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 (Pancreozymin) is catalysing the breakdown of the substrate (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.
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 33 and 40ºC, the graph shows an increase in the rate at which the insoluble casein was formed from the
Pancreozymin-milk enzyme substrate complex; it rose from 8.3% to 49.0%. 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. Once the temperature had been raised to 40ºC, the optimum rate of 49.0% had been reached for the Pancreozymin 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 Pancreozymin-caseinogen complexes within a shorter space of time.
However, although the temperature increased up to 50ºC, the rate began to decrease rapidly after 40ºC. This is because the Pancreozymin 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. This caused a decrease in the amount of enzyme-substrate complexes formed.
Evaluation:
The 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 bonds would break causing the active site of the enzyme to denature and this would prevent the Pancreozymin from binding to the milk protein. The anomalous result occurring at 30oc which was too high at 12% on the third time when repeated could be due to this. I have ranked this first 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. To overcome this I believe using a ph8 buffer tablet would stop the fluctuation of the PH.
The Second most significant reason for any anomalous results could be due to not mixing the enzyme and substrate accurately. As a result some enzyme molecules would have just remained at the bottom of the solution and when heated would not have been able to bind with the caseinogen. This may have been the cause for the anomaly at 30oc which was too high at 12% on the third time when repeated the way to overcome this problem if to make sure that each mixture is shaken thoroughly for a set time. However, the reason for ranking this second is that the heat would have caused the molecules to gain more kinetic energy and to therefore the probability of them colliding to form Pancreozymin-milk complexes would be dependent on this and not on how well the mixture is mixed.
A scratched cuvette could also cause anomalies, as it may affect the path of light. It would lessen the intensity of the transmitted light and would result in an error in the reading of the absorbance. This may have been the reason for the anomalous result at 35oc where the value was too low at 18% when first doing it. To overcome this problem you must make sure that the cuvette it not scratched and make sure that there is nothing on the cuvette that would affect the path of light.
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. This may have caused the anomaly at 50oc which was too high when first tried. However I believe this is unlikely to cause any major anomalies the milk would be from a batch which would have been mixed to produce a homogenous solution.
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