The rate at which enzymes catalyse reactions varies with their environment. Enzymes work best within an optimum range of physical and chemical conditions.
In all, enzymes are very efficient. Small quantities at low temperatures are able to produce results, which would require high temperatures and a violent reaction from any normal chemical means. Although increases in temperature may speed up the reaction, enzymes are unstable when heated.
Enzyme function can also be explained by the Lock and Key Hypothesis: the active site of an enzyme (the lock) has a specific shape in which only the precise amount of substrate (the key) will fit – forming an enzyme-substrate complex. Therefore producing a product.
DIAGRAM
All enzymes have four specific properties:
- All enzymes are proteins
- Enzymes are catalysts
-
Enzymes are denatured by high temperatures
- Enzymes work best at a certain pH (normally 7)
Hypothesis
Hydrogen peroxide will breakdown to oxygen and water in the presence of Catalase. The reaction will increase with increasing enzyme concentration when molecules of hydrogen peroxide are freely available. However, when molecules of the substrate are in short supply, the increase in rate of reaction is limited and will have little effect. And also the rate of reaction could also be affected by temperature.
Prediction
I predict that a rise in temperature increases the kinetic energy of molecules, which therefore move around more rapidly and collide with one another more often .In an enzyme-catalysed reaction, this means that the enzymes and substrate molecules come together more often in a given time, so that the rate of reaction is increased.
Diagram.
As shown on the graph above, this gives a rising curve. However, the temperature rise also increases the energy of the atoms, which make up of the enzyme molecule. Its atoms begin to vibrate and cause the hydrogen and other bonds, which holds it shape to brake. Gradually, the shape of the active site is disrupted, until they no longer fit their substrate molecules. At the point usually around 40 degree centigrade, the enzyme stops working and it is said to be denatured. Also on the above graph, the rate of this reaction follows a falling curve.
I also predict that, the reaction will begin very swiftly. As soon as the enzyme and substrate are mixed, bubbles of oxygen are released very quickly, but as the reaction continues the rate at which oxygen is released gradually slows down and it eventually stops because the rate at which reaction occurs will depend only on how many enzyme there are and at the speed at which the enzyme can then bind with another substrate molecules.
Variables
In this investigation, the variables that affect the activity of the enzyme, Catalase, were considered and controlled so that they would not disrupt the success of the experiment.
As temperature increases, molecules move faster (kinetic theory). In an enzyme catalysed reaction, such as the decomposition of hydrogen peroxide, this increases the rate at which the enzyme and substrate molecules meet and therefore the rate at which the products are formed. As the temperature continues to rise, however, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, are broken. If the molecular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is denatured.
To control this variable, the temperature was maintained at a fairly constant level that allowed the enzyme to work effectively (room temperature, approximately 23ºC).
Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.
In this experiment, the pH was kept constant using a pH 72 (buffer), selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (yeast).
Substrate Concentration
When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes´ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate.
To control the substrate concentration, identical quantities of the substrate were used for each reading. To ensure that this was measured precisely, 2ml syringes were used to accurately gauge to exact quantities.
Enzyme inhibitions are substance, which directly or indirectly interfere with the functioning of the active of an enzyme and so reduce its activity.
Sometimes, the inhibitor binds itself so strongly to the active site that it cannot be removed and so permanently prevent the enzyme functioning. These are called non-reversible or permanently inhibitors; they include metal ions such as mercury and silver. Most inhibitor only make temporally attachment to the active site. These are called reversible inhibitors and are of two types;
Competitive (active site directed)
Non-competitive (Non-active site directed)
Diagram.
Inhibitors therefore slow the rate of reaction. They should not have affecting this investigation, however, as none were added.
Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration will not have the rate of reaction effect, because there is already enough enzyme to cope with the amount of substrate.
Diagram.
The rate of reaction will then stabilise at a constant level. As shown as above graph.
Safety Precautions
- I will have to be careful when using the Hydrogen Peroxide, as it is a corrosive chemical, so I will attempt to overcome this problem by wearing goggles.
- Hydrogen Peroxide is a bleaching agent, so I will be wearing a lab coat so it doesn’t bleach my clothes.
- I will also wear gloves to prevent copper sulphate and Hydrogen peroxide from reaching my hand use
- I will clear up immediately after breakage occurs.
- Keep all apparatus away the desk to prevent from the edge of breakages.
- No run around or rush about
PRELIMINIARY WORK
There was some mistake s that caused an error in my preliminary experiment.
Firstly, my dilution table with copper sulphate exceeded the normal total volume, because I initially added 2cm3. Therefore, I had to change volume of copper sulphate to 1cm3, which finally gave me the same total with the rest of the second table.
Secondly, there was an approximately 50% of inaccuracy in my timing and recoding of amount of oxygen released during each reaction.
Also, my partner and I was very slow at the beginning of our experiment, so we had to increase the rate at which we carry out our experiment. Therefore, this hurry ness affected our result.
Finally I have really learnt many skills from my having this premiliary work. I will make sure that I use my mistake to have a better result when I have my next experiment.
MATERIALS AND METHODS
USED
Aim of the Experiment
To see how different concentrations of yeast affect how much oxygen is given off in every 10 seconds, when every mixture with yeast (which contains catalyse) is mixed with 10cm3 of hydrogen peroxide. Also the effect of Copper sulphate on catalase
Suggested Apparatus For Measuring Catalase Activity
Diagram
PROCEDURE
-
Set the apparatus up in the way shown above, making sure that no (or as little as possible) air leaks out of the beaker when it is connected with the Rubber connector.
Clamping the apparatus in place enables me to concentrate on running the experiment and not have to hold anything.
- Measure the solutions accurately (water, buffer and hydrogen peroxide) and pour into the beaker.
- Cover the beaker properly with the rubber bung and tube seal. To avoid the leakage of oxygen from the system. Any pressure build up could also lead to inaccuracies.
- Collect some yeast and inject it into the mixture after it has been covered and start the stopwatch immediately
A stop clock is most suitable because it is accurate and can easily be stopped and started without looking at it.
- After every 10 seconds enter the amount of gas collected in the gas syringe on the table, making sure that this point is at eye level to eliminate parallax error.
I decided to collect as much gas as possible if I had time, because this would reduce the percentage errors. If the point on the gas syringe is not at eye level, there will be accuracy in my readings or results.
- Stop the stop clock as soon as the gas syringe is full of gas (again, make sure that this point is at eye level to eliminate parallax error). The stop clock should be stopped the moment the oxygen collector reaches the edge of the gas syringe.
- Enter this time and the amount of oxygen collected in, the correct column for which repetition of the experiment it is on the table.
- Take out the beaker and rinse with distilled water before continuing the experiment.
- Repeat the experiment three times for deferent concentration of hydrogen peroxide, and water.
I am repeating these experiment three times because, the more repetitions the better, the result.
Notes:
First come the sheets of the table in which I recorded my results.
Then the sheet on which I put the information to be transferred to graphs.
Then come the graphs in a numbered order. They need no explanation as
they have complete headings.
The computer plotted the lines of best fit and some are more roughly sketched than others are.
