chains can form a protein. The tertiary structure of an enzyme determinds its unique shape and so this then forms an active site, like the one in Fig. 1[a].
This part of an enzyme contains the catalytic and binding properties to enable the substrate to bind to the active site shown in Figure 1[b]
below.
Once the enzyme-substrate complex has been formed the enzyme catalyses the chemical reaction to make the product. The product molecules then leave the active site. From Figure 1[b], you can see that during the reaction the enzyme has been unchanged and so the enzyme can be used again in the same process. However, the active site has a specific shape and so only the correct shape substrate is able to form an enzyme-substrate complex, if the enzyme is denatured by high temperatures, then the active site’s shape changes and so the substrate can no longer form the enzyme-substrate complex, reducing the rate of the reaction.
The temperature easily affects enzymes; the rate of reaction is very slow at low temperatures, as the collisions between the substrate and enzymes have slowed down. As the temperature increases the rate of reaction increases as the collisions have increased. The rate of reaction can only rise up to an optimum temperature. This is because the active sites in all enzymes have a substrate reacting.
The active site can change shape slightly to fit the substrate; this is called the “Induced Fit Theory”. Figure 1[c] shows this process.
If the temperature becomes too high then the enzyme can then become denatured. If this happens then the active site will change shape completely, resulting in substrates not being able to fit in the active site. This will then result in a lower rate of reaction. The enzyme can also be affected by the pH, if it’s too high or too low the active site will, again, change shape, resulting in a lower rate of reaction.
Method: -
Equipment list: -
- Standard acidified sucrase solution.
- Gelatine jelly.
- Standard laboratory glassware, beakers, test tubes etc.
- A ruler
- Stop clock
- Thermometer
- Pen and paper.
Variables: -
-
Independent variable: - My independent variable is the different concentrations of substrate.
- Dependant variable: - My dependant variable for this experiment is the measurement of the rate of reaction in different concentrations.
- Control variables: - My control variables are the variables that I need to keep the same to make sure that this experiment is a fair test. These are: -
- The temperature. It is important that I control this as this will denature the enzymes.
- pH. It is important that I control this as this will denature the enzymes.
- Time that the mixture is in the beaker before and during the experiment.
Procedure
- Half fill a glass beaker with water to act as a water bath. Adjust its temperature to between 35 C and 40 C. (Note: Using a Bunsen burner may force the temperature of the water to rise a few degrees above the stated temperature after the burner has been taken away.)
-
Then label six test tubes A to F. Add 5 cm3 of sucrose solution to each test tube as shown in the table on the next page. (Make sure you rinse out the filling instrument after filling each test tube.)
- Place all of the test tubes containing the sucrose solution into the water bath. Make sure that the temperature of the water bath does not fall below 35 C or rise above 40 C. Leave the test tubes in the water bath for two minutes.
-
After two minutes, add to each test tube quick succession 1 cm3 of the sucrase solution using a 10 cm3 syringe.
- Immediately start a stopwatch or stopcock and leave the test tubes in the water bath for ten minutes, swirling them gently every few minutes. Continue to maintain the temperature of the water bath, but do not let the temperature rise above 40 C.
- When the period of ten minutes is ended: -
- Stop the stopwatch or clock and return it to zero.
- Heat the water bath with the test tubes to 60 C (or place them in a water bath already at 60 C.)
- Leave the test tubes in the water bath at 60 C for a further two minutes.
- When the time period of two minutes is ended: -
- Arrange the test tubes so that they are standing in the water bath in order of sucrose concentration, A to F.
- Rinse a 10 cm3 syringe with distilled water and then fill it with Benedict’s solution.
- Start the stopwatch or clock.
- Immediately 1 cm3 Benedict’s solution to each test tube in quick succession starting with the lowest concentration of sucrose (Test tube A) and finish with the highest concentration (test tube F).
- As soon as you have finished adding the Benedict’s solution to the last test tube, carefully observe the contents of the tube in the water bath and record the time it has take the solution to turn a thick green cloudy colour.
Results
Below are the relative rates of cloudiness formation. I have worked this out by dividing 100 by the time taken in seconds for each tube as follows:
Relative rate of cloudiness formation = 1000
T
Graphs
Conclusion
In this experiment, my results show that as the concentration of sucrose increases then the rate of reaction decreases. This suggests that as an increase in substrate is added the enzymes can not catalyse these as they have reached their active peak. The graph shows this as a steady curve. This then backs up my hypothesis and disproves my null hypothesis.
Evaluation
To improve this experiment I would have to increase the number of tests to see if my results are repeatable and accurate. I would also want to make sure that the timing was quicker when adding the Benedict’s solution so that each tube would get the solution in a much shorter time to avoid anomalous results.
I would also take more care when conducting the experiment to ensure that there can be no random errors produced.
References:
[1a] & [1c] Taken from Enzyme slideshow by Joe Wright – Portsmouth College
[1b] Taken from the S.N.A.B. website -