Enzymes share common properties:
- They work rapidly
The ‘turnover number’ refers to the enzymes speed of action. This refers to the number of molecules of which one molecule of the enzyme turns into a product per minute. This varies from 100 to several million but is generally thousands. One of the fastest enzymes is catalase, this is found in the liver and it speeds up the decomposition of hydrogen peroxide (H²O²→ 2H²O + O²). Hydrogen peroxide is a toxic by-product of metabolism and needs to be rapidly converted to water. (Roberts et al., 1993, 2000)
- They are not destroyed by the reactions they catalyse
Enzymes are not destroyed and can be used again. This does not mean that an enzyme can be used indefinitely as it can be denatured by; changes in temperature, PH levels and also by certain chemicals such as ; urea, alcohol and detergents. Denaturizing is normally irreversible. Enzymes do wear out and the raw materials to replace the enzymes come from the organisms’ food. (Roberts et al., 1993, 2000)
- They can work in either direction
Enzymes are capable of working in either direction. This depends on the amount of substrates and products present. The reaction will change direction until equilibrium is reached. (Roberts et al., 1993, 2000)
- They will be denatured by high temperatures
Temperature will affect the rate of reaction. The optimum temperature is 37°C - 40°C. A 10°C rise in temperature will cause the reaction to double. Above the optimum temperature the enzyme reaction rate will begin to fall and then rapidly decline, stopping altogether at about 60°C. The enzymes are then denatured. (Roberts et al., 1993, 2000)
- They are sensitive to PH levels
Each enzyme has an optimum PH level e.g. Pepsin, which is found in the stomach and is acidic, has an optimum PH level of 1-3 whereas amylase which is found in the liver, works better at a neutral PH level (7), Trypsin has an optimum PH level of 8-10 as it is alkaline and is found in the duodenum. (Roberts et al., 1993, 2000)
- They will only catalyse one type of reaction
Most enzymes will only work on one particular substrate and will be ineffective on any other natural substrate, e.g. Catalase will only work on hydrogen peroxide whereas lipase, a digestive enzyme will digest a variety of different fats. (Roberts et al., 1993, 2000)
There are two differing theories concerning enzymes, one by Emile Fisher who suggest that enzyme activity is similar to a lock and key (As shown in FIG 1-Lock and Key Mechanism) and the particular site where the substrate fits, is called the active site and is a fixed shape,
Fig 1- Lock and Key Mechanism (www.1)
Whereas, Koshland suggests that the active site can change shape when the substrate molecule enters it (As shown in FIG 2-Induced Fit Hypothesis) enabling a tighter fit as the enzyme molecule closes around the substrate molecule.
Fig 2-Induced Fit Hypothesis (www.2)
Enzymes also need non-proteins to make them work. These are called cofactors. They maybe simple inorganic molecules such as iron, copper or magnesium or they may be complex organic molecules known as a coenzyme. The function of a coenzyme is to transfer chemical groups or atoms from the active site of an enzyme to an active site of another enzyme. There are certain substances which inhibit enzymes causing them to either slow down or to stop enzyme reactions altogether. These inhibitors can be split into different categories
- Irreversible inhibitors- which combine with the enzyme molecule, which makes it impossible for the substrate molecule to react with it
-
Reversible inhibitors- which can become detached from the enzyme, if and when needed (Roberts et al., 1993, 2000)
Activation energy is required by the enzyme to enable the reaction to begin and to speed up the reaction. A small amount of energy must be applied to the molecule to start the process of reaction. Enzymes work by lowering the amount of activation energy to make a reaction work.
The use of enzymes in industry is very important and there are advantages in using enzymes, they are less likely to produce unwanted by-products and they are specific in what they do .Enzymes are often used in medicine, food industry, manufacture of rubber and also the paper industry. Biological washing powder is very effective at lower temperatures as it usually contains the enzyme protease which helps to remove ‘biological stains’ such as blood and food. Amylase which converts starch to sugars is used in the manufacturing of syrups, fruit juice and chocolate Protease is used to tenderize meat and for skinning fish. Amylase is used in bread making as it reacts with the starch in the flour breaking it down to sugar: the sugar then reacts with the yeast
producing carbon dioxide thereby causing the bread to rise. Protease is also used in the manufacture of foods to lower the level of protein in biscuits. (Roberts et al., 1993, 2000)
The enzyme catalase is found in high concentration in the liver where its job is to speed up the decomposition of hydrogen peroxide. The effect of this chemical reaction can be seen by the presence of bubbles as the hydrogen peroxide is being rapidly split by the enzyme into water and oxygen. (Roberts et al., 1993, 2000)
Hypothesis
It is predicted that the reaction rate will increase as the temperature is increased up to the point that the amylase solution will be denatured. I hypothesis that the enzyme amylase will become denatured at 60°c. I believe that the ice bath will also cause the enzyme to become denatured. The optimum temperature for amylase is 40°c as amylase is found in the mouth cavity in saliva, so it functions best at body temperature.
It will be shown that enzymes have an optimum temperature and that by altering the temperature we can change the reaction rate. When amylase bonds with starch it forms a product. The concentration of the enzyme and the substrate will remain constant; the only variable to alter will be the temperature. Iodine is used as an indicator; to show the presence of iodine turns starch blue.
Apparatus
Test tubes x 7
Sample trays x 7 (Fig 3)
Test tube rack (Fig 4)
Pipette
Glass rods x 7
Water baths maintained at 20°c, 40°c, 50°c, 60°c, 80°c and 100°c
Catalase solution (enzyme)
Starch solution (substrate)
Iodine solution (indicator)
Method
Each test tube was labeled as follows;
room temperature, 20°c, 40°c, 50°c, 60°c,80°c,100°c
Sample trays were labeled with appropriate temperature.
Fig 3
To each test tube 5 cm³ of catalase solution was added.
The test tube marked ‘room temperature’ was placed in test tube rack
The other 6 test tubes were placed into appropriate water bath for 5 minutes exactly
Each of the test tubes were removed from water baths and placed in test tube rack.
Fig 4
To each test tube 5 cm³ of starch solution was added and mixed with a clean glass rod
At intervals of one minute, one drop of product was placed into sample tray and one drop of iodine was added to each sample
Observations were recorded, noting how long it takes before a blue colour ceases to be shown.
Risk assessment
All bags and coats must be kept in lockers. Lab coats and goggles must be worn to protect eyes and clothing Care must be taken with water baths to prevent scalding and spillage. Care must be taken when handling glass: test tubes and glass rods. Care must be taken when working with chemical solutions.
Results
Results as shown in Table 1 and Fig 5
Table 1 showing the results of group results and also showing the average results for the whole group.
Table 1
Fig 5 is showing the reaction rate of the enzyme solution at different temperature ranges. This is shown as average overall class results.
Fig 5
Conclusion and analysis
The enzyme appeared to show signs of denaturing at 60°c as the heat had broken down
the enzyme, altering the active site. The results in the graph show that the enzyme worked best at 50°c. The reactions of an enzyme begin to decrease at temperatures of above 40°c because the enzyme is denaturing. When the enzyme is denatured, the enzyme action stops which is what has appeared to have happened. This experiment proved that enzymes either increase or decrease the rate of reaction because of the change in temperature. Enzymes have their own functional temperature and will not function as well or will denature if the temperature is too high.
Appraisal
The results support the conclusion and hypothesis although it would be helpful to repeat the experiment several times to prove these results. Although it would seem to be an easy experiment to carry out, it turned out to be quite confusing as there were too many samples going on at the same time. It was difficult to be sure that you were timing each sample correctly. The temperature of the water baths need to be maintained as during this experiment they were not always constant due to variables within the laboratory eg doors/ windows being opened and closed and other students adding or removing test tubes to the water baths. The slight drop in temperature may have made a difference to the final results of the reaction rate within the enzyme solution.
Bibliography
Roberts, Reiss and Monger. Biology Principles and Processes. 1993 Nelson Surrey
Roberts, Reiss and Monger. Advanced Biology 2000 Nelson Cheltenham
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