FAT + water lipase fatty acids + glycerol
Enzymes are proteins and temperature sensitive. They have an optimum pH and temperature, which affects their ability to speed up reactions.
Until recently it was thought that all biological catalysts were enzymes, but now we know that other substances, such as abzymes and ribozymes may carry out catalytic functions in living organisms. Enzymes are not used up or altered in chemical reactions so they may be used over and over again. They are therefore effective in small amounts. Enzymes cannot cause reactions to occur, but only speed up ones, which would otherwise take place extremely slowly. Enzymes are complex three-dimensional globular proteins, some of which have other associated molecules. While the enzyme molecule is normally larger than the substrate molecule it acts upon, only a small part of the enzyme molecule actually comes into contact with the substrate. This region is called the active site. Only a few of the amino acids of the enzyme molecule make up the active site. These catalytic amino acids are often some distance apart in the protein chain but are brought closer together by the folding of that chain.
Enzymes operate on a lock and key mechanism. In the same way that a key fits a lock very precisely, so too, the substrate fits accurately into the active site of the enzyme molecule. The two molecules form a temporary structure called the enzyme-substrate complex. The products have different shape from the substrate and so when they are formed they escape from the active site leaving it free to become attached to another substrate molecule. As there are many different lock and key mechanisms, each one specifies for its own substrate and some locks can only be opened by one key while other locks can be opened by a number of familiar keys.
In my experiment I will be investigating if there is a change of rate at which hydrolysis occurs if the concentration of substrate is varied. I will be investigating how much gas is given off over a period of time when an enzyme is used to speed up a reaction as I know from scientific knowledge that the faster a reaction takes place, the more gas is given off. I will be investigating how the varied concentrations of hydrogen peroxide react with the enzyme catalase, which I will be obtaining from potatoes.
The reaction is:
catalase
Hydrogen peroxide (l) water (l) + oxygen (g)
catalase
2H2O2 (l) 2H2O (l) + O2 (g)
Catalase is an enzyme present in living cells. It breaks down hydrogen peroxide into water and oxygen. This is important because a build up of hydrogen peroxide in cells is toxic. The breakdown of hydrogen peroxide will occur very slowly on its own, but catalase speeds it up so that the toxic levels do not get dangerous in cells. In my experiment I will be using the following equipment:
- Hydrogen peroxide
- Potatoes
- Conical flask
- White tile
- Knife
- Stop clock
- Trough
- Beehive shelf
- Delivery tube
- Measuring cylinder
- Apple corer
- Pen and paper
I will start my experiment by measuring out five equal tubes of potato and placing them into a conical flask. I will set up the trough and measuring cylinder filled with water and I will put the measuring cylinder on a beehive shelf and insert a delivery tube. I will note how full the measuring cylinder is. I will pour 100 ml of 20% concentration hydrogen peroxide into the conical flask and start the stop clock as soon as I have put the bung with the delivery tube in position. I will record the level of the volume of the water inside the measuring cylinder every twenty seconds for a period of 5 minutes. After that I will repeat the experiment again, but this time I will use 10% concentration of hydrogen peroxide. I will do the experiment with a partner so that it will be easier to get precise measurements. One person will record the measurements as the other keeps track of the time.
In order to understand the experiment that I am carrying out, it is necessary that I take certain factors and variables into account. There are four possible factors affecting the reaction of hydrogen peroxide that need to be noted.
For a given amount of enzyme, the rate of an enzyme-controlled reaction increases with an increase in substrate concentration. At low substrate concentrations, the active sites of the enzyme molecules are not all used – there are simply not enough substrate molecules to occupy them all. As the substrate concentration is increased, more and more sites are used. A point, however, is reached when all the active sites are occupied and the amount of enzyme is the limiting factor, as the increasing of substrate concentration cannot increase the rate of reaction.
If the temperature increases, it can affect the rate of an enzyme-controlled reaction in two ways:
1) As the temperature increases, the kinetic energy of the substrate and enzyme molecules increases and so they move faster. The faster these molecules move, the more often they collide with one another and therefore the rate of reaction is faster.
2) As the temperature increases, the more the atoms, which make up the enzyme molecules, vibrate. This breaks the hydrogen bonds and other forces, which hold the molecules in their precise shape. The three-dimensional shape of the enzyme molecules is altered so much that their active sites no longer fit the substrate. The enzyme is said to be denatured and loses its catalytic properties.
The optimum temperature for an enzyme varies considerably, depending on its surroundings. For many enzymes the optimum temperature is 400C and denaturation occurs at 600C.
Altering the pH can also break the bonds of the three-dimensional molecular shape. This is because the bonds may be broken by the concentration of hydrogen ions and pH is a measure of hydrogen ion concentration. Any change in the pH can denature enzymes. Each enzyme work best at its own particular pH.
Changing the size of the surface area of the potato can also alter the rate of reaction. If there is a larger surface area, then there are more active sites where the enzymes can work and the reaction will speed up. A larger surface area can be achieved by cutting up the potato into smaller pieces.
One can also affect the rate of reaction by varying the substrate concentration used each time in the experiment. The more concentration used in the experiment the faster the hydrogen peroxide will react.
I will be varying the concentrations of hydrogen peroxide used in my experiment to find out if that affects the rate at which the enzymes cause the reaction to speed up. I will be controlling the temperature and the pH at which I conduct my experiment. I will also be controlling the surface area of my potatoes by making sure they are cut with the same size cork borer and that they are of equal length.
In order to make my results as precise as possible, I will use the stop clock to help get the exact time and readings and I will use a measuring cylinder that has accurate markings. I will measure all my readings on the same scale – ml. and I will make sure that I only start the stop clock once the bung has sealed off the conical flask. I will get one of my friends to help me in telling me when it is the exact time to read off the measurements and write them down. I must also make sure that I have diluted the substrate precisely in order to get accurate and reliable results.
In order for my investigation to be a worthwhile and conclusive experiment, I have to make sure that it is a fair test. Certain factors that are able to affect the rate at which catalase speeds up the reaction of hydrogen peroxide must remain the same throughout the entire experiment.
The temperature has to be constant and on room temperature the whole time in order that the results should not be affected.
I must also keep the pH of the substrate as neutral as possible so as not to kill off the enzymes or make them denatured. This pH must remain the same throughout the whole experiment so that it should be a fair test. This will be done by measuring out the acid from the same pH.
The surface area of the potatoes must also be kept the same throughout so that there are the same amount of active sites available to the substrate. If there is a larger surface area and more active sites, the reaction will be speeded up and the experiment will not be a fair test in relation to the other times that I will be performing the experiment.
I predict that there will be a faster reaction with the more concentrated substrate, as it will use more of the active sites that are available. This is because the concentrated solution is concentrated enough to react with as many sites as there are provided. The more active sites that are used, the faster the reaction. Therefore, more gas will be given off in the reaction with the more concentrated substrate, as the amount of gas given off is proportional to the concentration of the acid. The water level in the upside down measuring cylinder will decrease. This is because oxygen will be produced in the reaction as well as water. The water will not need to use up space in the measuring cylinder as it is able to overflow into the trough, but the oxygen gas will rise upwards causing the water level to “decrease”. The amount of water will not decrease but it will overflow into the trough.
Preliminary results:
I did a preliminary experiment in order that I will be able to improve my final method based on the results that I obtain from this preliminary.
From my preliminary I have discovered a few corrections that need to be made in order to perfect my plan.
I will also take readings from concentrations of 5% and 15% to give me a larger range from which to draw my conclusions and I will also repeat the experiment twice or three times in order to make sure my results are reliable. I will take an average from which to plot points on my graph.
I didn’t repeat the experiment twice for each concentration, as I did not have enough time. In my final experiment I will make sure that I have enough time to perform the experiment slowly and carefully.
I will also practice starting the stop clock at the precise moment that I put the bung in place so as to get accurate results.
I can now reinforce my prediction with the results that I got from my preliminary. I predict that the 5% concentration will react slower than the 10% concentration, as there will be less substrate to make use of the active sites of the enzyme. So too, the 15% concentration will react faster than the 10% concentration but slower than the 20% concentration as it has more substrate than the 10% concentration, but less substrate than the 20% concentration to fill the active sites of the enzyme.