas would have escaped in that period and the accuracy of my data would be lowered. The delay in starting my stopwatch may also explain why the first five seconds were so much quicker than the rest, and extra half second may have been added on in this time. This period would not have really mattered if I was able to do this in the same amount of time every time. However sometimes the cork was more difficult to get on the flask and took a longer time. This would have affected particular results, possibly the slightly anomalous results.
A modification I would make to improve the experiment is to have the hydrogen peroxide already in the flask as normal, but then I would also have the catalase in the flask as well also inside a test tube so they would not react. I could then attach the cork onto the flask before the experiment starts. Then all I have to do is shake the flask to knock the test tube over to start the experiment. I would also be able to start the stopwatch with my other hand to I could eliminate the problem I stated above. On top of that I would have also stopped the problem of not pouring all the yeast into the flask, as it would already be there.
If I could expand the experiment I would like to test a smaller range around 40˚c to see what the exact optimum temperature really is. I would use the modification I stated above but would do more repeats of each temperature, to make sure I had done it correctly.