Diagram
Induced Fit Theory - However, more advanced biological techniques have shown that the active sites of enzymes are not the rigid shapes once thought. In the induced fit theory, the active site is thought of as having a distinctive but flexible shape. Once the substrate enters the active site, the shape of that site is modified around it to form the complex. Once the products have left the complex, the enzyme reverts back to its original shape until another substrate molecule binds. The latter process is sometimes termed dynamic recognition.
Diagram of induced fit
Enzymes are sensitive to changes in their environment. Changes in temperature or pH can cause changes in the shape of the molecule, which affects its activity. Changes in the concentration of both the enzyme and its substrate will also affect the rate of an enzyme-catalysed reaction.
As I am concentrating on the relationship between temperature and the rate of activity of dehydrogenase enzymes in different yeast cells, I shall explain why this occurs. A rise in temperature increases the rate of reaction until the enzyme denatures. As the temperature increases, the kinetic energy of the substrate and the enzyme molecules increase, so they move faster and with more force, so there is a greater chance of successful collisions, leading to a greater rate of reaction (collision theory). At the optimum temperature of an enzyme (normally 45 oC), the reaction rate is at a maximum. But, if the temperature reaches too high a point (i.e. over optimum temperature), most enzymes lose their tertiary structure and they start to denature. The enzymes will have gained so much kinetic energy, they start to change shape themselves so that the substrates no longer fit into the active sites. They change shape because the bonding becomes irreversibly changed so the active site is permanently damaged. At this point, the rate of reaction rapidly starts to decrease. Even though at very high temperatures, the number of collisions is extremely high, without the active sites, no products can be formed.
The effect of temperature on the rate of reaction can be expressed as the temperature coefficient, Q10. For every 10 oC rise in temperature, the rate of reaction will have doubled:
Q10 = Rate of reaction at (x +10) oC
Rate of reaction at x oC
At decreasing temperatures (lower than the optimum), the rate of reaction decreases because of reduced enzyme/substrate collisions.
There are exceptions to these statements though. Thermophile enzymes have an optimum above 45oC, mesophile enzymes have an optimum between 20 oC and 45 oC and psychrophile enzymes are still efficient below 20 oC.
Graph of rate of reaction against temperature with explanation.
Prediction: For the practical I will be carrying out, I predict, that as I increase the temperature up to the optimum point (around 45 oC), the rate of reaction will increase in direct proportion to the temperature. So the colour change of the solution will steadily take a shorter time to occur. When the temperature hits the optimum point, the rate of reaction will be at its maximum. This will show the fastest colour change. As the temperature continues to rise above the optimum, the enzymes will start to denature and it will take longer for a colour change to occur. The rate of reaction will rapidly decrease as the enzymes are given so much kinetic energy, they start to change shape so the substrates don’t fit in exactly anymore. They change shape because the bonding becomes irreversibly changed so the active site is permanently damaged. The experiment will eventually reach a temperature where the enzymes will be totally denatured and will have completely changed shape, so the rate of reaction will be zero.
Outline Method: This experiment is designed to show the relationship between temperature and the rate of activity of dehydrogenase enzymes in different yeast cells. Triphenyl tetrazolium chloride (TTC) is an artificial hydrogen acceptor, or redox indicator. When oxidised, TTC is colourless, but when it is reduced, TTC will form red, insoluble compounds called formazans. This colour change shows the presence of active dehydrogenase enzymes in yeast cells. The temperature of the TTC solution and yeast suspension will affect the rate at which this colour change occurs, which in turn will show how the activity of dehydrogenase enzymes in different yeast cells changes.
Set up of apparatus
Proposed Experimental Method:
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Set up a water bath at 10 oC, adding crushed ice if necessary.
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Put 10cm3 using a pipette of ‘bakers’ yeast suspension into a test tube labelled A.
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Put 1cm3 using a pipette of TTC solution into another test tube labelled B.
- Place both test tubes A and B into the water bath using tongs, and leave for several minutes to reach the temperature of the water bath. Check the temperature by using a thermometer.
- By using tongs mix together the two solutions by placing the contents of test tube A into test tube B (using tongs) and return to the water bath.
- Start the stop clock immediately.
- Observe the test tube carefully and record the time taken (in a table) of the duration it took for a colour change to occur. Stop the stop clock immediately.
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Repeat steps 1 – 7 using different temperatures for the water bath (in point 1.): 20 oC, 30 oC, 40 oC and 50 oC.
- Repeat steps 1 – 8 except in point 2. use ‘brewers’ yeast instead of ‘bakers’ yeast
Repeat the experiment 3 times for each yeast, ‘bakers’ and ‘brewers’, over the 5 different temperatures.
- Take averages of all the data and record in appropriate column of table.
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Plot a graph of temperature, oC, (x axis) against rate of reaction, 1/time, (y axis) using the average values.
Variable Identification and Control:
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Temperature – I am going to vary the temperature of this experiment using set values of 10 oC, 20 oC, 30 oC, 40 oC and 50 oC. To get these set temperature values, I will change the temperature of the water bath by either heating it up or adding crushed ice. This will help me to draw a conclusion of the relationship between temperature and the rate of activity of dehydrogenase enzymes in different yeast cells.
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Substrate Concentration – The substrate concentration must remain the same throughout the whole experiment. At high substrate levels the number of collisions increases, as the reacting particles are closer together and more active sites are used up. At low substrate levels, the active sites are not used up as there are not enough substrate molecules to occupy them all. Increasing the concentration of the substrate increases the rate of reaction until the enzyme concentration limits the rate of reaction. I can control this by using the same concentration of TTC from the same bottle of solution.
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Enzyme Concentration - The enzyme concentration must remain the same throughout the whole experiment. As the concentration of the enzyme increases, the number of active sites increases, so there are more sites for substrate molecules to combine with. As the concentration of the enzyme decreases, the number of active sites decreases, so there are more uncombined substrate molecule. Increasing the enzyme concentration increases the rate of reaction until the substrate concentration limits the rate of reaction. I can control this by using the same concentration of actively respiring yeast suspension from the same bottle of solution.
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pH – Each enzyme has its own characteristic pH at which the reaction will proceed at the fastest rate. Above and below the optimum value, the reaction will proceed more slowly so I must keep the pH value constant throught the experiment. At extreme pH values the enzyme will become denatured, and the shape of the protein molecules are altered as the hydrogen bonds and sulphur bridges are broken or formed. I can control this by using the same pH of enzyme and substrate from the same bottles of their solutions, and double check by using universal indicator paper.
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Volume – I must keep the volume of the yeast suspension and the TTC solution the same. If I put a large volume of TTC into a test tube and react it with the yeast at a set temperature, the rate of this reaction would be faster than if I put a small volume of TTC into a test tube and reacted it with the same yeast at the same temperature. To control this I will measure the volume (in cm3) using a measuring cylinder of each solution.
Reliable results:
Range of temperatures (oC): 10 oC, 20 oC, 30 oC, 40 oC and 50 oC. (Measured with thermometer)
Types of yeast: ‘Bakers’ and ‘Brewers’.
Concentration of enzyme and substrate (mol):Kept the same throughout. Recorded at each set temperature.
Volume of enzyme and substrate (cm3): Kept the same throughout. Recorded at each set temperature. (Measured with measuring cylinder)
pH of enzyme and substrate: Kept the same throughout. Recorded at each set temperature.
Number of repetitions of each yeast at
the set temperature: 3
Risk Assessment and Ethical Considerations:
See ‘Material Data Safety Sheet’ for TTC.
Always wear safety goggles to prevent solutions splashing into eyes.
