All enzymes are globular proteins, each with a specific three- dimensional tertiary structure that determines its function. Enzymes are usually specific to the substance- or substrate- they work with and so the shape of the enzyme’s active site- where the substrate binds - has to be complementary to the shape of the substrate.
In the case of this reaction, the enzyme (rennin) is catalysing the breakdown of the substrate (milk protein- caseinogen)
Whether it is induced fit or the lock and key which occurs, the result is the same: once the enzyme has served its purpose, it is released from the product formed:
Enzyme + Substrate Enzyme-substrate complex Enzyme + Product
As previously mentioned, in order for an enzyme-substrate complex to form, the shapes of the active site and the substrate have to be complementary. The shape of the active site is determined by the tertiary structure which is the three- dimensional arrangement of polypeptides. This arrangement is stabilised by ionic, disulphide and weak hydrogen bonds.
In this investigation, temperature was the independent variable and is known to have a great influence over enzyme activity. For example, until the temperature was raised between 35 and 39ºC, the graph shows an increase in the rate at which the insoluble casein was formed from the rennin-milk enzyme substrate complex; it rose from 4.4 to 7.5/s-1 x1000. This occurred as a result of the molecules gaining more kinetic energy causing them to move faster, and therefore increasing the chances of the enzyme colliding with the substrate and producing casein. Similarly between 39 and 44ºC, the mean rate rose from 7.5 to 9.2/s-1 x1000 and although this followed the general trend of an increasing rate, it did not increase as much as it did between the previous range. Once the temperature had been raised to 49ºC, the optimum rate of 13.9/s-1 x1000 had been reached for the rennin and here we could say that the molecules had been given the maximum amount of kinetic energy. As a result the enzyme molecules were colliding with the substrate molecules faster to produce a greater number of rennin-caseinogen complexes within a shorter space of time.
However, although the temperature increased up to 60ºC, the rate began to decrease rapidly after 49ºC. This is because the rennin particles began to vibrate and this caused strain upon the ionic bonds which became so great that the bonds broke. Consequently, this caused the shape of the tertiary structure to alter- and therefore the shape of the active site. As the shape of the enzyme’s active site had been altered- or denatured- it was no longer complementary to the substrate- or caseinogen and so no enzyme-substrate complexes could form, and in turn no products- insoluble casein- could form, therefore bringing the rate down to 0/s-1 x1000.
Evaluation:
Although the graph shows the general trend expected when temperature is the independent variable on enzyme activity, there were some anomalous results within the mean times used to calculate the rates.
For the reactions at 35ºC I did not highlight any anomalies as although the times recorded ranged from 180 seconds to 255, the mean result was 226 and this falls somewhere in the middle of this range. Furthermore, although 180 seconds seems very low in comparison with a mean result of 226 seconds, two groups recorded this time and so I believe it is no completely anomalous. For the rennin-caseinogen complexes incubated at 39ºC, there were two clear anomalies in comparison with the mean time of 134 seconds; one anomaly was 90 seconds and the other 285 seconds. The result of 285 seconds seems especially slow considering that at 39ºC you would expect the molecules to have a large amount of kinetic energy. The next anomalous results were for the complexes incubated at 44ºC where the mean time taken for the milk to solidify was 109 seconds. The anomalous results were 180 and 225 seconds which again are very high, especially the rest of the results for this temperature ranged from 60 to 120 seconds. The final anomaly came from group 2 who were the only group to record a time of 105 seconds for solidification of milk when incubated at 49º.
There were no anomalous results for 60ºC as all groups recorded ‘no reaction’ for this temperature.
The most significant reason for these anomalous results came from the variation of end points between groups. For example some groups may have waited until all the milk protein had gone solid before recording the time whereas others may have waited until the milk began to show signs of solidifying. One such high anomalous result – or low anomalous in terms of the rate- came from groups 4’s reading of 285 seconds when the milk was in a water bath of 39ºC. This group may have waited until all the milk solidified. The reason for ranking this first is that it impacts on the mean rate so much. The entire investigation is based on the effect of temperature on enzymes through observing the rates of product formation and if there are differences on the way the times are recorded, this influences the outcome and the findings more than a small range of temperature etc. An improvement which would help prevent this would be to standardise this by telling each group to tilt the test tube 90º and only record the time when there is no liquid left.
The second most significant procedural error would have been the exclusion of using a pH buffer. Similarly, as with temperature, pH affects enzyme activity as if it is too high or too low, the ionic and hydrogen bonds within the tertiary structure break causing the active site to denature and this would prevent the rennin from binding to the milk protein. This would result in a low rate- or even no reaction at all- and a high anomalous recorded time. For example this could explain the anomalous result which came from groups 2 and 4 who recorded times of 180 and 225 seconds respectively for 44ºC. I have ranked this second as pH has a significant effect on enzyme activity and variations between the pH values of solutions would cause variation in the rate of enzyme activity and for this investigation, only temperature is supposed to be an independent factor. As the optimum pH for many enzymes is pH7, the solution for this could be to ensure that all groups added a buffer tablet of pH7 to their rennin before combining and incubating with the milk.
The third most significant reason for any anomalous results could again be down to differences in procedural technique between groups. The rennin and milk were combined after incubating separately but whereas some groups would have shaken the tube to ensure a homogenous solution, others may not have. As a result some enzyme molecules would have just remained on top of the solution and when heated would not have been able to bind with the caseinogen. This may have been the case for group 2 who recorded 105 seconds for 49º whereas the mean was 72 seconds. The only way of improving this would be to ensure that all groups made an attempt to homogenise their solutions by shaking thoroughly before heating. However, the reason for ranking this third is that the heat would have caused the molecules to gain more kinetic energy and to therefore the probability of them colliding to form rennin-milk complexes would be dependant on this.
The least significant reason for anomalous results would be biological variation between the milk samples used. This could be an error as here may be differences in pH between different breeds or individuals. However I believe this is unlikely to cause any major anomalies as all groups were told they were using full fat milk and furthermore, the samples of milk would not have some from single cows, but from a batch which would have been mixed to produce a homogenous solution.
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