Mechanism Of Enzyme Action
Enzymes are taught to operate on a lock and key mechanism. In the same way that a key fits a lock very precisely, so the substrate fits accurately into the active site of the enzyme molecule. The two molecules form a temporary structure called the enzyme-substrate complex. The products have a different shape from the substrate and so, once formed, they escape from the active site leaving it free to become attached to another substrate molecules. The sequence is summarized in the figure below.
Factors Affecting Enzyme Activity
A number of external factors affect the activity of enzymes in speeding conversion of reactants to products. The factors affecting enzyme activities are: -
- Substrate Concentration
- Enzyme Concentration
- pH
- Temperature
Effect Of Temperature On Enzyme Action
The temperature of a system is to some extent a measure of the kinetic energy of the molecules in the system. Thus the lower the temperature, the lower the kinetic energy of the system and likewise the higher the temperature the greater the kinetic energy of the system. Increase in the kinetic energy of a system results from increase in the kinetic energy of the system. This has several effects on the rates of reaction.
More energetic collisions.
When molecules collide, the kinetic energy of the molecules can be converted into chemical potential energy of the molecules. If the chemical potential energy of the molecules becomes great enough, the activation energy of an exergonic reaction can be achieved and a change in chemical state will result. Thus the greater the kinetic energy of the molecules in a system, the greater is the resulting chemical potential energy when two molecules collide. As the temperature of a system is increased more molecules per unit time will reach the activation energy. Thus the rate of reaction may increase.
The number of collisions per unit of time will increase.
In order to convert substrate into product, enzyme must collide with and bind to the substrate at the active site. Increasing the temperature of a system will increase the number of collisions of enzyme and substrate per unit of time. Thus the rate of the reaction will increase.
The heat of the molecule inside the system will increase.
As the temperature of the system is increased the internal energy of the molecules in the system will increase. The internal energy of the molecules may include the translocation energy, vibration energy and rotational energy of the molecules, the energy involved in chemical bonding of the molecules as well as the energy involved in nonbonding interactions. Some of this heat would be converted into chemical potential energy. If this chemical potential energy is high enough some of the weak bonds that determine the three dimensional shape of the active proteins maybe broken. This could lead to a thermal denaturation of the protein and thus inactivate the protein. Thus too much heat can cause the rate of an enzyme-catalysed reaction to decrease because the enzyme becomes denatured and inactive due to high temperature.
Optimum temperature is the temperature at which the enzyme works the best and the rate of the reaction is the fastest.
If temperature is reduced to near or below freezing point, enzymes are inactivated, not denatured. They will regain their catalytic influence when higher temperatures are restored.
Hypothesis: - I hypothesize that as the temperature increases the rate of enzyme catalysed reaction increases.
Apparatus Used
The following are the apparatus, which I will be using for my practical: -
- Thermometer: - To measure the temperature of hydrogen peroxide in the water bath.
- Water bath: - To bring the solution of hydrogen peroxide to desirable temperature.
- Measuring cylinder: - To measure the volume of oxygen release.
- Gloves: - To protect my hands from the spillage of hydrogen peroxide.
- Pipette: - To take out the specific volume of hydrogen peroxide from the test tube.
- Test tubes: - To keep the potato piece and hydrogen peroxide for the reaction to take place.
- Rubber tubing: - To stop the oxygen release during the reaction to go out into the atmosphere.
- Knife: - To cut the potato into pieces.
- Ruler: - To measured the size of the potato piece to be cut.
- Stopwatch: - To measure the time to allow for the reaction.
- A cork borer: - To take the potato slice out of the whole potato.
- Boiling tubes: - To keep the hydrogen peroxide inside the water bath to raise the temperature of it.
- Goggles: - To protect my eyes from hydrogen peroxide.
- Trough: - I will place water so as to keep water filled measuring cylinder inverted to collect oxygen in it by the downward displacement of water.
- Delivery tube: - For the passage of oxygen between the test tube containing hydrogen peroxide and potato piece to the water filled measuring cylinder in the trough.
- Lab coat: - I will be wearing lab coat so that no chemical spills on my clothes.
Chemicals Used
The following are the chemicals, which I will be using for my practical
- Hydrogen peroxide: - It will be the substrate, which I will be using. I will be using 15% concentrated hydrogen peroxide.
- Potato: - For getting the enzyme Catalase.
- Water: - To fill the water bath with water.
- Buffer solution: - To maintain the pH of the solution.
Controlled Factors
These are the factors, which I will be controlling during my practical: -
- pH
A change in pH can also affect enzyme activity. Each enzyme has an optimal pH range that help maintain its normal configuration in an environment which it operates. Pepsins is a proteolytic enzyme found in the stomach and functions at pH 2. At pH 2 the tertiary structure of pepsin is not altered and will catalyse the reaction. Trypsin is an enzyme found in the small intestine and prefers a pH of about 8. The tertiary structure of a protein depends on interactions such as hydrogen bonding, between R groups. A change in pH can alter the ionisation of these side chains and disrupt the normal configuration and in some case denature the enzyme. A denatured protein cannot combine with a substrate.
I will keep the pH of the hydrogen peroxide constant by using the buffer solution. I will keep the pH of the solution constant because the enzyme works best at an optimum pH. If the pH is increased or decreased the enzymes’ structure is affected due to which the enzyme is not able to work at the efficient rate.
- Enzyme concentration
Provided that substrate concentration is maintained at a high level, and other conditions such as pH and temperature are kept constant the rate of reaction is proportional to the enzyme concentration. Normally the reactions are catalysed by enzyme concentrations, which are much lower than substrate concentration. Thus as the enzyme concentration is increased, so will be the rate of the enzyme action. If the amount of substrate is restricted it may limit the rate of reaction. The addition of further enzyme cannot increase the rate of reaction and therefore the reaction becomes constant.
By using the same length and mass of the potato from where I will be getting the enzyme Catalase. I will be using the same length and mass because increasing or decreasing the enzymes’ concentration will increase or decrease the rate of enzyme-controlled reaction. So if the concentration of the enzyme is not maintained I will not know whether the effect was due to the temperature or enzyme.
- Substrate concentration
Enzymes react distinctively to alteration in the concentration of reacting molecules. At very low substrate concentration, collisions between enzyme and substrate molecules are infrequent and reaction proceeds slowly. As the substrate concentration increases, their reaction rate initially increases proportionately as collisions between enzyme molecules and reactants become more frequent. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration diminish. At the point at which the enzymes are cycling as rapidly as possible, further increases in substrate concentration have no effect on there reaction rate. At this point the enzyme is saturated and the reaction remains at the saturation level.
By using the same volume of hydrogen peroxide. I will be using the same volume of hydrogen peroxide because increasing or decreasing the substrates’ concentration will increase or decrease the rate of enzyme-controlled reaction. So if the concentration of the substrate is not maintained I will not know whether the effect was due to the temperature or enzyme.
- Time
I will keep time constant through out the experiment. This I will do by using a stopwatch. I will allow the reaction to take place for only 180 seconds. If time is not kept constant than it will have an affect on the volume of oxygen produced. If some particular experiment is allowed for more time the volume of oxygen produced will be more because than the enzyme Catalase will be able to breakdown more amount of hydrogen peroxide than it was broken down before, thus producing more oxygen. Likewise if some particular experiment is allowed for less time the volume of oxygen produced will be less because than the enzyme Catalase will be able to breakdown less amount of hydrogen peroxide than it was broken down before, thus producing more oxygen. So the rate of reaction is directly proportional to the time.
- Temperature
The temperature of a system is to some extent a measure of the kinetic energy of the molecules in the system. Thus the lower the temperature, the lower the kinetic energy of the system and likewise the higher the temperature the greater the kinetic energy of the system. If the kinetic energy of the molecules is low than the rate of reaction will be low. Likewise if the kinetic energy of the molecules is high than the rate of reaction will be high. So the rate of reaction is directly proportional to the temperature.
I will keep the temperatures other than that on which I am working constant. This I will do by keeping the air conditioner off, so that the room doesn’t become cold and lowers the temperature of the solution.
- Volume Of Substrate
This I will keep constant by taking the same volume of hydrogen peroxide for the experiments. I will do this because if there is any change in the volume of substrate than it will have an effect on the number of substrate molecules present in the solution. This in turn will affect the number of enzyme-substrate complex being formed, which will affect the rate of reaction.
- Surface Area Of The Potato
I will be keeping the surface area of the potato constant. This I will be doing by using the cork borer, knife and ruler to make sure that all the potato pieces are of equal length and width. This is because if there is any change in the surface area of the potato than it will have an effect on the number of enzyme molecules present in the solution. This in turn will affect the number of enzyme-substrate complex being formed, which will affect the rate of reaction.
As I am studying the effect of temperature on enzyme-controlled reaction it is necessary that I keep the factors mentioned above constant. As changes in any of them will change the rate of the enzyme controlled reaction and thus affect my readings.
Safety Precautions
These are the safety precautions, which I will be taking during my practical: -
- While measuring the volume of hydrogen peroxide in the pipette I will take the lower meniscus.
-
While using the measuring cylinder to transfer 2H2O2 from bottle to the beaker I will use the lower meniscus.
- I will ensure that the measuring cylinder is completely filled with water and that there isn’t any air bubbles inside the measuring cylinder.
- I will close the stopper on the test tube as soon as I put the potato into the test tube containing hydrogen peroxide.
- I will start the stopwatch as soon as I will close the test tubes so that no oxygen is release into the atmosphere.
- I will make sure that the potato pieces are of the same length by taking them of the same length.
- I will also make sure that the tube in which the reaction is taking place is airtight and that there is only one way out from where I will be collecting the oxygen.
- I will use digital thermometer to get accurate readings of hydrogen peroxide.
- I will cut the potato slice into smaller pieces so that the surface area is increased and more reaction can take place.
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I will make sure that the potato is fully emerged in the hydrogen peroxide (H2O2) so that all the Catalase present can act on hydrogen peroxide.
- I will be careful using the electrical appliances (water bath).
- While measuring the temperature I will make sure that the thermometer is not touching the boiling tube.
- I will keep the hydrogen peroxide in water bath for 2 to 3 minutes.
Risk Assessment
These are the risk assessments of the practical: -
- I will wear the gloves so as to ensure that hydrogen peroxide doesn’t get spilled on my hand.
- While taking hydrogen peroxide in the pipette I will ensure that I don’t drink it by mistake.
- I will wear goggles on my eyes so as to protect them.
- While handling the electronic devices such as water bath I will be careful that I don’t get electrical shock.
- I will be tying my hair.
- I will be keeping the solutions at distance from electrical devices.
Method
First I will make the 15% concentrated solution of hydrogen peroxide. This I will do by first taking 100ml of distilled water in the measuring cylinder from the bottle and pour into the beaker. Getting over with this I will take 30% concentrated solution of hydrogen peroxide from the bottle. I will also be taking 100ml of hydrogen peroxide using the measuring cylinder. Then I will pour the hydrogen peroxide from the measuring cylinder into the beaker containing water.
After that I will transfer 100 ml of hydrogen peroxide solution from bottle to the beaker using a measuring cylinder. Than I will use the pipette to take 4 ml of hydrogen peroxide from the beaker and put it in the boiling tube. Than I will use the cork borer and cut the whole cylindrical piece of potato. After that using the ruler I will measure the length of the potato and make it 4 cm by cutting it using a knife. Then I will fill trough and measuring cylinder with water. I will place one end of the delivery tube inside the measuring cylinder and the other end inside the hole of rubber stopper. After doing so I will fill the water bath with water and then I will switch on the water bath after which I will keep the boiling tube in the water bath. I will place the thermometer inside the boiling tube where the solution of hydrogen peroxide is present. As soon as the temperature is reached I will take the boiling tube out of the water bath and remove the thermometer from the boiling tube. Then I will invert the measuring cylinder and put it inside the water filled trough. As soon as I finish doing it I will put the potato inside the boiling tube and put on the stopper and start the stopwatch. I will keep shaking the boiling tube. After three minutes I will measure the volume of the oxygen obtained in the measuring cylinder by the downward displacement of the water.
I will be carrying out my practical in the method described above. I will be carrying out the experiment on the following temperatures: - 25 °C, 30 °C, 35°C, 40 °C, 45 °C, 50°C and 55 °C. I will be taking readings thrice for each temperature so as to ensure that I get the accurate result.
Observation Table
I will be using the following table to record my readings: -
Prior Test
Method
Before starting the experiments I had wore the lab coat, goggles and the gloves so as to ensure my own safety. Besides that I had also tied my hair.
First I had made the 15% concentrated solution of hydrogen peroxide. This I had done by first taking 100ml of distilled water in the measuring cylinder from the bottle and poured into the beaker. Getting over with this I had taken 30% concentrated solution of hydrogen peroxide from the bottle. I had taken 100ml of hydrogen peroxide using the measuring cylinder. Then I had poured the hydrogen peroxide from the measuring cylinder into the beaker containing water. While measuring the water and hydrogen peroxide I had used the lower meniscus. And while pouring the solution I had been careful that I don’t spill any water or hydrogen peroxide on myself or outside the beaker. After that I had transferred 100 ml of hydrogen peroxide solution from bottle to the beaker using a measuring cylinder, while doing so I made sure that I measured the volume using the lower meniscus. Than I had used the pipette to take 4 ml of hydrogen peroxide from the beaker and put it in the boiling tube. Like in the measuring cylinder I had taken the lower meniscus and also made sure that I don’t take in hydrogen peroxide by mistake. Than I had used the cork borer and cut the whole cylindrical piece of potato. I had placed the cork borer on the table so that I won’t get hurt. After that using the ruler I had measured the length of the potato and make it 4 cm by cutting it using a knife. Then I had filled trough and measuring cylinder with water. While filling the measuring cylinder I had ensured that the measuring cylinder was completely filled with water and that there wasn’t any air bubbles inside the measuring cylinder. I had then placed one end of the delivery tube inside the measuring cylinder and the other end inside the hole of rubber tubing. After doing so I had filled the water bath with water and then I had switched it on. After that I kept the boiling tube in the water bath for 2 minutes so that heat is supplied nicely to it. I had than place the thermometer inside the boiling tube where the solution of hydrogen peroxide is present making sure that it wasn’t touching the boiling tube. As soon as the temperature was reached I had removed the boiling tube out of the water bath and taken the thermometer out from the boiling tube. Then I had inverted the measuring cylinder and put it inside the water filled trough making sure that the measuring cylinder is full of water and there isn’t any air bubbles in it. As soon as I had finished doing it I had putted the slice potato inside the boiling tube and putted on the stopper and started the stopwatch. I had than continuously shaken the boiling tube. After three minutes I had measured the volume of the oxygen obtained in the measuring cylinder by the downward displacement of the water.
While carrying out the final experiment I will do the following improvements: -
- I will cut the potato into smaller pieces so as the surface area is increase and more Catalase is expose.
- I will use small delivery tube so that the distance to be travelled for oxygen is reduced and the maximum amount of oxygen is obtained.
- I will use a wider range of temperatures so as to obtained better results.
OBTAINING EVIDENCE
List Of Apparatus & Chemicals
The following are the apparatus and chemicals, which I will be using to obtain my final readings: -
- Thermometer
- Lab Coat
- Water bath
- Measuring cylinder
- Gloves
- Pipette
- Test tubes
- Rubber tubing
- Knife
- Ruler
- Stopwatch
- A cork borer
- Boiling tubes
- Goggles
- Trough
- Delivery tube
- Lab Coat
- Hydrogen peroxide
- Potato
- Water
- Buffer Solution
Method
Before starting the experiments I had wore the lab coat, goggles and the gloves so as to ensure my own safety. Besides that I had also tied my hair.
First I had made the 15% concentrated solution of hydrogen peroxide. This I had done by first taking 100ml of distilled water in the measuring cylinder from the bottle and poured into the beaker. Getting over with this I had taken 30% concentrated solution of hydrogen peroxide from the bottle. I had taken 100ml of hydrogen peroxide using the measuring cylinder. Then I had poured the hydrogen peroxide from the measuring cylinder into the beaker containing water. While measuring the water and hydrogen peroxide I had used the lower meniscus. And while pouring the solution I had been careful that I don’t spill any water or hydrogen peroxide on myself or outside the beaker. After that I had transferred 100 ml of hydrogen peroxide solution from bottle to the beaker using a measuring cylinder, while doing so I made sure that I measured the volume using the lower meniscus. Than I had used the pipette to take 4 ml of hydrogen peroxide from the beaker and put it in the boiling tube. Like in the measuring cylinder I had taken the lower meniscus and also made sure that I don’t take in hydrogen peroxide by mistake. Than I had used the cork borer and cut the whole cylindrical piece of potato. I had placed the cork borer on the table so that I won’t get hurt. After that using the ruler I had measured the length of the potato and make it 4 cm by cutting it using a knife. Then I had filled trough and measuring cylinder with water. While filling the measuring cylinder I had ensured that the measuring cylinder was completely filled with water and that there wasn’t any air bubbles inside the measuring cylinder. I had then placed one end of the delivery tube inside the measuring cylinder and the other end inside the hole of rubber tubing. After doing so I had filled the water bath with water and then I had switched it on. After that I kept the boiling tube in the water bath for 2 minutes so that heat is supplied nicely to it. I had than place the thermometer inside the boiling tube where the solution of hydrogen peroxide is present making sure that it wasn’t touching the boiling tube. As soon as the temperature was reached I had removed the boiling tube out of the water bath and taken the thermometer out from the boiling tube. Then I had inverted the measuring cylinder and put it inside the water filled trough making sure that the measuring cylinder is full of water and there isn’t any air bubbles in it. As soon as I had finished doing it I had putted the slice potato inside the boiling tube and putted on the stopper and started the stopwatch. I had than continuously shaken the boiling tube. After three minutes I had measured the volume of the oxygen obtained in the measuring cylinder by the downward displacement of the water.
Results
The table below shows the result, which I have got for the experiment: -
ANALYSE SECTION
Summary Result
The table above shows the average volume and the rate of the oxygen produced. First the rate of reaction increases as the temperature increases until the optimum temperature. At the optimum temperature the volume and rate of the oxygen produced are maximum. Then as the temperature is increased the rate of the reaction decreases.
Graphs
Trends And Patterns In The Graphs
For the temperatures 25 ºC to 40 ºC the volume of oxygen produced increases. Then the volume of the oxygen produced decreases from the temperature 45 ºC to 55 ºC. The maximum volume of oxygen produced is at 40 ºC. The graph doesn’t have a perfect curve does showing that there are some errors in the results.
Graph
Trends And Patterns In The Graphs
For the temperatures 25 ºC to 40 ºC the rate of oxygen production increases. Then the rate of the oxygen produced decreases from the temperature 45 ºC to 55 ºC. The maximum volume of oxygen produced is at 40 ºC. The graph doesn’t have a perfect parabola does showing that there are some errors in the results.
Scientific Explanation Of Result
The above results show that enzyme (Catalase) works best at the temperature 40 ºC, which is the optimum temperature for it. This is shown by the maximum volume and rate of the oxygen production. At temperatures below the optimum temperature the enzyme (Catalase) doesn’t work at maximum because the enzyme and substrate doesn’t have enough kinetic energy, which can be converted into chemical potential energy of the molecules. The number of collisions between the enzyme and substrate molecules is also less due to which less number of enzyme–substrate complex are formed. Which reduces the number of water and oxygen molecules being formed. When heat is supplied the rate of reaction increase because more molecules gets an increase in kinetic energy due to which more of the energy is converted into chemical potential energy. As the molecules had got more kinetic energy there is an increase in the movement of the particles due to which there are more collision. As the number of collision has increased there are more enzyme and substrate molecules colliding with each other thus forming more enzyme substrate complex. Due to which more hydrogen peroxide molecules are broken down to water and oxygen at a faster rate.
When the optimum temperature is reached there are maximum number of collisions and the number of enzyme-substrate complex are the greatest, due to which the rate of reaction is the greatest.
As the temperature is increased the internal energy of the molecules (enzyme and substrate) increases. If the internal energy is high enough some of the weak bonds that determine the three-dimensional shape of the active proteins are broken. This could lead to a thermal denaturation of the protein and thus inactivate the protein. Due to which the rate of reaction decreases.
Conclusion
The results show that the rate of reaction increases as the temperature increases and that the enzyme works best at a particular temperature and then if the temperature is increased the rate of reaction decreases. Because I got the results the way I had hypothesised. I believe that my results prove my hypothesis.
Evaluation
I carried out the experiment on how the temperature affects the rate of enzyme-controlled reaction. For this experiment I used the enzyme Catalase obtained from the potato and hydrogen peroxide as a substrate. I maintained the pH, enzyme concentration, substrate concentration, time and temperature, through out the experiment. For each new experiment I had changed the temperature of the enzyme.
I have got the results as I have expected though there are some errors in the results. These errors are shown by the curve not been a smooth parabola as it should have been. At the low temperatures the rate of reaction is slow but as the temperature is increased the rate of reaction for the enzyme increases. At 40 ºC the rate of reaction is maximum because it is the enzyme’s optimum temperature. After that temperature the rate of reaction decreases, as the enzyme’s molecules starts to get denature.
Limitations
My results were accurate and reliable but not 100%. I can say that the results weren’t accurate because the results of my repeated experiments are exactly the same. This is because of the limitations, which I had during the practical. The first limitation is the usage of measuring cylinder instead of gas syringe. This I believe because the inverting of the measuring must have lost small volume of oxygen. Thus I didn’t get the exact volume of the oxygen produced
I believe my second limitation was the usage of long delivery tube. The long delivery also affected the volume of oxygen produced. This is because in the long deliver tube it took the oxygen molecules more time to come to the measuring cylinder than it would have taken if the delivery tube had been short. So in more time less oxygen was collected
The third limitation I believe was the less range of temperatures, which I took for the experiment. I believe that if I had taken more range of temperatures with out regular intervals, I would have gotten more accurate optimum temperature.
Reliability And Accuracy
I believe that my results are reliable and though not completely accurate. This is because the majority of my results are the way the should be i.e. to say that the rate of reaction increases with temperature; works best at an optimum temperature; and then the rate of reaction decreases as the temperature is increased. Though they are reliable I won’t say that they are completely accurate. This is because the graph curves are not completely smooth as they should be.
Improvements
The following are the improvements I will be doing if I carried out the experiment again. The first improvement will be that I will use a permanent set-up instead of the set-up, which I had used. So that time is not wasted while inverting the measuring cylinder due to which some oxygen molecules would have been lost.
Second improvement will be doing is the usage a shorter delivery tube instead of long delivery tube. So that oxygen can easily be collected in the measuring cylinder.
The third improvement I will like to do is take greater range of experiment instead of taking the temperatures of fixed regular intervals. This is will give me more better results.