- Concentration of the enzyme – at low concentrations of the enzyme, there are more substrate molecules than enzyme molecules to catalyse the decomposition. This means that all the active sites are filled with substrate molecules, and there are substrate molecules ‘waiting’ for an empty active site to bind with, so there will be a fairly low rate of reaction. At higher concentrations, there are more active sites available, so there are more catalytic events/second, so the rate of reaction will increase. Doubling the number of enzyme molecules will double the number of active sites available for the substrate to bind with, therefore a doubling of the rate of reaction up to a certain point. However, as higher concentrations of enzyme are used, over half the substrate molecules will be bound to enzymes, so the rate of reaction will not double with enzyme concentration, but the reaction will still speed up. At a certain point of enzyme concentration, the reaction will not get faster, even if more enzyme is added, because all the substrate molecules will be bound to the active sites of enzymes. The graph to show the effect of varying enzyme concentration on an enzyme controlled reaction is as follows:
Graph to show the effect of enzyme concentration on an enzyme controlled reaction (Cambridge Biology 1)
-
Substrate concentration – as substrate concentration increases, the initial rate of reaction also increases. This is again obvious, as the more substrate molecules there are around, there more often an enzyme’s active site can bind with one. However, the more the substrate concentration there is, keeping enzyme concentration constant, there comes a point where every enzyme active site is working continuously. Even if more substrate is added, there enzyme cannot work any faster as it is all being used. The substrate molecules are ‘queuing up’ for an active site to become vacant. The enzyme is working at its maximum possible rate, known as Vmax. The graph to show the effect of varying substrate concentration on an enzyme controlled reaction is shown below:
Graph to show the effect of varying substrate concentration on an enzyme controlled reaction
All the possible variables for this experiment have now been discussed fully. The variable to be investigated in this investigation is the concentration of the enzyme catlase, found in yeast in this case. In order that the experiment is fair, all the other variables must be kept the same throughout the whole experiment. The temperature will be kept at room temperature, which is approximately 21°C. The pH will not be adjusted at all during the experiment so the pH will remain the same throughout. The concentration of the substrate hydrogen peroxide will be determined in the trial investigation.
The fact that enzymes combine briefly with their reactants makes them susceptible to inhibition by unreactive molecules that resemble the substrate. The inhibiting molecules can combine with the active site of the enzyme but tend to remain bound without change, blocking access by the normal substrate. As a result, the rate of there reaction slows. If the concentration of the inhibitor becomes high enough, the reaction may stop completely. Inhibition of this type is called competitive because the inhibitor competes with the normal substrate for binding to the active site.
Some inhibitors interfere with enzyme-catalyzed reactions by combining with enzymes at locations outside the active site. These inhibitors, rather than reducing accessibility of the active site to the substrate, cause changes in folding conformation that reduce the ability of the enzyme to lower the activation energy. Because such inhibitors do no directly compete for binding to the active site, their pattern of inhibition is called noncompetitive. Some poisons or toxins exert their damaging effects by acting as enzyme inhibitors. For example, the action of cyanide and carbon monoxide as poisons depends on their ability to inhibit enzyme important the utilization of oxygen in cellular respiration. Poisons and toxins typically act irreversibly by combining so strongly with enzymes, either covalently or nocovalently, that the inhibition is essentially permanent. Some irreversible poisons, rather than combining with the enzyme, destroy enzyme activity by chemically modifying critical amino acid side groups.
The cell has built in mechanisms to control directly both enzyme concentration and activity. First cells are able to regulate whether an enzyme is present at all. This type of control regulates protein synthesis and will be discussed in a later chapter. Cells also have ways to control the level of activity of enzymes that have already been synthesized and are present in the cell.
In noncompetitive inhibition, a molecule binds to an enzyme but not at the active site. The other binding site is called the allosteric site (allo - other and steric -structure or space). The molecule that binds to the allosteric site is an inhibitor because it causes a change in the 3-dimensional structure of the enzyme that prevents the substrate from binding to the active site. In cells inhibition usually reversible; that is the inhibitor isn't permanently bound to the enzyme. Irreversible inhibition of enzymes also occurs, due to the presence of a poison. For example, penicillin cause the death of bacteria due to irreversible inhibition of an enzyme needed to form the bacterial cell wall. In humans, hydrogen cyanide irreversibly bind to a very important enzyme (cytochrome oxidase) present in all cells, and this accounts for its lethal effect on the body.
The activity of almost every enzyme is a cell is regulated by feedback inhibition. Feedback inhibition is an example of common biological control mechanism called negative feedback. Just as high temperature will cause furnace to shut off, in a similar manner the product of an enzyme can inhibit a enzyme reaction. When the product is in abundance, it binds competitively with its enzyme's active site; as the product is used up, inhibition is reduced and more product can be produced. In this way the concentration of the product is always controlled within a certain range.
Activation of an allosteric enzyme by an activator is another form of feedback inhibition. Combination of the activator and the allosteric site cause a conformational change in the active site permitting substrate binding and the reaction will be caltalyzed. (ntri.tamuk.edu/cell/enzyme2.html)
Prediction
I predict that as the concentration of catalase increases, so too will the rate of reaction, up to the maximum possible value where a plateau will appear on the graph. The rate of reaction will increase in proportion to the concentration of the enzyme catlase.
Hypothesis
To explain the prediction, first the action of an enzyme needs to be explained. The first and most simple explanation is the ‘lock and key’ hypothesis. An enzyme is a globular protein, coiled into a precise 3D shape. This has an active site, which is a depression into which another molecule can bind. This molecule is the substrate of an enzyme, as it has a complementary shape which fits into the shape of the active site perfectly. Each type of enzyme will usually only act on one type of substrate molecule, as the shape of the active site will usually only allow one shape of molecule to fit in. For this reason the enzyme in said to be specific for this substrate. The enzyme then catalyses a reaction into which the substrate molecule is split into two or more molecules, called the products. A diagram of this is shown below:
However, the lock and key hypothesis is over-simplified, and not exactly how the process takes place. The more accurate hypothesis is the ‘induced fit’ hypothesis. This hypothesis is more based around the protein nature of the enzyme, allowing for flexibility and for the shape of the active site to be altered slightly. The binding of the enzyme and the substrate molecule because of the attraction between groups on the substrate molecule and complementary groups on the active site of the enzyme molecule. The substrate molecule does not fit perfectly with the active site, rather forces its way in and in doing so alters the shape of the active site, thus a better more tight fit. Once the products have been released, the enzyme and active site returns to its original shape.
The main reason for enzyme controlled reactions is to lower the activation energy of a reaction. As catalysts, enzymes increase the rate at which chemical reactions occur. Most of the reactions which occur in living cells would occur so slowly without enzymes that they would virtually not happen at all. In many reactions, the substrate will not be converted to a product unless it is given some extra energy. This is called the activation energy. One of the main ways to give a reaction energy is to heat it. However, in humans, where body temperature is 37°C, the temperature is not enough to give most substrates the activation energy which they need to change into the products. Enzymes are a good solution to this problem as the reduce the activation energy of the reactions they catalyse. They do this by holding the substrate or substrates in such a way that their molecules can react much more easily.
Enzymes also work on the basis of kinetic theory. In order that enzyme and substrate molecules bind, they must collide with the right amount of energy and at the correct angle. Therefore if the molecules have more kinetic energy, such as from heat, there will be more collisions because the molecules are increased in speed, therefore and increase in rate of reaction. Other factors involved in enzyme controlled reactions are inhibitors and activators, but neither occur in this reaction so the explanation of these factors is unnecessary.
For this experiment, catalase and hydrogen peroxide bind as per the ‘induced fit’ hypothesis. Therefore, if the concentration of catalase, the enzyme, is increased, then there are more active sites for the substrate molecules, hydrogen peroxide, to bind with, meaning an increase rate of reaction as there are more catalytic events per time. If there are twice enzyme molecules, obviously there will be twice as many active sites for the substrate molecules to bind with and therefore the rate of reaction will double. Also if there are double the number of enzymes, then the number of collisions between molecules will double, therefore again a doubling in rate of reaction. I predict the graph for this experiment will look as follows:
The graph plateaus because at a certain point all the substrate molecules will be to an active site, so the rate will be at its maximum. Even if more enzyme molecules are added, the rate of reaction will not increase.
Preliminary Investigation
A trial run was carried out in order that the range of variables used in the actual experiment would produce accurate results to support a firm conclusion. A number of points needed to be decided on from the trial investigation:
- Range of variant (concentration of catalase) to be used
- Substrate concentration
- Volume of substrate
- Number of readings to be taken
- Time taken to measure results
- Apparatus used
Before the trial run it was decided that 1cm3 20vol hydrogen peroxide should be used. It was also decided that the volume of gas produced for each concentration would be measured after one minute.
A dilution series was produced to show how much yeast should be mixed with water to produce varying concentrations of catalase:
Each solution was therefore 5cm3.
Table to show effect of concentration of catalase on the volume of oxygen gas produced from the decomposition of hydrogen peroxide:
These results produced results which did not produce a regular pattern as predicted. This is due to the fact that the decomposition had either finished or slowed down so much that the results did not differ between 3% and 5% catlase. For this reason it was decided that the volume of gas produced would be measure after 20 seconds rather than every minute. It was also decided that 3 readings would be taken for each concentration, and then the average taken in order to produce more accurate results. The concentrations seemed to be a good range in order to produce accurate results so the concentrations used in the actual investigation were as used in the preliminary investigation.
Apparatus
-
1cm3 syringe
- 1 litre beaker
-
25cm3 open ended syringe
- 10% yeast solution
-
5cm3 syringe
- 20 vol hydrogen peroxide
- Clamp stand and boss
- Test tube and bung
- Gas delivery tube
Diagram
Method
- The apparatus was set up as shown in the diagram above.
-
The 5cm3 of yeast solution was made up in different concentrations by using a syringe to measure out the water and yeast. Care was taken to view the syringes from the side to ensure the bottom of the meniscus was lined up properly with the gradations and there were no air bubbles in the syringe. The dilution series was as follows:
-
The hydrogen peroxide in the 1cm3 syringe was then added to the 5cm3 yeast solution. The stopclock was then started. The decomposition started to occur, and oxygen gas bubbled into the 25cm3 syringe. After 20 seconds, the volume of gas produced was recorded.
-
When the results were taken, care was taken to ensure that the 20cm3 syringe was perpendicular to the bottom of the beaker so the volume of gas recorded was entirely accurate.
- This was repeated 3 times for each concentration of catalase (from yeast) and the results recorded.
Safety
In order that the experiment was carried out in a safe way, safety goggles were worn as hydrogen peroxide is a bleach and an irritant so can cause great damage to the eyes. Great care was taken and if any hydrogen peroxide was spilled it was cleaned up immediately. If any hydrogen peroxide came into contact with skin, the skin was rinsed thoroughly with water so that no burning of the skin occurred.
Justification of Method
-
1cm3 syringe – used because 1cm3 of hydrogen peroxide was used in this experiment, and as the graduations were a lot smaller, it was easier to obtain the correct amount of hydrogen peroxide more accurately.
-
1 litre beaker – a large amount of water was needed for the 20 cm3 syringe to be completely submerged, and this volume was perfect because the syringe was completely submerged, and there was room for hands to be put inside to adjust the syringe if necessary. This meant it was easier to fill the open ended syringe with water completely more easily.
-
25cm3 open ended syringe – from the trial readings, the largest amount of gas produced was 18 cm3 so this gives a bit of room for error above this reading, but not so large that it was difficult to get an accurate measurement of the volume of gas produced. An open ended syringe was used as opposed to an upturned measuring cylinder because if there was a gas bubble at the top it could easily be removed by opening the top of the syringe, meaning more accurate and reliable results could be used because the water always filled the whole syringe.
- 10% yeast solution – this solution provided enough catlase for the reaction to produce quite a lot of gas so the final pattern was more easy to discern. This made it easier to make conclusions as the results were spread apart more so it was easier to spot a pattern in the results.
-
5cm3 syringe – a number of these were used. They were used to measure out the volumes of water and yeast to be mixed. This volume was used because it was the closest to the volumes needed, but small enough to mix accurate quantities of the water and yeast. A clean syringe was used each time to ensue accuracy.
- 20 vol. hydrogen peroxide – there was a choice between 5 vol. and 20 vol. It was decided that the reaction would occur more quickly with 20vol hydrogen peroxide so this was used because it allowed for more readings to be taken in the time available, meaning that conclusions could be drawn more reliably as there were more results.
- Clamp stand and boss – this set-up was needed for support of the test tube, and this set-up is reliable and easy to adjust.
- Test tube and bung – the yeast solution needed to be contained in something, but gas needed to be able to escape from the top. The bung had a hole in which was connected to the delivery tube, so the gas could travel quickly from the test tube into the syringe.
- Gas delivery tube – the gas needed to be transferred from the test tube to the syringe, and the only way to do this was by gas delivery tube.
Bibliography
- Cambridge Biology 1 (Mary Jones, Richard Fosbery. Dennis Taylor. © Cambridge University Press 2000, reprinted 2001)
- ntri.tamuk.edu/cell/enzyme2.html