One the products have been formed; they leave the active site of the enzyme, which is left free to combine with a new substrate molecule. Enzymes, like chemical catalysts, are not used up in the reaction they catalyse so they can be used over and over again. The active site of an enzyme is the region that binds the substrate and contributes the amino acid residues that directly participate in the making and breaking of chemical bonds. The amino acid residues are called the catalytic groups. Enzymes differ widely in structure, function & mode of catalysis so active sites vary, but possible to make some generalizations. Enzymes are usually very large in comparison with substrate, so only small portion of amino acid residues are near or in direct contact with substrate in enzyme-substrate complex. Most of the enzyme involved in control and maintaining correct structural configuration of enzyme i.e. structural backbone. Active site is a 3-dimensional entity. Not a point or a plane usually an intricate pocket or cleft structurally designed to accept the structure of the substrate in 3-D terms. Substrate is bound by relatively weak forces. The free energy of interaction between enzyme and substrate ranges -12 to -36 kJ/mole compare this with the strength of a covalent bond up to -450 kJ/mole. Most are clefts or crevices designed to exclude water from the active site and are surrounded with non-polar amino acid residues, which give the active site a non-polar environment. This appears essential for both binding and catalysis. Essential to exclude water (unless water involved in the reaction) because water disrupts bond breaking and making processes. Specificity, active site provides specificity for its particular substrate, which is a characteristic feature of enzymes. The overall reaction between the enzyme and substrate can be represented by the following equation:
ENZYME + SUBSTRATE ENZYME-SUBSTRATE Complex ENZYME + PRODUCTS
There are several factors, which affect enzyme activity. Enzymes, being proteins, are sensitive to changes in their environment. Changes in temperature and pH can cause changes in the shape of the enzyme molecule and will therefore affect its activity. Changes in the concentration of both enzymes and its substrate will also affect the rate of and enzyme-catalysed reaction.
There are three main factors, which affect enzymes and they pH, concentration and temperature. I will be looking closely at the temperature factor, as it is associated with this experiment.
Temperature has a complex effect on enzyme activity. On one hand, a rise in temperature will increase the kinetic energy of enzyme and substrate molecules, and therefore will increase the chemical reaction. However, increase increases in temperature will affect the stability of the enzyme molecule. The precise shape of the active site is essential for catalytic activity so, if the shape changes, the enzyme will unable to combine with its substrate. The overall rate of activity will depend on a balance between these two factors and the enzyme will have an apparent optimum temperature at which it functions most rapidly. At temperatures above this optimum the enzyme rapidly loses its activity and becomes progressively denatured, that is, it is unable to combine with the substrate and therefore has lost its catalytic properties. Denaturation is time-dependent, so exposure to a high temperature for a brief period of time will have less effect on the enzyme than prolonged exposure.
I predict that the optimum temperature of trypsin added to the casein will have an optimum temperature of about 40 ºC when heated. That means that the casein suspension will clear the fastest at around 35 ºC – 40 ºC, it will be as its fastest around 35 ºC – 40 ºC because otherwise it will begin to denature. Enzymes work best at around body temperature.
This is how I expect the graphs to look during the experiment.
Graph 1. Graph 2
Optimum Temp
Rate of
Reaction
Time/
Min
Temperature/ ºC Temperature/ºC
The first experiment to take place was with two other enzymes and they were pepsin and protease. However, during the experiments, the pepsin and protease were working with the casein substrate at a very slow rate, so doing the experiment with the protease and pepsin would have been very time consuming. The point of doing trypsin, pepsin and protease was to make a comparisons about their optimum temperature. So I carried out the experiment just looking at the effect of temperature on the enzyme trypsin. I used trypsin at 0.5% molar concentration because if I were to anymore the reaction would go quick and if I was to add any less, it would be very slow. I also used to 4% molar concentration of casein because if I was to use too much, there would more interactions and therefore the experiment would finish quickly. If I used to little the amount of interactions would be slow so the experiment would be slow.
Method:
Apparatus:
1.Casein Suspension, 4%
2. Trypsin solution, 0.5%
3. Distilled water
4. Test tubes and rack
5. Graduated pipette
6. Water bath
7. Thermometer
8. Black card
9. Stopwatch
- I set up a water bath at 30 ºC
- I then made a solution of 4% (4 gdmˉ³) casein mixed with water.
- Pipette 5 cm³ of casein suspension into test tube and 5 cm³ of trypsin solution into another tube.
- Stand both tubes in the water bath and leave them for several minutes to reach the temperature in the water bath.
- Meanwhile, set up a control tube containing 5 cm³ of casein suspension plus 5 cm³ of distilled water. Stand this tube in the water bath.
- Mix the enzyme and substrate together and replace the tube in the water bath. Start stopwatch immediately.
- Observe the contents of the tube carefully, checking against a piece of black card, and record the time taken for a suspension to become clear.
- Repeat this procedure at a range of temperatures, between 30 ºC and 60 ºC. Using the same volumes of casein suspension and enzyme solution each time.
Diagram of Experiment:
Results:
I decided to do this experiment because I wanted to investigate the optimum temperature of the enzyme trypsin. This table shows the rate of interaction for the enzyme trypsin with the casein when heated in the water bath.
I showed the 1/Time to allow me to draw the Rate of Reaction graph.
Conclusion:
From the experiment, I noticed that trypsin was temperature resistant. Trypsin is temperature resistant because the trypsin solution became clear when it was added to the casein; this means that the casein substrate was ‘locked’ with the trypsin.
The optimum temperature of trypsin was 40 ºC this means that the casein substrate interacted with the trypsin fastest at 40 ºC. However, as I mentioned in my hypothesis that biological enzymes work best at body temperature this is shown by the fact that the optimum temperature of trypsin is 40 ºC. Trypsin is used in the digestion system, therefore it would need to work best at around 37 ºC (Body temperature). If I had tested trypsin at 37 ºC I may have got a slightly shorter time.
Enzymes react with temperature in a very complex way. When increasing the temperature, the kinetic energy of the enzyme and the substrate molecules increase and therefore interact more frequently, thus increasing the rate of reaction. So at 40 ºC the casein and trypsin were interacting the most frequently, thus the 40 ºC is the optimum temperature at 40 ºC. At 40 ºC the solution became clear that means the trypsin had reacted with the casein. The solution will be at its clearest at 40 ºC. The purpose of the black card was to see it is visible behind the solution this would indicate the end of the trial.
When the temperature begins to increase beyond the optimum temperature the rate of reaction starts to decreases, this is because the stability of the enzyme molecule is changed. The precise shape of the active site is essential for catalytic activity so, if the shape changes, the enzyme will be unable to combine with its substrate, this is known as denaturing. This was shown on the graph after 40 ºC, when the slope began to go down. If the enzyme is denatured it cannot be used again. However, when the enzyme is working at its optimum temperature it can be used to again in the same experiment. Denaturation is time-dependent, so exposure to a high temperature for a brief period of time will have less effect on the enzyme than prolonged exposure.
The purpose of the control tube was to see if the casein was to turn clear without the enzyme trypsin.
Enzymes are large proteins that speed up chemical reactions. In their globular structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place. Enzyme and substrate fail to bind if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate.
Evaluation:
Sources of error were inevitable. The following are precautions taken whilst doing the experiments to decrease the level of inaccuracy: -
The equipment was well washed so that any impurities could be washed off and therefore not contaminate the water and the solution, which may alter the optimum temperature of the enzyme and change my results. I did not hold the thermometer as I would conduct heat into the thermometer and alter the readings so therefore it was left to stand in the beaker. To get a more accurate set of results, using shorter temperature intervals would show me the optimum temperature more clearly. I was using 5 ºC if was to use a 2 ºC I think I would get a more accurate set of results. Also, the concentration of trypsin, if it was changed it could show the optimum temperature more accurately. The experiment, with an increased amount of trypsin concentration, would have gone faster.
The following sources errors, in my opinion have affected my results, they are: -
The rounding up of temperatures and measurements. A lot of the heat could have been conducted into the desk from the beaker, causing our temperature readings to fluctuate. Some of the heat could have been radiated away. Also some of the heat heated up the thermometer therefore maybe a digital thermometer could have been used or maybe a calorimeter to get an accurate reading of the temperature. Also some the heat may have been lost by convection currents.
Bibliography:
Textbooks: Molecules and Cells by John Adds, Erica Larkcom & Ruth Miller
The Internet:
Teacher’s Notes
