The effect of an inhibitor on sucrase activity.

Nang

The effect of inhibitor on sucrase activity

Introduction

Sucrase is an enzyme which catalyses the hydrolysis of sucrose into glucose and fructose. The products of the reaction are reducing sugars (glucose and fructose). Like many enzymes, sucrase is a globular protein and its structure is maintained by hydrogen bonds, disulphide bridges and the ionic bond. Sucrase has a specific site called active-site where substrate molecules (sucrose) bind to form enzyme-substrate complex. The shape of the active-site is so specific that only sucrose can fit in. This is called lock and key mechanism and suggests that one enzyme can only catalyse one reaction. However, enzyme slightly changes its shape when substrate binds to its active- site. This process is known as an induced fit. The presence of these molecules can be tested by using Benedict’s solution. The reducing sugar means that it can reduce the copper (II) in Benedict’s solution into copper (I) which is red precipitate. In this experiment, we are looking at the effect of inhibitor on sucrase activity. Inhibitor is a substance which slows down the rate of enzyme controlled reaction.

Hypothesis

The rate of formation of glucose and fructose will slow down with the presence of silver nitrate solution compared with the rate of reaction which does not contain silver nitrate.

Reason

Silver nitrate is used as inhibitor in this experiment. Silver nitrate contains a heavy metal ion silver ion which acts as an inhibitor. This kind of inhibitor is know as non-active-site directed inhibitor because it does not compete with sucrose for the active-site, but binds to the other site on enzyme known as allosteric site. This alters the overall shape of molecule of enzyme and thus changes the specific shape of the active-site. So sucrose can no longer combine with enzyme to form enzyme-substrate complex, resulting in the decrease in the formation of glucose and fructose.

Method

The experiment is carried out according to the procedure.
Two test tubes of the same volume are prepared. One test tube contains silver nitrate but another test tube contains the same volume of distilled water. The one with distilled water acts as a control. This test tube will undergo at the normal rate of enzyme controlled reaction. The test tube with silver nitrate will have a slower rate in forming reducing sugar because silver nitrate will slow down the activity of sucrase.
Enzymes are sensitive to the change in temperature. At very low temperature, the kinetic energy of molecules will be so small that the rate of reaction will be very slow. At very high temperature, the heat energy supplied will starts breaking the bonds holding the tertiary structure of the enzyme. Finally, the enzymes will loose their specificity and become denatured and can no longer catalyse the reaction. So it is important to keep the temperature as constant as possible at the optimum temperature. It is the temperature at which enzymes work most efficiently that is the rate of reaction is the fastest. In this experiment, the temperature is kept at 35°C which is assumed to be the optimum temperature for sucrase.
The pH of the solution is also need to be constant at the optimum pH for sucrase. Change in pH alters the ionisation of R-groups in amino acid in enzymes. At extreme pH, the bonds holding the tertiary structure will starts to break and so will the shape of the active-site. So the enzyme is said to be denatured because it can no longer bind with substrate molecules to form enzyme-substrate complex. In this experiment, pH is kept constant by using buffer solution which is pH 5. This is assumed to be the optimum pH for sucrase, pH at which sucrase work best and the rate of reaction is the fastest.
The concentrations of enzyme and substrate also affect the rate of enzyme controlled reaction. These also need to be kept under controlled. Increase in concentration of enzyme or substrate will increase the rate of the reaction and vice versa. By using the same volume for both enzyme and substrate, the variations are controlled.
The reaction media are incubated in water bath until they reach 35°C. This is done to ensure that both the reactions are taking place at known temperature. The temperature of water bath is kept as constant as possible to minimise the temperature fluctuation.
The same volume of Benedict’s solution is used for every test for the presence of reducing sugar. The interval of time for heating Benedict’s solution and the reaction time for sucrase on sucrose are both kept constant. So that we can compare the amount of reducing sugar being formed from the two test tubes (one with silver nitrate and the other without silver nitrate) over the same period of time. Different pipettes are used for the water and silver nitrate tubes so the control tube does not become contaminated with silver nitrate.

Tables to show the concentration of reducing sugar in (moldm-3) produced over a period of time

Table 1

Table 2

Analysis

This is a preview of the whole essay

Tables to show the concentration of reducing sugar in (moldm-3) produced over a period of time

Table 1

Table 2

Analysis

The concentration of reducing sugar being formed increases as the reaction proceeds. The gradient of the graph of water containing test tube (T1) is much greater than that of the graph of silver nitrate containing test tube (T2) because (T1) graph is steeper than (T2) graph. This shows that more reducing sugar is being formed from non-inhibited reaction compared with inhibited reaction over the same period of time. The rate of formation of enzyme-substrate complex is higher without the presence of silver nitrate because all the active-sites are available for sucrose molecules to bind and form enzyme-substrate complexes. Enzymes are working at their maximum rate so more and more products are formed as the reaction proceeds. But with the presence of silver nitrate, very few products are formed. This is because silver nitrate binds to sucrase at allosteric sites and causes the change in shape of the enzymes which in turn alter the specific shape of the active-sites. Fewer and fewer active-sites are available for sucrose as the reaction proceeds, resulting in fewer enzyme-substrate complexes. Therefore fewer glucose and fructose are formed.

For the reaction in the medium without silver nitrate, we expect the graph to flatten off after a certain period of time. But this reaction cannot give the graph this shape because it needs a longer time for the experiment. At this particular point where the graph flattens off, there can be three possible reasons.

All the substrate molecules are used up during the reaction. The volume of the substrate is constant in the experiment. So this acts as the limiting factor for the reaction. All the sucrose molecules are broken down into glucose and fructose at this point and thus no more products are formed beyond this point.
The reaction might reach the equilibrium. This occurs when the rate of forward reaction (formation of glucose and fructose) balances the rate of reverse reaction (the break down of glucose and fructose). In this case, there is no net movement forward or reverse if the equilibrium is reached. So there is a constant rate of reaction which would be indicated by the horizontal line of the graph.
Another possible condition is that the reaction reaches the end-point inhibition. This would also be indicated by the horizontal line of the graph.

For the reaction with silver nitrate, first the graph increases slowly because silver nitrate binds to the allosteric sites of enzymes and thus the active-sites of enzymes are being changed. Therefore the rate of formation of enzyme-substrate complexes is much slower than that without the silver nitrate. This indicates that fewer glucose and fructose are being produced from the reaction with silver nitrate which in turn shows that the activity of enzyme is being inhibited by the inhibitor (silver nitrate). The graph flattens off after 20 minutes. This might be that all the sucrase molecules are bound with silver nitrate and their active-sites have been changed. So they can no longer bind with sucrose to form enzyme-substrate complexes and no glucose and fructose are produced.

There are slight deviations for the graph without the silver nitrate at 15 minutes and 20 minutes. It is slightly less than the expected result. This might be due to the time delay between transferring the solution into the Benedict’s solution. The test tubes might not be left in the boiling water long enough which is supposed to be 5 minutes. The volume being added into the Benedict’s solution might not be exactly 0.3 cm3.

There are two anomalies for the graph with silver nitrate. One is at 10 minutes and the other is at 15 minutes. The two values are the same for both timings. We expect both of the valued to be more. This might be that the result is taken as a group average and so it is not an individual value. Different students will get different values for the same interval of time. There might be a timing delay between transferring the solution into the Benedict’s solution and setting the clock. The time allowed for the reaction between the sucrose and sucrase and/or for boiling the Benedict’s containing test tubes is not accurate. The number of trails is not the same for all the students, some might do the experiment for 5 trails but some do it for 6 trails.

Evaluation

There was a difficulty in controlling the temperature of the water bath at 35°C. Although the hot water from the tap was used, the temperature of the water kept falling over a period of time. If some more hot water was added, the temperature rose more than 35°C. So there was a temperature fluctuation in the experiment. This can be minimised by using the electronic water bath.

We could not transfer the enzyme into the two media at the same time. So there was a time delay between them. So the reaction time was not the same for the two test tubes. This problem can be overcome by doing in a group. One student can do one test and another can do the other test so that enzyme would be added into the two test tubes simultaneously.

The enzyme was not incubated to the same temperature as the media. When enzyme was added, the temperature of the enzyme was lower than that of the media. This might give some anomalies for the graph. This can be overcome by incubating all the enzyme, substrate, buffer, distilled water and silver nitrate before mixing. So the reaction would be taking place at the same temperature.

We did both the experiment and the control at the same time. This caused inaccurate timing. For example, we marked the time of the control while forgot to mark the time for the experiment. If the experiment and the control were done separately, we only needed to measure the time for only one experiment which would lead to more accurate result.

The experiment was not repeated and thus the average could not be taken for each temperature. If we could taken the average reading for every interval of time, we would get a more reliable graph. But we didn’t have enough time to repeat the experiment. We also could not get the result until the graph is flatten off which we expect to get. All this could be done if we were allowed a longer time.

There was another uncertainty where we had to compare the colour of our result to that of the standard solution. Some of our result did not perfectly match with the standard colour (e.g. the colour lies between 0.22 and 0.17moldm-3) and thus we took the reading between those two readings. This could possibly give us the anomalous points on our graphs. A more accurate reading could be obtained if we used the colorimeter to measure the colour of the solution. This would give us a more reliable result because the colorimeter would be calibrated each time the reading was taken.

There was the other way of measuring the concentration of reducing sugar. The precipitate could be filtered out and dried. We could measure the dry mass of the precipitate and work out the concentration of reducing sugar.

Other possible coursework

This experiment can be adapted to investigate the effect of the following factors on the enzyme activity.