2H2O2+Enzyme 2H2O+O2+Enzyme
Enzyme- Substrate Complex
Aim
Now that I have covered the functions of the enzyme, I am going to investigate certain factors that affect the rate at which Catalase can decompose Hydrogen Peroxide to Water and Oxygen. The factors that I have chosen to investigate are as follows:
- Temperature
- Substrate Concentration
- Enzyme Concentration
- pH Levels
I have chosen to study these four variables because they will give me a thorough understanding on when an enzyme, here Catalase, is at it’s most efficient and the causes that reduce it’s efficiency. Before I start to plot down apparatus that I will require for these four different categories, I want to study some relevant background information on each to give me an idea for expected results.
- Temperature
Enzymes in the body have been found to work best at an ‘optimum temperature’ of around 40 degrees Celsius. Generally as the temperature increases, so does the rate of the chemical reaction, but, at temperatures above 45 degrees Celsius, enzymes in the body will start to denature, this is because as the temperature rises, the atoms within the enzyme vibrate and gain energy, so at high temperatures the atoms vibrate too much causing the weaker bonds to break, thus the tertiary structure of the protein is no longer maintained, causing the active site shape to change, so no further enzyme-substrate complexes can be formed. These Hydrogen bonds cannot reform; therefore the enzyme is rendered useless, and has become denatured.
From this and the graph below, I can deduce the temperature values I want to test the enzyme Catalase with, I should expect my graph to be quite similar to the one below, the rate of reaction gradually increasing as the temperature increases, and peaking around 40 degrees Celsius, then there should be decrease in activity as the enzyme cannot tolerate much higher temperatures before it’s denatured.
- Substrate Concentration
Increasing the amount of substrate will increase the likelihood of an enzyme-substrate complex to be formed. So therefore increasing the amount of substrate will increase the rate of reaction, as long as the enzyme concentration is kept constant, because more substrate molecules can attach to the active sites of the enzymes and thus more products are formed. But adding too much substrate doesn’t mean that the rate of reaction will continue to increase, this is because the enzymes will only have a limited amount of active sites, so when the maximum amount of reactions are taking place, the enzymes are said to be saturated as no further active sites are available until some of the reactions have taken place. Therefore the graph for this experiment should show gradual increase in rate as the substrate increases, but reach a maximum point and level out, when all the enzymes active sites are in use, as the graph shows below.
From this information I know that my graph should definitely show an increase in rate of reaction as I increase the substrate concentration. I know that if I increase the substrate concentration by double, the rate of reaction will increase by double, so the rate is proportional to the substrate concentration, until the saturation point.
- Enzyme Concentration
The enzyme concentration is obviously as vital as the substrate concentration. This is because the enzyme provides the active site to which the substrate joins to form an enzyme-substrate complex. The enzyme most importantly catalyses a reaction, meaning it reduces the energy required to start a reaction, the first graph below shows this reduction of activation energy. So therefore increasing the enzyme concentration will provide more active sites for the substrate, Hydrogen Peroxide to form a complex with, allowing more products to be formed. Therefore the rate of reaction increases as the concentration of the enzyme increases, if the substrate concentration is kept constant, but again there will be a saturation point when all the active sites are being used, so the graph should be very similar if not exactly the same as the substrate concentration one, showing gradual increase of the rate of the reaction as the concentration of the enzyme increases, reaching a maximum when the enzymes are saturated, then levelling off, as the second graph below shows.
From this information I know that my graph should definitely show an increase in rate of reaction as I increase the enzyme concentration. I know that if I increase the enzyme concentration by double, the rate of reaction will increase by double, so the rate is proportional to the enzyme concentration, until the saturation point.
- pH Levels
Just as an enzyme has an optimum temperature level, it also has an optimum pH level. Overall, most enzymes are denatured in solutions that are strongly acidic or alkaline. The cause of this is that the Hydrogen ions (H+) present in an acid, and the Hydroxyl ions (OH-) present in an alkali, are attracted to the charges on the amino acids of the enzyme. The amino acids are the sequence that makes the polypeptide chains of the enzyme, once they interact with the Hydrogen and Hydroxyl ions, the bonds that maintain the enzymes tertiary structure are disrupted, therefore causing it’s shape to change, so substrates can no longer fit into the active site and the reaction stops, as the enzyme is denatured. The graph below shows the expected shape of the graph under various pH levels.
From this information I can gain an idea of what values oh pH will increase the rate of reaction in my experiment, knowing that most enzymes don’t work in extreme acidic or alkaline conditions, and peak around pH 7.
Prediction
From studying the above background information, I have come to the following predictions:
- Increasing the temperature will increase the rate of reaction to an optimum, where all Catalase active sites are in use with Hydrogen Peroxide, further increase will decrease the rate of reaction rapidly, as the enzyme will start to denature.
- Catalase will function more efficiently at around pH 7, and at a decreased rate around conditions of pH 4 and pH 9.
- Increasing the concentration of Hydrogen Peroxide will increase the rate of reaction to an optimum, where all the Catalase active sites are in use, where the rate will level off.
- Increasing the concentration of Catalase will increase the rate of reaction, until a saturation point is reached, where no further reaction can take place and the rate will level off.
In all of the above variables, the rate of the reaction will always depend on the number of successful collisions between the enzyme and the substrate, caused by random thermal motion. Therefore I predict an increase in rate with higher substrate/enzyme concentration. For low concentrations I think that the rate of the reaction will be directly proportional to the concentration of hydrogen peroxide/enzyme in the solution. This is because if double the amounts of substrate/enzyme molecules are in the solution, double the amount will find an enzyme/substrate molecule at the same time, if all the substrate/enzyme molecules are moving at the same speeds (with the average speed being directly proportional to the temperature). Therefore if there are double the amount of substrate/enzyme molecules in a solution, double the amount of reactions will take place at once and the rate will therefore double as well.
Safety
Risk Assessment:
Apparatus
- Potatoes (Are the source of the enzyme Catalase)
- Cork Borer ( To cut same sized potato rings)
- Hydrogen Peroxide (Substrate forms enzyme-substrate complex with Catalase)
- Ruler (To measure out equal sized potato pieces)
- Scalpel (To cut the measured sized potato pieces)
- Stopwatch (To measure the rate of reaction in each experiment)
- Test Tubes and Rack (To hold the enzyme-substrate solutions)
- Delivery Tube with Bung (To let Oxygen to pass through test tube into burette)
-
50 cm3 Burette (To measure amount of Oxygen released at regular time intervals)
- Water Bath (To use at different temperatures with enzyme-substrate solution)
- pH Levels 2 to 7 (To use with enzyme-substrate solution under various pH’s)
- Thermometer 50°C (To measure room temperature and solution temperature)
-
Pipette Filler 10cm3 (To measure amount of Hydrogen Peroxide used)
- Retort stand, Boss and Clamp (To support and hold the burette and test tube)
Methods:
Temperature
Substrate Concentration