Enzymes are extraordinarily efficient. Minute quantities of an enzyme can accomplish at low temperatures what would require violent reagents and high temperatures by ordinary chemical means. About 30 g (1 oz) of pure crystalline pepsin, for example, would be capable of digesting nearly 2 metric tons of egg white in a few hours.
The kinetics of enzyme reactions differ somewhat from those of simple inorganic reactions. Each enzyme is selectively specific for the substance in which it causes a reaction and is most effective at a temperature peculiar to it. Although an increase in temperature may accelerate a reaction, enzymes are unstable when heated. The catalytic activity of an enzyme is determined primarily by the enzyme's amino-acid sequence and by the tertiary structure—that is, the three-dimensional folded structure—of the macromolecule. Many enzymes require the presence of another ion or a molecule called a cofactor, in order to function.
As a rule, enzymes do not attack living cells. As soon as a cell dies, however, it is rapidly digested by enzymes that break down protein. The resistance of the living cell is due to the enzyme's inability to pass through the membrane of the cell as long as the cell lives. When the cell dies, its membrane becomes permeable, and the enzyme can then enter the cell and destroy the protein within it. Some cells also contain enzyme inhibitors, known as antienzymes, which prevent the action of an enzyme upon a substrate.
Practical Uses of Enzymes
Alcoholic fermentation and other important industrial processes depend on the action of enzymes that are synthesised by the yeast's and bacteria used in the production process. A number of enzymes are used for medical purposes. Some have been useful in treating areas of local inflammation; trypsin is employed in removing foreign matter and dead tissue from wounds and burns.
Prediction:
The factors that will affect the rate of enzyme reactions, are; temperature, pH, enzyme concentration, substrate concentration and the effect of inhibitors on enzyme activity.
I will only be investigating what the effects of temperature are on enzyme activity; therefore I will have to keep all the other factors the constant.
I predict that the breakdown of gelatine will increase as the temperature increases until it reaches about 50 oc when the enzymes will get denatured. The reaction will be faster as the temperature increases because as with any chemical reaction increasing the temperature will give the molecules more energy to collide with. However when the temperature reaches 50 oc the enzymes will be denatured which means that the shape of the enzyme will be changed by heat and therefore it will no longer fit into the lock and key process and would'nt be able to catalyse the breakdown of gelatine. So I predict that 50 oc is about the optimum temperature for the enzymes to work fastest
Reasons:
Temperature, concentration and inhibitors are the main factors which effect the rate at which enzymes react, these are explained below
Temperature:
A rise in temperature increases the rate of most chemical reactions; a fall in temperature slows them down. In many cases a rise in 10 oc will double the rate of reaction in a cell. The collision theory states that as the temperature increases the particles move around more quickly. This causes them to collide more often, and therefore react more quickly. At first the activity of an enzyme increases with temperature, but then the activity falls as the enzyme is denatured. All enzymes have an optimum temperature. This is the temperature of maximum activity. The Kinetic theory also states that when a substance is heated, energy is given to the particles and they speed up. Therefore when heat is applied to an enzyme and substrate, the particles speed up, increasing the rate at which they bind with each other. This would suggest that the rate of reaction should increase as the temperature is increased until it reaches the optimum temperature, which is about 50c when the enzyme is denatured.
Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5·C or below is generally the most suitable. Some enzymes lose their activity when frozen.
pH:
Acid or alkaline conditions alter the chemical properties of proteins, including enzymes. Most enzymes work best at a particular level of acidity or alkalinity (pH). The protein digesting enzyme in your stomach, for example, works well at an acidity of pH 2. At this pH the enzyme amylase , from your saliva, cannot work at all. Inside the cells most enzymes work best in neutral conditions, Extremely high or low pH values generally result in complete loss of activity for most enzymes. The optimum pH value will vary greatly from one enzyme to another.
Concentration:
There are two ways that concentration can affect the rate of reaction, these are concentration of substrate and concentration of enzymes. The concentration of substrate can alter the rate of reaction because if it is higher then there will be more frequent collisions with enzymes and therefore a higher rate of reaction. If the concentration of the substrate is lower then collisions of enzymes and substrates will be less frequent and the rate of reaction will be slower.
The concentration of enzymes affects the rate of reaction in a similar way because if there is a lower concentration of enzymes then there will be less frequent collisions and the rate of reaction will be slower. If there is a higher concentration of enzymes then there will be more frequent collisions and therefore a faster rate of reaction.
Effect of inhibitors:
Enzyme inhibitors are substances, which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition.
Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex ES. The existence of temporary ES structures has been verified in the laboratory.
Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can be used to explain why inhibition occurs.
The lock and key theory utilises the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favourable. If we consider the enzyme as the lock and the substrate the key (Figure 9) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally.
Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate.
Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11 shows the reaction velocity decreasing after the maximum velocity has been reached.
Additional amounts of substrate added to the reaction mixture after this point actually decrease the reaction rate. This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from occupying them.
This causes the reaction rate to drop since the entire enzyme present is not being used.
Apparatus:
- Test tube
- Water bath
- Syringe
- Splint
- Thermometer
- Stop watch
- Pieces of Photographic film
- Beaker containing trypsin.
Method:
- Heat the water bath to 30c
- Measure 10ml of trypsin using a syringe, hold it at eye level to make sure its accurate then pour it into a test tube
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When the water bath is at the right temperature e.g. 30 oc, put in the test tube containing trypsin and measure the temperature with a thermometer until it reaches 30 oc.
- When it reaches the right temperature Attach the piece of photographic film to the end of a splint and put it inside the test tube leaving the test tube in the water bath, and start the stopwatch at the same time.
- Stir the photographic film inside the test tube with the other end of the splint continuously and check the film every few seconds.
- Once the layer of plastic film and the silver compound have separated and the plastic film becomes transparent, stop the stopwatch and record the results.
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Repeat this experiment with different temperatures, ranging from room temperature to 60 oc increasing the temperature by about 10c every time. (the water bath wouldn't be needed for room temperature)
- Repeat each of the temperatures three times to keep it fair.
For this test I will have to make sure everything is done with safety and fairness. Throughout the whole experiment safety glasses must be worn, and all other Lab rules must be followed as well. To make sure the experiment is fair I must make sure nothing is changed for different experiments.
The equipment should be kept the same to ensure all results are taken without any advantages or disadvantages. Everything in the experiment should be kept the same apart from the temperature, which is what we're investigating.
Results: -
From my results I drew two graphs (which can be seen on the next page) one shows the temperature against time and the other one shows rate of reaction against time. I worked out the rate of reaction by calculating 1/time, 1 stands for the amount of trypsin we used. Therefore the units for the rate of reaction is ml/s as we kept the amount of trypsin the same.
Conclusion: -
My results supported my prediction and proved that it was correct to a certain extent. I predicted that the rate of reaction of the enzymes would increase until it reaches 50 oc when it denatures and therefore no longer catalyse the breakdown of gelatine. My graph is a curve showing that the rate of reaction between trypsin and gelatine does not increase proportionally. But the slope becomes steeper as the temperature rises i.e. the rate of reaction increases faster at higher temperatures, however the slope would be a lot lower if we had tried lower temperatures e.g. at 10 oc there would have been a clear curve. I think this is because as the enzymes get closer to their optimum temperature they work more effectively than before and the rate of reaction increasec. At 50c however the enzymes start to denature which means the shape of the enzyme changes and is therefore no longer able to catalyse the breakdown of trypsin, this is explained by the 'lock and key' mechanism. The graph shows that as the temperature reaches 50 oc the rate of reaction slows down but the enzymes do not stop working. This contradicts my prediction as I expected there to be a sharp decline and that the enzymes will stop working all together after 50 oc. This may be because our experiment was not accurate enough due to human error or that maybe because trypsin may work slightly higher than 50c as my research referred to enzymes in general. Although my graph clearly shows that my prediction was correct I cannot make a firm conclusion because I don't have enough results. If we had done the experiments for 10 oc and 70 oc I would have been able to draw a better conclusion, however we were given 3 lessons to complete all the experiments and we could've used our time a lot more efficiently.
Evaluation: -
Although I conducted the experiment as accurately as I could there were many sources of error in the method we used.
We were told to stop the stopwatch when the piece of photographic film was almost clear as it takes a very long time for the entire silver compound to separate completely, and it often doesn't. This wasn't a very good method as different people conducted the experiments and so everyone may have different impressions of almost. This is very inaccurate and could be improved if only one person conducted each experiment.
It was also inaccurate because the test tube was stirred and shook by different people during the experiment. One person could've stirred more vigorously than another. This could've also been improved if one person shook and stirred the test tubes in turn.
Another inaccuracy was that we had to take the splint out of the test tube every few seconds to check how much of the silver compound (if any) had been dissolved. Even the same person may have done this at different intervals and may cause the results to be inaccurate. This could've been prevented if there was a set time at which we would check the film e.g. every 30seconds.
The pieces of photographic film being used weren't all exactly the same size. They were only cut roughly the same size and were not measured. This would have caused inaccuracy because the concentration of the substrate was to be kept exactly the same for each experiment in order for it to be fair. If the pieces of photographic film were not exactly the same size then this means that the concentration was slightly altered which may cause inaccurate results. This could've been improved if the pieces of photographic film were accurately measured exactly the same size each.
The experiments were conducted on three different days. This is inaccurate because the efficiency of the enzymes would decrease with time and may cause the trypsin not to catalyse the breakdown of gelatine as efficiently the second time as it did the first time. This could've been improved if a new solution of trypsin was used each lesson.
Our experiment could have also been more accurate and time efficient if we had organised things before hand and allocated each person in the group with one or two things to do i.e. like the construction line in factories. This would be more precise as the same person is doing the same thing each time and it prevents human error because different people may do things slightly differently. It would've also been a lot less time consuming and we may have had time to do more experiments e.g. 10 oc or 70 oc, it would give us clearer results and a more accurate conclusion.
