The ‘lock and key’ hypothesis states that the enzyme is like a lock, which will only have one key. The substrate shown is the only substance that fits the enzyme. An enzyme substrate complex is the compound formed when the substrate is attached to the active site, where it is only in this form for a short period of time while the substrate is being broken down.
The activity of an enzyme can be affected by many different factors. I am investigating the affects of temperature on the rate of reaction of Amylase on Starch. The Kinetic Theory states that with increased temperature, molecules receive more movement energy, which results to faster movement. This means that the enzymes are likely to collide with substrates more often. Hence, the increased frequency of collisions means that the rate of reaction will inevitably increase. And therefore, as they have more energy, when they do collide, it is likely that they will be able to overcome the activation energy barrier and form a product with great success.
On the other hand, enzymes are proteins, which means at high temperatures enzymes will begin to denature. The structure of the enzyme molecule vibrates so energetically that some of the bonds holding the enzyme molecule in its precise shape begin to break. This is especially true of hydrogen bonds and the hydrophilic interactions holding together the shape of the enzyme. Subsequently, the active site will lose its unique shape, hence, the enzyme will be denatured and will no longer be able to react with its substrate. The temperature at which the enzyme works best is known as the Optimum temperature. The optimum temperature for most enzymes is 37°C, after this they begin to denature. The enzymes in our body have this optimum temperature and the body has adapted to control its temperature so the enzymes are working at their best. It would be dangerous to maintain a body temperature of 40°C, as even a slight rise above this would begin to denature the enzymes.
The rise of rate of reaction is governed by the Q10 coefficient, which states that a 10ºC raise will result in an approximate double of the rate of reaction, up to its limiting optimum temperature.
Enzymes also have an optimum pH level, where they work best, any changes to this level will cause the enzymes to begin to denature. In other words pH changes can affect the structure of an enzyme molecule and so affect its ability to bind and act on its substrate. Most enzymes work best at pH 7, which are in neutral conditions. Some, however, such as the protease pepsin works best in acidic conditions as it is found in the acidic conditions of the stomach and used along with stomach acid. It has an optimum of pH 2; hence it is well adapted for working in the very acidic environment of the stomach. On the other hand, Lipase works best in alkali conditions as its found working with bile in the intestines. Changes in pH affects the ionic bonds, which hold the enzyme in its shape. Enzyme contains many positive and negative charges. If extra charges are added, i.e. pH, the enzyme will denature as different charges will repel or attract each other. It may also affect the R groups in the active site, which form temporary bonds with the substrate.
The pH is a measure of the concentrations of hydrogen ions in a solution. Lower the pH, the higher the hydrogen concentration. Hydrogen ions can interact with the R groups of amino acid; hence pH can affect the way in which they bond with each other and therefore affecting their 3D arrangement.
At low enzyme concentration there is great competition for the active sites and the rate of reaction is therefore low. However, as the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. This is basically what should be expected; the more enzyme molecules there are, the more often a substrate molecule will collide with it. Nevertheless, eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor.
At a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. Alternatively, when more substrate molecules are added, more enzyme-substrate complexes can be formed, as the unoccupied active sites begin to be used. And therefore the initial rate of reaction increases. Nonetheless, eventually increasing the substrate concentration even more will have no effect. If the substrate concentration is increased, while keeping the enzyme concentration constant, there comes a point where every enzyme active site is working persistently, and soon substrate molecules begin to ‘queue up’ for an active site to become vacant. This means the active sites will be saturated so no more enzyme-substrate complexes can be formed. Hence, the enzyme is working at its maximum possible rate, known as Vmax.
Hypothesis
As the enzymes and substrates are likely to collide more frequently with higher temperature, I predict that with increased temperature the rate of reaction will inevitably increase. Usually, rises of 10ºC will double the rate of reaction. However, I also predict that, temperature rises above the enzyme’s optimum temperature (37ºC - 40ºC) will radically decrease the rate of reaction.
Variables
In this experiment I have decided that temperature will be the independent variable. Hence, most of the temperatures will test the effectiveness of the reaction. On the other hand, the fixed variables are the substrates and enzyme concentrations. The dependant variables will be reaction volume. A list of these will be made for each experiment.
Method
To investigate if temperature affects the rate of reaction of Amylase on Starch, I will adapt a pilot experiment, in which I will examine if the enzyme amylase breaks down starch to maltose. The trial run will be carried out encase I make or notice any mistakes, from which I can learn and prevent from occurring in the original investigation. While I am carrying out the experiment, I will observe and measure the disappearance of starch.
I will be using the following steps to carry out the experiment:
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I will place 30cm² of 0.5% (500mg. 100cm³) starch solution in a boiling tube
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Place 10cm³ of amylase of 1% concentration in a 2nd boiling tube.
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Label both tubes and place in the water bath at 10°C to equilibrate.
- To begin the digestion I will mix the contents of the two tubes and immediately start the clock.
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Take 4cm³ of the mixture, place in a colorimeter and add 0.4cm³ of iodine in potassium iodine solution, shake, put in the colorimeter and record the absorbance.
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I will repeat steps 1-5 with different temperatures (20°C – 60°C) at suitable time intervals (2mins).
- Finally, I will record my results in an appropriate result table.
Apparatus
I was advised to use a pipette to measure the volumes of substances in use; however instead, I prefer to use a syringe to measure the volumes of substances in use, as I believe a measuring cylinder is more accurate than a pipette. Nonetheless, I will also use a syringe to collect the substances; hence it will assist in measuring the amount of substances collected.
I will use an electronic water bath to equilibrate the temperature of the mixtures as it can maintain the temperature to what it is, where as a Bunsen burner couldn’t.
I will also use the following equipments in the experiment:
- Test tubes
- Boiling tubes
- Beakers
- Test-tube rack
- Thermometer
- Colorimeter - A colorimeter measures the light absorbed by a coloured/cloudy solution. This is known as absorbency and is shown in arbitrary units. The colorimeter measures the light that is transmitted through a solution. This is known as transmission and is expressed in a percentage form.
- Goggles
Risk Assessment
Before I can begin the investigation and set-up any equipment for the experiment, I must ensure that during the experiment my fellow classmates and I will be safe at all times. I will be using many corrosive and irritant solutions, such as Iodine, which can stain hands or clothing. Therefore, I have decided that I must wear safety goggles throughout the experiment, and I should only remove them when I am 100% positive that I am far away from any form of danger.
To ensure that I keep my clothes in a good condition and avoid any form of damage, I will be wearing a safety laboratory coat. I have also decided that I will be using a test tube holder for any test tubes or boiling tubes used during the experiment, and furthermore, I will use forceps if I decided to pick up test tubes from the water bath. I should always be very cautious while handling hot water.
Results
Once I have completed the preliminary experiment, I will use a Calibration curve to work out the Absorbance.
Using the Calibration curve I can work out the rate of reaction, time taken in 2mins. I will use the following formula:
Rate = substrate concentration ÷ 2
e.g. if substrate concentration is 200cm¯³
200 ÷ 2 = 100mg¯³ min¯¹
I believe that one set of results will not be enough to reach a valid conclusion, therefore I will repeat the experiment at least 2 more times.
(Bibliography: ‘Biology 1’, endorsed by OCR, ‘Letts Study Guide’, by Glenn and Susan Toole, ‘Biology of the mammal’, by P. Catherine and Arthur G. Clegg, ‘On Course’, Bob McDuell, Keith Hirst, the Internet (, , , ), ‘GCSE Biology Revision Guide’, edited by Richard Parsons.
