The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H+ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.
Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H+ and OH- ions. The hydrogen ion (H+) left over can replace that lost by Glu-35.
In either case, the chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location on the bacterial cell wall and continue its work of digesting it.
FACTORS AFFECTING ENZYME ACTION
The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of
-
(i.e. shape) in enzyme function and
- noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.
Examples:
- the protease pepsin works best as a pH of 1-2 (found in the stomach) while
- the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).
Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalytic action of lysozyme would cease.
Increasing temperature easily disrupts hydrogen bonds. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become at high temperatures.
REGULATION OF ENZYME ACTIVITY
Several mechanisms work to make enzyme activity within the cell efficient and well coordinated.
Anchoring enzymes in membranes
Many enzymes are inserted into cell membranes, for examples,
-
the
-
the membranes of and
- the endoplasmic reticulum
- the nuclear envelope
These are locked into spatial relationships that enable them to interact efficiently.
INACTIVE PRECURSORS
Enzymes, such as proteases, that can attack the cell itself are inhibited while within the cell that synthesizes them. For example, pepsin is synthesized within the (in gastric glands) as an inactive precursor, pepsinogen. Only when exposed to the low pH outside the cell is the inhibiting portion of the molecule removed and active pepsin produced.
FEEDBACK INHIBITION
If the product of a series of enzymatic reactions, e.g., an amino acid, begins to accumulate within the cell, it may specifically inhibit the action of the first enzyme involved in its synthesis (red bar). Thus further production of the enzyme is halted.
PRECURSOR ACTIVATION
The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.
In the case if a molecule is regulating feedback inhibition and precursor activation, the activity of the enzyme which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.
- In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.
- In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.
REGULATION OF ENZYME SYNTHESIS
The four mechanisms described above regulate the activity of enzymes already present within the cell.
What about enzymes that are not needed or are needed but not present?
Here, too, control mechanisms are at work to regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on - or off - the .
If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.
Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase - by transcribing and translating the necessary gene(s) - and so can begin to metabolize the sugar.
E. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA.
SOUDEH MASHAYEKHI
Preliminary (BIOLOGICAL & NON-BIOLOGICAL WASHING POWDERS)
Aim: To compare the action of two powders to help decide whether biological or non-biological enzymes are better at removing stains.And to also see how temperature affects their activity.
Hypothesis
I predict that a non-biological enzyme will be better at removing the stain. A non-biological enzyme reacts quicker and I believe will help get rid of the stain faster.
Quantitative Prediction
I predict that the more I warm the enzymes, the faster the enzymes start to work on the stain. However, after 40 degrees celsius enzymes begin to denature and it is at this point in which I believe the speed of enzyme action will decrease and eventually stop removing the stain.
Apparatus
- Stop Watch
- Bio washing powder
- Non-bio washing powder
- Top pan balance
- Bunsen burner
- Large ball of string
- beaker
- thermometer
- goggles
Method
Place the apparatus on the table and begin to cut the ball of string into 15, 5cm pieces. Place five pieces aside as your controls. Then cover the remaining 10 pieces with 3cm of ketchup each.
Fill a beaker with 250 ml of water and begin to heat. Place a thermometer in the beaker.
Place the string in the water and pour 10g of biological washing powder inside, stirring for around 3 minutes.
Raise the temperature to 30, 40, 50, 60 and 70degrees, whilst looking at the thermometer at eye level. Remember to wear safety goggles and keep well away from the flame. Also take care when handling hot items like beakers and tongs. And wear a lab coat during the whole of the experiment.
Write down your results and continue this experiment for a non-biological washing powder.
Compare these results to the control and repeat the experiment for more accuracy.
Conclusion:
After looking at my results it is clear to me that my prediction was correct. A non-biological washing powder is better at cleaning stains and acts faster on them. However, after 40 degrees celsius, the enzymes begin to denature and no longer have any affect on the stain and by 70 degrees celsius the stain has returned to it's original colour.
TEMPERATURE AS A FACTOR
Aim: -To investigate how temperature effects enzyme activity.
Hypothesis:
I predict that the optimum temperature of enzyme activity will be about 40 degrees Celsius. I have reached this conclusion from the kinetic theory. The kinetic theory states that when more energy is given to molecules, they move faster and collide more.
And in order for a substrate to be broken down by an enzyme collision is needed. As the temperature increases, the speed of collision will increase and far more substrate will be broken down. However, after 40 degrees celsius, the enzyme will denature and lose it's shape. This way it will no longer fit into the active site shape and the activity will come to a halt.
Dependant variable: - Time (minutes) we changed the time to see how long the reaction took.
Control Variables: - pH of substance
Concentration of Diastase
Concentration of starch solution
Independent variable: - We measured the temperature (degrees celsius)
Apparatus
- Tongs
- Glass rod
- Water
- Iodine
- Starch(25cm3)
- Diastase(25cm3)
- 10 Test tubes
- Thermometer
- Test tube rack
- Syringe
- Stop watch
- Gauze
- Tripod
- Clay mat
- Bunsen burner
METHOD
Take 5cm3 of starch solution and also 5cm3 of diastase. Add 3 drops of iodine to the starch solution and shake completely until the colour changes to a blue- black colour.
Get the stop -watch ready and start timing after adding the diastase to the starch solution. As the lab is already around 20 degrees celsius, there is no need to heat the first solution. The timing must be quick and accurate as the enzyme will have an affect on the starch and break the active sites.
Continue timing until you see that the solution has become completely colourless.
Now you must continue the experiment for 30,40,50,60 and 70 degrees celsius. This will offer you with a wide range of results from cold to hot temperatures and you may see any obvious changes. Remember to wear safety goggles and keep well away from the flame. Also take care when handling hot items like beakers and tongs. And wear a lab coat during the whole of the experiment.
Heat a water bath on top of the bunsen burner until 30 degrees. Place the diastase and starch test tubes into the water bath and allow 3 minutes for the temperature to change, to make sure they are all the same temperature for a fair test.
Then pour the diastase into the starch test tube and begin timing again.
Remember to wear safety goggles whilst using the bunsen burner and record the time it takes for the mixture to go colourless.
Check the temperature at eye level and continue the experiment one by one till 70 degrees celsius.
Conclusion
After doing this experiment it is clear that my prediction was correct. The optimum temperature of enzyme activity was about 40 degrees Celsius. When more energy was given to the molecules, they moved faster and collided more. And in order for the substrate to be broken down by an enzyme collision was needed. As the temperature increased, the speed of collision also increased and far more substrate was broken down. However, after 40 degrees celsius, the enzyme denatured and lost it's shape. This way it no longer fit into the active site shape and the activity came to a halt.
Evaluation
My results were Ok as a whole. They showed that the optimum temperature was 40 degrees celsius. However, like most experiments there was an anomalous value. At around 50 degrees celsius, the enzyme took around 10 minutes to break down the starch. I believe our timing may have been inaccurate or the temperature reading might have been wrong too. We could have used more computerised equipment for our experiment like digital thermometers to produce more reliable and accurate results.
We could have also been more precise when measuring the enzyme (there may have been too little) or even the substrate (may have been too much.) We could have tried harder in checking the temperature correctly. In one 0f the occasions I remember we only wrote down a rough temperature and did not look at the thermometer at eye level which may explain the anomalous result. In the future I would like to repeat the experiment using a different enzyme like Trypsin or pepsin to see if my conclusion follows for any of the other known enzymes and try to reach a more clear overall conclusion towards enzyme behaviour in the presence of temperature.
My graph is trough shaped. This shows the optimum value is 40 degrees, and it is at this point that the time starts to increase again. We could have been more precise in different measurements. But because we repeated the experiment our results are now more reliable. We could have also used a broader range of temperatures to enable even better results. And repeating this experiment a few more times could help prevent inaccuracies.
CONCENTRATION AS A FACTOR
Aim: - To investigate how the concentration of a substrate and enzyme effects enzyme activity.
Hypothesis
I predict that the more starch solution, the slower the reaction. And the less starch solution, the faster the reaction and the results are proportional. If there are more starch molecules, there are only a few active sites present to break them down. This way the enzyme must break down the starch molecules one by one and will take far longer.
Dependant Variable : -
We measured the Time (minutes)
Independent Variable: -
We changed the concentration (cm3)
Control Variables: -
pH
Concentration of diastase
Temperature (degrees celsius)
Apparatus
- 10 test tubes
- Syringe
- Iodine
- Starch solution(25cm3)
- Diastase(25cm3)
- Test- tube rack
- Stopwatch
- Glass stirring rod
Method
Measure out 5cm3 of diastase and 10cm3 of Starch solution. Then mix them together with iodine and stir thoroughly. Keep the temperature at 20 degrees (normal lab temperature). Continue timing until you notice the solution has become completely colourless. This shows that the enzyme has broken down the starch solution.
Now change the concentration of the starch solution to 20cm3 and repeat the experiment.
Continue until 60cm3, as this will provide you with a wide range of varying results that can help you draw to a correct conclusion.
Conclusion
By looking at the results in the table above, I have come to the same result as my hypothesis. My results show that the greater the concentration of the substrate, the longer it takes for the enzyme to break down the substrate. This is due to the fact that, when there are more substrates, there are less active sites to break them down. This way the enzyme must break down the starch molecules and lock into them one by one and there are fewer collisions taking place at this time. Therefore there will be less kinetic energy and the reaction will take far longer.
Evaluation
My results have turned out better than I expected. The results seem to be correct and there are no anomalous results. However, I noticed that the experiment took very long. This may have been due to the fact of the low temperature that we used. I believe if we changed the temperature to 40 degrees (which is the optimum) the experiment would be faster and with better results. We could also repeat the experiment to make our results more accurate. A few repetitions would help draw to a good conclusion.
A number of our measurements were rough and imprecise which could cave also affected our findings. I also noticed that we were not careful when using the syringe and we were not 100% careful not to mix the different substances with the same syringe. If they had been mixed, the enzyme would work quicker and give wrong results. We should have used separate syringes to ensure better results.
In future I would like to use a broader range of concentrations for this experiment to see how the results would further change. I would like to carry on the experiment till 120cm3 to find out when the enzyme becomes saturated. I also look forward to doing other experiments using different enzymes to see if concentration change has the same affect on them.
pH as a factor
Aim: - To investigate how pH level effects enzyme activity.
Hypothesis
I predict that to a certain level, an increase in pH will increase the speed of enzyme activity. This is because as proteins, enzymes are sensitive to pH levels. As pH levels decrease (to extreme acidic conditions), the enzymes will denature from their active sites and will no longer fit in the substrate shape. I believe if the environment is too alkali for the enzyme it will also denature. Neutral or slightly acidic/alkali conditions would be the best for the enzyme.
Dependant Variable: -
We measured the Time of reaction (minutes)
Independent Variable: -
We changed the pH
Control Variables: -
Concentration of starch solution
Concentration of diastase
Temperature (degrees celsius)
Apparatus
- 10 test tubes
- Syringe
- Iodine
- Starch solution(25cm3)
- Diastase(25cm3)
- Test- tube rack
- Stopwatch
- Glass stirring rod
- Acid
- Alkali
Method
Pour 5cm3 of diastase in each of the 5 test tubes and 5 more cm3 of starch in the other 5 test tubes. Take the starch solutions and start pouring the acids and alkalis. You must be extremely careful whilst dealing with these extreme acidic and alkali conditions. Thoroughly clean the work area to ensure you are far from harmful conditions. Take care of your eyes and hands when dealing with acids and alkalis.
The first one should be filled with 2cm3 of pH 3acid.
The second one should be filled with 4cm3 of pH 4 acid.
One should be 3cm3 of neutral liquid.
And the other 2 should have pH 8 (1cm3) and pH 11 (0.5cm3). (I have used a wide range of values for a more accurate result)
Now pour 3 drops of iodine into all the beakers, and pour the diastase once they have turned black. Make sure all test tubes have been filled with diastase and time how long it takes for them to go colourless again.
Conclusion
By looking at my results, it is clear to me that my prediction was correct. There is a trough in my graph, which shows that the optimum pH for diastase is indeed 7 and this enzyme works best in neutral form. This is because enzymes are made of proteins in their active sites, which are sensitive to pH level. The enzyme did not work particularly well in the very acidic or alkali form. However, each enzyme has a specific optimum pH. So, the use of different enzymes would give completely different results.
Evaluation
This experiment went rather well and we did not have any anomalies. However, a lot can be done to help further improve our results. We could repeat this experiment to make our results more accurate. We could also use more pHs, so as to have a broader range of results and to fully understand how different pHs might affect the enzyme. To ensure our results were correct, we could have done a better job in measuring. A number of our measurements were rough and imprecise which could cave also affected our findings. I also noticed that we were not careful when using the syringe and we were not 100% careful not to mix the different substances with the same syringe. If they had been mixed, the enzyme would work quicker and give wrong results. We should have used separate syringes to ensure better results. I also look forward to doing other experiments using different enzymes to see how temperature, concentration and pH affect other enzymes. This way we could make a rule as a whole for all enzymes and we cannot completely say that the optimum values for diastase apply for all other enzymes. Repetition always helps in producing better results.
FURTHER WORK
Aim: - To further investigate the effect of temperature on the enzyme Trypsin.
Hypothesis:
I predict that the optimum temperature of enzyme activity will be about 40 degrees Celsius. I have reached this conclusion from the kinetic theory. The kinetic theory states that when more energy is given to molecules, they move faster and collide more.
And in order for a substrate to be broken down by an enzyme collision is needed. As the temperature increases, the speed of collision will increase and far more substrate will be broken down. However, after 40 degrees celsius, the enzyme will denature and lose it's shape. This way it will no longer fit into the active site shape and the activity will come to a halt.
Dependant variable: - Time (minutes) we changed the time to see how long the reaction took.
Control Variables: - pH of substance
Concentration of Diastase
Concentration of starch solution
Independent variable: - We measured the temperature (degrees celsius)
Apparatus
METHOD
Take 5cm3 of starch solution and also 5cm3 of Trypsin. Add 3 drops of iodine to the starch solution and shake completely until the colour changes to a blue- black colour.
Get the stop -watch ready and start timing after adding the diastase to the starch solution. As the lab is already around 20 degrees celsius, there is no need to heat the first solution. The timing must be quick and accurate as the enzyme will have an affect on the starch and break the active sites.
Continue timing until you see that the solution has become completely colourless.
Now you must continue the experiment for 30,40,50,60 and 70 degrees celsius. This will offer you with a wide range of results from cold to hot temperatures and you may see any obvious changes. Remember to wear safety goggles and keep well away from the flame. Also take care when handling hot items like beakers and tongs. And wear a lab coat during the whole of the experiment.
Heat a water bath on top of the bunsen burner until 30 degrees. Place the diastase and starch test tubes into the water bath and allow 3 minutes for the temperature to change, to make sure they are all the same temperature for a fair test.
Then pour the diastase into the starch test tube and begin timing again.
Remember to wear safety goggles whilst using the bunsen burner and record the time it takes for the mixture to go colourless.
Check the temperature at eye level and continue the experiment one by one till 70 degrees celsius.
Conclusion
After doing this experiment it is clear that my prediction was correct. The optimum temperature of enzyme activity was about 40 degrees Celsius. When more energy was given to the molecules, they moved faster and collided more. And in order for the substrate to be broken down by an enzyme collision was needed. As the temperature increased, the speed of collision also increased and far more substrate was broken down. However, after 40 degrees celsius, the enzyme denatured and lost it's shape. This way it no longer fit into the active site shape and the collisions and kinetic energy decreased and the activity came to a halt.