AS1 Chapter 2 - Enzymes Flashcards
1
Q
What is an enzyme?
A
An enzyme is a biological catalyst that speeds up metabolic reactions. The enzymes themselves are not changed by the reaction and can be reused. Enzymes are globular proteins.
2
Q
Why do enzymes speed up the rate of a reaction?
A
Enzymes speed up the rate of a reaction by lowering the activation energy required for a reaction to take place.
3
Q
What is activation energy?
A
Activation energy is the energy required to overcome the energy barrier in a reaction.
4
Q
What is catabolism?
A
Catabolism is the breakdown of molecules
5
Q
What is anabolism?
A
Anabolism is the building up of molecules
6
Q
The substrate fits into a special site (groove) on the enzyme, the active site, and this forms the …
A
Enzyme-substrate complex (ES)
7
Q
How does the enzyme turn the substrate into its constituent products after the enzyme-substrate complex has been formed? What is the product formed?
A
The enzyme and substrate interact with bonds forming between some of the amino acids of the enzyme and parts of the substrate molecule. The reaction changes the substrate, it becomes an enzyme-product complex (EP), so that the product(s) no longer fit the active site and are released.
8
Q
What is enzyme specificity?
A
Enzyme specificity is the term used to describe the fact that each enzyme is specific to a particular substrate(s).
9
Q
What are the two models for enzyme action?
A
Lock and key model
Induced fit model
10
Q
What is the lock and key model?
A
The lock and key model of enzyme action proposes that the enzyme active site is an exact match to the shape of the substrate, ie the two are complementary shapes.
11
Q
What is the induced fit model?
A
The induced fit model proposes that rather than being an exact fit, the active site of the enzyme very closely matches the shape of the substrate.
The induced fit model proposes that the active site can mound itself around the substrate, forming a precise fit. The active site is therefore flexible and as it changes shape to fit the substrate, the enzyme is able to put pressure on the substrate, breaking particular bonds and therefore lowering the activation energy required for the reaction to take place. The products will be released, as they are a different shape to the active site when it returns to its ‘pre-reaction’ shape.
12
Q
What are cofactors?
A
Cofactors are non-protein substances that some enzymes require in order to function. Examples include metal ions which form attachments to the enzyme and change the shape of the active site, enabling the reaction to take place.
13
Q
What are the two types of cofactors?
A
Prosthetic groups
Coenzymes
14
Q
Give some examples of cofactors
A
Metal ions (Mg2+, Ca2+, Fe3+) Chloride ions (Cl-) in salivary amylase Haem in the enzyme catalase
15
Q
What are coenzymes?
A
Coenzymes are a particular type of cofactor. They are non-protein, organic molecules necessary for enzyme action. Unlike some other cofactors, they are not permanently attached. Coenzymes are very important in the biochemistry of respiration and photosynthesis. The coenzymes NAD and FAD act as hydrogen acceptors in respiration.
16
Q
What factors affect the rate of enzyme activity?
A
Enzyme concentration Substrate concentration Temperature pH Enzyme inhibitor concentration (if any)
17
Q
How and why does temperature affect the rate of enzyme activity?
A
Increasing temperature gives both the substrate and the enzyme molecules more kinetic energy. This increased kinetic energy means that the molecules are able to move around faster, increasing the possibility of collisions between enzyme and substrate, and the formation of enzyme-substrate complexes.
Above the optimum, the increasing temperature causes some of the bonds (especially the weak hydrogen bonds) to break. As the temperature rises, more and more bonds break and the shape of the enzyme and, in particular, it’s active site changes. The enzyme will become denatured (this is a permanent and irreversible change).
18
Q
How and why does pH affect the rate of enzyme activity?
A
Changes in pH disrupt the bonds that are important in determining protein shape (especially the active site). Ionic bonds, in particular, are subject to disruption when in a non-optimal pH.
19
Q
What is an enzyme inhibitor?
A
Enzyme inhibitors are substances that interfere with enzyme action. They reduce enzyme activity by directly or indirectly affecting the functioning of the active site.
20
Q
What is the name given to enzymes which have a second site where (non-substrate) molecules can attach?
A
Allosteric enzymes
21
Q
What are allosteric enzymes?
A
Enzymes that have a second site where (non-substrate) molecules can attach are described as allosteric enzymes.
22
Q
What is competitive enzyme inhibition?
A
Competitive enzyme inhibition is when the inhibitor substance directly competes with the usual substrate for the active site. Competitive inhibitors are very similar in shape to the usual substrate.
23
Q
What is non-competitive enzyme inhibition?
A
Non-competitive inhibition is when the inhibitor attaches itself to a part of the enzyme other than the active site. The presence of the non-competitive inhibitor leads to the active site changing shape so that it is no longer complementary to the substrate molecule.
24
Q
The effects of most competitive and non-competitive inhibitors are …
A
Reversible
25
Practical work - Enzyme investigations
Effect of temperature on enzyme activity
- What are the important features in this type of investigation?
* There should be an appropriate number and range of temperatures used. For example, a minimum of five temperatures should give enough values to allow meaningful conclusions to be drawn. A range of temperatures such as 20°C, 30°C, 40°C, 50°C and 60°C should show clear differences in enzyme activity.
* It is important to be able to control and monitor the experimental temperatures used. The normal method will involve thermostatically controlled water baths. It is also important to monitor the temperature of the water baths at intervals to check that they remain at their required temperatures.
* Other variables, other than temperature, that affect reaction rates need to be controlled. pH can be controlled using buffers (although many school/college practical activities are carried out without using buffers on the assumption that the pH will not significantly change). Enzyme volume and concentration need to be controlled as does substrate volume and concentration.
Note: the temperature of both the substrate and the enzyme should be brought to the required experimental temperature separately in the water bath before being mixed together and starting to time the reaction.
26
Practical work - Enzyme investigations
Effect of substrate concentration on enzyme activity
- What are the important features in this type of investigation?
• It is important to use a suitable number and range of substrate concentrations.
Note: it may be necessary in advance of the investigation to carry out a 'pilot' investigation to check a suitable range to use. This is important as if a number of concentrations are selected in which the substrate concentrations are all 'in excess' at the end of the investigation then there will be no or little difference between concentrations. The same principle applies to testing for suitable enzyme concentrations.
• In this investigation temperature, enzyme volume and concentration, substrate volume and pH are controlled variables. A water bath set at 35°C will keep the temperature constant and ensure that temperature is not a limiting factor on enzyme activity.
27
Practical work - Enzyme investigations
Effect of enzyme concentration on enzyme activity
- What are the important features in this type of investigation?
• In this investigation it is important to use a suitable number and range of enzyme concentrations.
Note: it may be necessary in advance of the investigation to carry out a 'pilot' investigation to check a suitable range to use. This is important as if a number of concentrations are selected in which the enzyme concentrations are all 'in excess' at the end of the investigation then there will be no or little difference between concentrations. The same principle applies to testing for suitable substrate concentrations.
• Temperature, substrate volume and concentration, enzyme volume and pH are controlled variables. A water bath set at 35°C will keep the temperature constant and ensure that temperature is not a limiting factor on enzyme activity.
28
Practical work - Enzyme investigations
Effect of pH on enzyme activity
- What are the important features in this type of investigation?
* In this type of investigation enzyme activity is investigated for a suitable number and range of pH values.
* Controlled variables are temperature, enzyme volume and concentration, and substrate volume and concentration. A water bath set at 35°C will keep the temperature constant and ensure that temperature is not a limiting factor on enzyme activity.
* Buffers are used to change (and maintain) the pH of a solution. Therefore in this type of investigation a range of pH buffers are used to ensure that the enzyme reaction under investigation takes place at particular pH values.
Note: it is important to add the buffer to the enzyme before the enzyme and substrate are mixed and if the reactions are being carried out at, for example, 35°C in a water bath, that the enzyme (and buffer) and substrate are brought to the reaction temperature separately before mixing. This applies to all the enzyme investigations where the experimental temperature is anything other than room temperature.
29
Why are enzymes often used as biomarkers of disease?
As some enzymes are only present or active during disease processes.
30
Name three clinical samples used to detect the presence of enzymes concerning disease progression/recovery.
Blood
Urine
Sputum (Also known as phlegm)
31
What can these clinical samples be used for?
Disease diagnosis/monitoring
32
Give an example of an enzyme which is only present/active during disease processes
Elastase is an enzyme released by white blood cells as a consequence of lung infection. Elastase breaks down bacterial pathogens.
However, elastase also breaks down (hydrolyses) the structural protein elastin.
33
Why can enzyme inhibitors be used as therapeutic drugs?
If enzyme inhibitors are used to target enzymes involved in causing diseases, then disease progression can be reduced or stopped.
34
For enzyme inhibitors to be effective in their role as therapeutic drugs they need to be ...
1. Specific in that they will only target an enzyme involved in disease progression (and not normal metabolic pathways).
2. Able to work at low doses (to prevent a build-up of the inhibitor to levels which may be toxic).
35
The enzyme inhibitors used as therapeutic drugs are ...
Active site directed
36
What are immobilised enzymes?
Immobilised enzymes are physically or chemically trapped within or attached to appropriate inorganic or organic insoluble support materials such as fibres, gels or plastic beads.
37
Why are enzymes immobilised?
To maximise efficiency through cost-effective enzyme applications.
38
Name the five principle methods of enzyme immobilisation
1. Adsorption
2. Covalent bonding
3. Cross-linkage
4. Encapsulation (enmeshment or membrane entrapment)
5. Entrapment (gel entrapment)
39
What is adsorption?
Adsorption - The enzymes are attached by weak forces to an inert substance such as glass or a matrix.
40
What is covalent bonding?
The enzymes are covalently bonded onto a solid support structure.
41
What is cross-linking?
Cross-linking - The enzymes are bonded covalently to a matrix as a consequence of chemical reactions (or even to each other using linking/bonding chemicals).
42
What is encapsulation?
Encapsulation (enmeshment or membrane entrapment) - The enzymes are trapped inside a selectively permeable membrane.
43
What is entrapment?
Entrapment (gel entrapment) - The enzymes are trapped within polymers or gels.
44
What are the benefits of enzyme immobilisation?
1. Increased enzyme stability
- The enzymes are thermostable. They remain stable and are more effective over a wider range of temperatures than enzymes that are not immobilised.
- The enzymes are more resistant to changes in pH.
- The enzymes can be retained and reused.
2. Facilitation of continuous flow processes
- Commercial processes can be continuous, which is faster and produces less wastage (i.e. Continuous flow column reactors).
3. Enzyme-free products
- There is no contamination of the end product with enzymes, simplifying the downstreaming process, reducing purification costs and avoiding possible allergic reactions in consumers.
45
What are the disadvantages of enzyme immobilisation?
1. The gels or materials involved in some types of immobilisation may reduce the speed of diffusion between substrate and enzyme.
2. The immobilisation process may also hold some enzymes in physical positions that make some of the enzyme active sites inaccessible.
46
What is a biosensor?
A device that uses an immobilised enzyme (or antibody) to detect the presence of a particular chemical.
47
What is a diagnostic tool?
A diagnostic tool allows the identification of medical or other concerns.
48
How do biosensors work?
The general principle is that the molecule being monitored reacts with immobilised enzymes and the reaction produces a colour change or is converted into an electrical signal.
49
Give two examples of diagnostic reagent strips being used as biosensors.
1. Pregnancy tests
- In some pregnancy tests, antibodies in the test strip react with specific proteins (hormones) to give a positive test.
2.
50
Practical Work
Describe an experiment which illustrates enzyme immobilisation. What factors should be kept constant during the experiment?
Place some milk (eg 25 cm^3) containing lactose into a syringe or column containing immobilised lactase enzymes within alginate beads. Use gauze to stop the alginate beads from blocking the tip of the syringe or column. The milk will initially give a negative result when tested with Clinistix, but after one or two runs the milk (now containing galactose and glucose) will give a positive result.
Factors:
1. Flow rate
2. Number of beads in column
3. Bead size
4. Volume of milk used
51
Past Paper Question - June 2015 AS1
Q7 Starch is broken down by the enzyme amylase. A simple experiment involves mixing starch and amylase, after which samples are taken at intervals and tested with iodine. The time at which the iodine shows no colour change is taken as the end point of the reaction. However, this simple type of experiment gives no idea of the rate of the reaction over time.
In an alternative experiment to more accurately track the progress of the reaction, a colorimeter can be used. Iodine is added to the starch and amylase. As the reaction progresses, the amount of light transmitted through the starch-amylase-iodine mixture changes, as shown in the table below.
Time after mixing starch and amylase/min, Light transmitted through
sample/ %
0 2
5 38
10 72
15 86
20 94
25 98
b) The solutions used in the colorimeter experiment are more dilute than those used in the simple end-point experiment described at the start of this question. Suggest and explain a reason for this. [2]
Q7 b) Lighter colour of blue allows light to be transmitted/ dark blue-black would not allow any light to be transmitted;
So that small changes in colour are picked up, even in the early part of the reaction. [2]
52
Past Paper Question - June 2015 AS1
Q7 Starch is broken down by the enzyme amylase. A simple experiment involves mixing starch and amylase, after which samples are taken at intervals and tested with iodine. The time at which the iodine shows no colour change is taken as the end point of the reaction. However, this simple type of experiment gives no idea of the rate of the reaction over time.
In an alternative experiment to more accurately track the progress of the reaction, a colorimeter can be used. Iodine is added to the starch and amylase. As the reaction progresses, the amount of light transmitted through the starch-amylase-iodine mixture changes, as shown in the table below.
Time after mixing starch and amylase/min, Light transmitted through
sample/ %
0 2
5 38
10 72
15 86
20 94
25 98
c) Explain precisely why a red filter should be used when measuring % transmission of light through a starch-amylase-iodine mixture. [1]
Q7 c) Results in increased sensitivity to small changes in blue colouration/ only wavelengths absorbed by starch-iodine mixture are being measured/ solution absorbs red light. [1]
53
Past Paper Question - June 2015 AS1
Q7 Starch is broken down by the enzyme amylase. A simple experiment involves mixing starch and amylase, after which samples are taken at intervals and tested with iodine. The time at which the iodine shows no colour change is taken as the end point of the reaction. However, this simple type of experiment gives no idea of the rate of the reaction over time.
In an alternative experiment to more accurately track the progress of the reaction, a colorimeter can be used. Iodine is added to the starch and amylase. As the reaction progresses, the amount of light transmitted through the starch-amylase-iodine mixture changes, as shown in the table below.
Time after mixing starch and amylase/min, Light transmitted through
sample/ %
0 2
5 38
10 72
15 86
20 94
25 98
d) Apart from using the correct colour of filter, identify one other procedure which would lead to more accurate readings of % transmission of light. [1]
Q7 d) Use clean cuvette for each solution/ only handle cuvette on sides not used for transmission/ reset colorimeter (using dilute iodine solution) before test reading.
54
Past Paper Question - June 2015 AS1
Q7 Starch is broken down by the enzyme amylase. A simple experiment involves mixing starch and amylase, after which samples are taken at intervals and tested with iodine. The time at which the iodine shows no colour change is taken as the end point of the reaction. However, this simple type of experiment gives no idea of the rate of the reaction over time.
In an alternative experiment to more accurately track the progress of the reaction, a colorimeter can be used. Iodine is added to the starch and amylase. As the reaction progresses, the amount of light transmitted through the starch-amylase-iodine mixture changes, as shown in the table below.
Time after mixing starch and amylase/min, Light transmitted through
sample/ %
0 2
5 38
10 72
15 86
20 94
25 98
e) A calibration curve can be used with the results of the colorimeter experiment. This allows values for % transmission of light to be converted into starch concentrations.
Suggest the procedure which would be used to produce this calibration curve. [3]
Q7 e) Any three from:
• Start with a standard solution of starch (i.e. a solution with a known concentration of starch).
• Make a range of starch concentrations (i.e. make serial dilutions of the standard solution and add iodine).
• Measure the % transmission (or absorbance, but in this case the question specifically asks for % transmission) of each starch concentration in the colorimeter.
• Plot a graph with % transmission on the y-axis and starch concentration on the x-axis. [3]
55
Practical Work
| How can one produce a calibration curve?
1. Start with a standard solution of the substance (i.e. a solution with a known concentration of the substance).
2. Make a range of concentrations of the substance (i.e. make serial dilutions of the standard solution and add appropriate chemicals, such as an indicator or reagent).
3. Measure the % transmission (or absorbance) of each concentration of the substance in the colorimeter.
4. Plot a graph with % transmission on the y-axis and substance concentration on the x-axis.
56
Practical Work
| Name the two methods for preparing dilutions.
1. Simple (arithmetic) dilution
| 2. Serial dilution
57
Practical Work
| What is simple (arithmetic) dilution?
When the solutions produced go down in steps of 10 or 20 % concentration.
58
Practical Work
| What is serial dilution?
In serial dilutions each solution across a series is less concentrated than the previous one in the series by a set factor, for example, a dilution factor of x2 is in effect doubling dilutions, where each concentration is half that of the previous one. When using a dilution factor of 10, 1 cm^3 of the solution is added to 9 cm^3 water and so on. This means that each time the solution is diluted, it is ten times less concentrated than the previous one.
59
Practical Work
| What are some things that should be taken into consideration when preparing dilutions?
1. When preparing a dilution series it is important to use fresh pipettes/tips/syringes/graduated cylinders etc for each dilution of the stock/standard solution to prevent the carry-over of solutions.
2. Solutions must be thoroughly mixed at each stage when preparing a dilution series.
60
Past Paper Question - January 2012 AS1
Q7 An oxidase enzyme found in apples is responsible for the flesh of the fruit discolouring and turning brown when it is exposed to air. This is because the enzyme oxidises chemicals called phenols which occur in the apple, eventually forming dark coloured products. Many recipes involving cut or crushed apple suggest adding an acid fruit juice, like lemon juice, to the pieces of apple to stop them from going brown.
An investigation was carried out to determine the effectiveness of various substances in preventing discolouration. Apple extract was used as the source of the enzyme and catechol, one of the natural phenols in apple tissue, was used as the substrate. When a solution of catechol is oxidised, an orange colour develops. The more active the enzyme, the more intense the colour.
The method used was as follows:
1. Using measuring cylinders add 4 cm^3 of Apple extract and 1 cm^3 of distilled water to a clean test tube.
2. Add 10 cm^3 of 0.1 M catechol solution to the test tube, and shake thoroughly.
3. After 5 minutes decant some of the liquid into a clean cuvette.
4. Using a colorimeter, with a blue filter, measure the % transmission through the mixture.
5. Repeat steps 1-4 with fresh solutions, but replace the distilled water with 1 cm^3 of decolourised lemon juice.
6. Repeat step 5, but replace the lemon juice with 1 cm^3 of decolourised orange juice.
a)i) Suggest how this procedure could be altered to improve its accuracy. [1]
Q7 a)i) Replace the measuring cylinder with a pipette/syringe/ other appropriate response. [1]
61
Past Paper Question - January 2012 AS1
Q7 An oxidase enzyme found in apples is responsible for the flesh of the fruit discolouring and turning brown when it is exposed to air. This is because the enzyme oxidises chemicals called phenols which occur in the apple, eventually forming dark coloured products. Many recipes involving cut or crushed apple suggest adding an acid fruit juice, like lemon juice, to the pieces of apple to stop them from going brown.
An investigation was carried out to determine the effectiveness of various substances in preventing discolouration. Apple extract was used as the source of the enzyme and catechol, one of the natural phenols in apple tissue, was used as the substrate. When a solution of catechol is oxidised, an orange colour develops. The more active the enzyme, the more intense the colour.
The method used was as follows:
1. Using measuring cylinders add 4 cm^3 of Apple extract and 1 cm^3 of distilled water to a clean test tube.
2. Add 10 cm^3 of 0.1 M catechol solution to the test tube, and shake thoroughly.
3. After 5 minutes decant some of the liquid into a clean cuvette.
4. Using a colorimeter, with a blue filter, measure the % transmission through the mixture.
5. Repeat steps 1-4 with fresh solutions, but replace the distilled water with 1 cm^3 of decolourised lemon juice.
6. Repeat step 5, but replace the lemon juice with 1 cm^3 of decolourised orange juice.
a)ii) Blue and orange are at opposite ends of the light spectrum. Explain precisely why a blue filter was chosen for use in the colorimeter when measuring the intensity of orange colouration. [1]
Q7 a)ii) The solution reflects (transmits) orange light/ absorbs blue light/ results in a wider/ more discriminate range of percentage transmission values. [1]
62
Past Paper Question - January 2012 AS1
Q7 An oxidase enzyme found in apples is responsible for the flesh of the fruit discolouring and turning brown when it is exposed to air. This is because the enzyme oxidises chemicals called phenols which occur in the apple, eventually forming dark coloured products. Many recipes involving cut or crushed apple suggest adding an acid fruit juice, like lemon juice, to the pieces of apple to stop them from going brown.
An investigation was carried out to determine the effectiveness of various substances in preventing discolouration. Apple extract was used as the source of the enzyme and catechol, one of the natural phenols in apple tissue, was used as the substrate. When a solution of catechol is oxidised, an orange colour develops. The more active the enzyme, the more intense the colour.
The method used was as follows:
1. Using measuring cylinders add 4 cm^3 of Apple extract and 1 cm^3 of distilled water to a clean test tube.
2. Add 10 cm^3 of 0.1 M catechol solution to the test tube, and shake thoroughly.
3. After 5 minutes decant some of the liquid into a clean cuvette.
4. Using a colorimeter, with a blue filter, measure the % transmission through the mixture.
5. Repeat steps 1-4 with fresh solutions, but replace the distilled water with 1 cm^3 of decolourised lemon juice.
6. Repeat step 5, but replace the lemon juice with 1 cm^3 of decolourised orange juice.
In addition to the fruit juices mentioned in the procedure, two enzyme inhibitors, PHBA and phenylthiourea, were also investigated. The results of the investigation are shown below.
Test substance % transmission through solution after 5 minutes
Distilled water 27
Lemon juice 90
Orange juice 74
PHBA 99
Phenylthiourea 92
b)ii) Lemon juice had a pH of 2.4 and orange juice has a pH of 3.5. Using this information and the information in the graph, describe and explain how the results for these two substances differ. [3]
Q7 b)ii) There is a lower transmission value with orange juice than lemon juice/ where pH is higher;
the enzyme is more active with orange juice/where pH is higher;
the higher pH of orange juice causes less denaturation of the enzyme; Accept converse references for lemon juice. [3]
63
Practical Work
| How can results obtained from a colorimeter be improved in terms of their accuracy and validity?
Increased accuracy ---> Improved validity
1. Make sure cuvettes are clean, any marks on the surface of the cuvette will affect light transmission.
2. Rinse and dry the cuvette between samples.
3. Ensure that the cuvettes are filled to the correct level.
4. Ensure that the orientation of the cuvette in the colorimeter is correct.
5. Re-calibrate every so often by placing the 'blank' back in the colorimeter, and re-calibrate if necessary.
64
Practical Work
Describe the shape of a starch-amylase reaction graph, showing time on the x-axis and the amount of starch remaining on the y-axis.
y=1/x
65
Practical Work
What are the two types of line of best fit?
What important points should be considered when drawing a line of best fit?
Straight line
Smooth curve
When drawing a line of best fit, be careful that you do not extend your line beyond the range of the points available (extrapolation) unless there is good reason to do so. Also do not always assume that it is appropriate to start at the origin (0,0 co-ordinate).
A line of best fit can be used if there are a sufficient number of points to be confident that intermediate points fall on the line or an understanding of the theory involved suggests that a best fit line is appropriate.
66
Practical Work
| How can a colorimeter be used to follow the course of a starch-amylase catalysed reaction?
Calibration - It is important to calibrate the colorimeter. For example, if the colorimeter is going to follow the course of amylase breaking down starch to maltose, a weak solution of iodine could be calibrated as the end-point or 'blank', i.e. 100% transmission.
Choosing a filter - An appropriate filter is used that will maximise the change in transmission/absorbance as the investigation progresses (provides the biggest contrast and range of colorimeter readings). i.e. the filter colour should be opposite to the colour of the test solution.
67
Past Paper Question - June 2017 Q1 AS1 (Legacy Material)
Q1 Complete the following paragraph using the most appropriate word(s).
Enzymes are (blank) proteins whose role is to lower the (blank) (blank) of a reaction. As a substrate binds, a specific region of the enzyme called the (blank) (blank) changes shape to become (blank) to the substrate. This is referred to as the (blank) (blank) model of enzyme action. [5]
```
Q1 Globular;
Activation energy;
Active site;
Complementary;
Induced fit;
```
68
Past Paper Question - June 2017 Q7 a)i) AS1 (Revised Spec.)
Q7 When a tadpole changes to an adult frog, it's tail is broken down by the enzyme collagenase, which has a zinc cofactor.
a)i) Define the term 'cofactor'. [1]
Q7 a)i) A non-protein substance that an enzyme may require to function. [1]
69
Past Paper Question - June 2017 Q7 b)i) AS1 (Revised Spec.)
Q7b) An investigation was carried out to determine how zinc availability affects the rate of collagenase activity and therefore the reabsorption of tadpole tails.
Tadpoles of the clawed frog (Xenopus laevis) were allowed to mature in water containing normal zinc concentrations and in water containing no zinc. The average diameters of the collagen fibrils in tadpole tails during the first 25 minutes of the investigation are shown in the table below.
Average collagen fibril diameter/ nm
Time/ min Zinc present Zinc absent
0 27 28
5 26 27
10 12 27
15 8 26
20 2 25
25 1 24
i) Plot the results, using an appropriate graphical technique. (Use the graph paper opposite). The caption is already included. [4]
Caption = The effect of zinc availability on average collagen fibril diameter due to collagenase action over time.
Mark Scheme June 2017 AS1 (Revised Spec.) Question 7 b)i)
Q7b)i) Independant variable (time) on X-axis;
Axes correctly labelled with units and scale;
Accurate plotting;
Points joined with short, straight lines including a legend;
70
Past Paper Question - June 2017 Q7 b)ii) AS1 (Revised Spec.)
Q7b) An investigation was carried out to determine how zinc availability affects the rate of collagenase activity and therefore the reabsorption of tadpole tails.
Tadpoles of the clawed frog (Xenopus laevis) were allowed to mature in water containing normal zinc concentrations and in water containing no zinc. The average diameters of the collagen fibrils in tadpole tails during the first 25 minutes of the investigation are shown in the table below.
Average collagen fibril diameter/ nm
Time/ min Zinc present Zinc absent
0 27 28
5 26 27
10 12 27
15 8 26
20 2 25
25 1 24
ii) Describe and explain the trend evident in the results. [3]
Q7b)ii) Average fibril diameter decreases much faster when zinc is present/ fibril digestion is much slower when zinc is absent;
Zinc makes active site more complementary to the substrate;
Hence in the absence of zinc, few enzyme substrate complexes form; [3]
71
Past Paper Question - June 2017 Q7c) AS1 (Revised Spec.)
Q7 When a tadpole changes to an adult frog, it's tail is broken down and reabsorbed into the body. A major component of the tail is the protein collagen, which provides support.
Collagen in the tail is broken down by the enzyme collagenase, which has a zinc cofactor.
c) Collagen is also found in joints in the human body. The breakdown of this collagen by collagenase is a factor in arthritis. Use the information provided and your knowledge of enzymes to suggest how a therapeutic drug could treat arthritis. Explain your answer. [3]
Q7 c) Targeted active site inhibitor specific to collagenase/ block zinc binding site on collagenase;
So collagen is not broken down; [2]
72
Past Paper Question - June 2016 Section B Q8 a) AS1 (Legacy Material)
Q8 a) The relationship between enzyme concentration and the rate of reaction is shown in the graph. Describe and explain the relationship shown. [4]
(Graph shows rate of reaction increase as enzyme concentration increases initially. Soon the rate plateaus)
Q8 a) Increasing enzyme concentration increases rate of reaction to a particular point and then further increase in enzyme concentration has no further effect on rate of reaction/rate remains unchanged (not reaction stops);
increase in enzyme concentration provides more active sites;
allowing more enzyme-substrate complexes to form;
at higher enzyme concentrations the number of substrate molecules will become limiting (as enzyme-substrate complexes form at maximal rate); [4]
73
Past Paper Question - June 2016 Section B Q8 b) AS1 (Legacy Material)
Q8 b) Enzyme immobilisation is a technique frequently used in industry. Describe two methods of enzyme immobilisation and give an account of the advantages and disadvantages of enzyme immobilisation. [9]
Quality of written communication [2]
Q8 b) Any nine from: (maximum four marks name and description)
• adsorption
• attaches enzyme to the surface of an inert material (such as glass or a matrix by weak forces)
• entrapment
• traps the enzyme within/between strands of a polymer (such as alginate beads or microspheres)
• encapsulation(enmeshment)
• traps the enzymes within a partial/selectively permeable membrane
(such as nylon)
• cross-linkage
• covalently bonds enzymes to a matrix (such as cellulose)
Advantages (maximum five marks from advantages/disadvantages)
• increased thermal/pH stability/thermostability
• can be recovered and reused easily
• allows for continuous/column reactors with faster production
• downstream processing of product is simplified and purification costs are reduced/reduces possibility of allergic response in consumer/ product is enzyme-free
Disadvantages
• when enzymes are immobilised the matrix or gels used for
immobilisation may reduce speed of diffusion of substrate/enzyme may
be wasted off/build-up of byproduct within the matrix
• enzyme active site may be in wrong orientation/partially blocked
• reactions used in the immobilisation process may result in shape
change of the active site
• initial set-up/research costs high [9]
74
Past Paper Question - June 2015 Q7 a) AS1 (Legacy Material)
Q7 Starch is broken down by the enzyme amylase. A simple experiment involves mixing starch and amylase, after which samples are taken at intervals and tested with iodine. The time at which the iodine shows no colour change is taken as the end point of the reaction. However, this simple type of experiment gives no idea of the rate of the reaction over time.
In an alternative experiment to more accurately track the progress of the reaction, a colorimeter can be used. Iodine is added to the starch and amylase. As the reaction progresses, the amount of light transmitted through the starch-amylase-iodine mixture changes, as shown in the table below.
Time after mixing starch and Light transmitted through
amylase/min sample/%
0 2
5 38
10 72
15 86
20 94
25 98
(a) Plot the above data on the graph paper opposite, using the most appropriate graphical technique. Your graph should include a caption.
[4]
Q7 a) Change in % transmission of light with time for a starch-amylase (and iodine) mixture;
independent variable (time) on X-axis;
axes correctly plotted with units and scale;
accurate plotting and points joined with short, straight lines; [4]
75
Past Paper Question - January 2014 Q4 a)i)+ii)+b) AS1 (Legacy Material)
Q4 Mature seeds contain an embryo plant and a store of energy-rich food. In some seeds, such as soybean, the main energy store is lipid. When seeds are planted in springtime they absorb water, which activates enzymes such as lipase.
The graph below shows the relative activity of soybean lipase at various temperatures.
a) i) Describe precisely the trends evident in this graph. [2]
ii) Using your understanding of enzyme action, explain the trends you have identified. [3]
b) The temperature range for soybean lipase activity, as shown in the graph, is different to that for mammalian lipase. Suggest a reason for this difference in temperature range and suggest how this is an advantage to the growth of the soybean plants. [2]
(Go do this past paper question)
Q4 a)i) As temperature increases from –5°C to 25°C the lipase activity increases;
above 25°C lipase activity decreases dramatically
[Not ‘decreases rapidly’ or other suggestions of link to time]; [2]
(ii) Up to 25 °C increasing kinetic energy increases the chance of collision between the active site (enzyme) and the substrate/formation of ES complexes;
above 25 °C vibrations within the lipase molecule break some of the hydrogen/ionic bonds; this distorts the shape of the active site so that
it is no longer complementary to the substrate [not simply ‘enzyme is denatured’]; [3]
b) Environmental soil temperatures would be low (possibly still around 0 °C); lipase activity in this range allows seeds to germinate early in the year to allow a long growing season/in springtime; [2]
76
Past Paper Question - June 2014 Q4 a)+b)i)+ii)+c)
Q4 Biological washing powders contain enzymes which help to break down stains on fabric. The enzyme activity in a biological washing powder at a range of different temperatures was investigated. The graph below shows the results.
a) Describe and explain the trend shown in the graph between 5°C and 45°C. [4]
b) i) Suggest an explanation for the two different peaks observed in enzyme activity. [2]
b) ii) Suggest why such a pattern of enzyme activity would be useful in biological washing powders. [1]
c) Suggest an explanation for the enzyme activity observed between 45°C and 55°C. [2]
(Go do this past paper question)
Q4 a) Activity increases between 5 °C and 40 °C, then falls sharply between 40 °C and 45 °C;
increase at lower temperatures is due to the greater kinetic energy of enzyme/substrate molecules;
causing more frequent formation of enzyme-substrate complexes;
above 40 °C, bonds within the tertiary structure are broken/the active
site is distorted; [4]
(b) (i) There are two different enzymes present;
each peak represents a different optimum temperature; [2]
(ii) Allows enzyme activity to take place over a wide range of (washing) temperatures; [1]
(c) From 45 °C to 50 °C, one enzyme is being denatured;
from 50 °C to 55 °C, the activity of the second enzyme is increasing; [2]
77
Past Paper Question - January 2013 Q6 a) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
a) Using the most appropriate graphical technique, plot the above data. (Use the graph paper opposite). [4]
Q6 a) Caption (the effect of increasing temperature on the rate of reaction of a protease enzyme);
independent variable (temperature) on x-axis and appropriate scaling; label on each axis, with appropriate units;
accurate plotting of points and points joined with short, straight lines; [4]
78
Past Paper Question - January 2013 Q6 b)i) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
b)i) Using the graph, estimate the optimum temperature for this enzyme. [1]
Q6 b)i) Somewhere between 50°C and 60°C (51°C to 60°C); [1]
79
Past Paper Question - January 2013 Q6 b)ii) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
b)ii) Describe how the experiment could be extended to obtain a more precise value for the optimum temperature. [1]
Q6 b)ii) Carry out experiment at smaller intervals between 50 °C and 60°C/70°C; [1]
80
Past Paper Question - January 2013 Q6 b)iii) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
b)iii) Protease enzymes such as trypsin, normally found in the human body, have an optimum rate of reaction at about 37°C. Suggest an explanation for the different optimum obtained from the graph. [1]
Q6 b)iii) Enzyme has been extracted from thermophilic bacteria (with more disulfide bonds)/enzyme has been immobilised/enzyme is synthetic/10 minutes may not be sufficient time to denature enzyme (below 60°C); [1]
81
Past Paper Question - January 2013 Q6 c) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
c) Suggest why the enzyme and casein suspension were incubated separately before mixing. [1]
Q6 c) To allow enzyme and substrate to reach the reaction temperature before being mixed; [1]
82
Past Paper Question - January 2013 Q6 d)i) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
d) To make the investigation valid, all other variables should be kept constant. For example, an optimum pH must be maintained.
i) State how pH could have been kept constant. [1]
Q6 d)i) Use of pH buffer; [1]
83
Past Paper Question - January 2013 Q6 d)ii) AS1 (Legacy Material)
Q6 Powdered milk contains a protein called casein, which forms a milky-white suspension when mixed with water. When a protease enzyme is added to the mixture it digests the casein and the mixture will become clear.
An experiment was carried out to investigate the effect of temperature on the protease enzyme.
* Two test tubes, one containing 10 cm3 of milk suspension and one containing 10 cm3 of protease enzyme solution were placed in a water bath at 20°C.
* After 10 minutes, the contents were mixed and the time taken for the mixture to clear was recorded.
* The experiment was repeated at a series of temperatures up to 80°C. The rate of reaction (min-1) was then calculated.
* The results are shown in the table below.
Temperature/°C. Rate of reaction/min-1
20 0.95
30 2.00
40 4.00
50 7.50
60 8.00
70 6.00
80 1.00
d) To make the investigation valid, all other variables should be kept constant. For example, an optimum pH must be maintained.
ii) Explain precisely why pH should be maintained at the optimum level for this enzyme. [2]
Q6 d)ii) At optimum pH the active site is most complementary (for the binding of the substrate)/at other pHs the active site is less complementary; reference to effect on ionic bonds within enzyme molecule; [2]
84
Past Paper Question - January 2012 Q7 b)i) AS1 (Legacy Material)
Q7 An oxidase enzyme found in apples is responsible for the flesh of the fruit discolouring and turning brown when it is exposed to air. This is because the enzyme oxidises chemicals called phenols which occur in the apple, eventually forming dark coloured products. Many recipes involving cut or crushed apple suggest adding an acid fruit juice, like lemon juice, to the pieces of apple to stop them from going brown.
An investigation was carried out to determine the effectiveness of various substances in preventing discolouration. Apple extract was used as the source of the enzyme and catechol, one of the natural phenols in apple tissue, was used as the substrate. When a solution of catechol is oxidised, an orange colour develops. The more active the enzyme, the more intense the colour.
The method used was as follows:
1. Using measuring cylinders add 4 cm^3 of Apple extract and 1 cm^3 of distilled water to a clean test tube.
2. Add 10 cm^3 of 0.1 M catechol solution to the test tube, and shake thoroughly.
3. After 5 minutes decant some of the liquid into a clean cuvette.
4. Using a colorimeter, with a blue filter, measure the % transmission through the mixture.
5. Repeat steps 1-4 with fresh solutions, but replace the distilled water with 1 cm^3 of decolourised lemon juice.
6. Repeat step 5, but replace the lemon juice with 1 cm^3 of decolourised orange juice.
In addition to the fruit juices mentioned in the procedure, two enzyme inhibitors, PHBA and phenylthiourea, were also investigated. The results of the investigation are shown below.
Test substance % transmission through solution after 5 minutes
Distilled water 27
Lemon juice 90
Orange juice 74
PHBA 99
Phenylthiourea 92
b)i) Using the most appropriate graphical technique, plot the above data. (Use the graph paper opposite). [4]
Q7 b)i) Caption(How % transmission/enzyme activity is affected by various substances);
label on each axis (% transmission and test substance);
independent variable (various substances) on x-axis and appropriate scaling (of y-axis);
accurate plotting of bars (bars should not touch); [4]
85
Past Paper Question - January 2012 Q7 c)i) AS1 (Legacy Material)
Q7 An oxidase enzyme found in apples is responsible for the flesh of the fruit discolouring and turning brown when it is exposed to air. This is because the enzyme oxidises chemicals called phenols which occur in the apple, eventually forming dark coloured products. Many recipes involving cut or crushed apple suggest adding an acid fruit juice, like lemon juice, to the pieces of apple to stop them from going brown.
An investigation was carried out to determine the effectiveness of various substances in preventing discolouration. Apple extract was used as the source of the enzyme and catechol, one of the natural phenols in apple tissue, was used as the substrate. When a solution of catechol is oxidised, an orange colour develops. The more active the enzyme, the more intense the colour.
The method used was as follows:
1. Using measuring cylinders add 4 cm^3 of Apple extract and 1 cm^3 of distilled water to a clean test tube.
2. Add 10 cm^3 of 0.1 M catechol solution to the test tube, and shake thoroughly.
3. After 5 minutes decant some of the liquid into a clean cuvette.
4. Using a colorimeter, with a blue filter, measure the % transmission through the mixture.
5. Repeat steps 1-4 with fresh solutions, but replace the distilled water with 1 cm^3 of decolourised lemon juice.
6. Repeat step 5, but replace the lemon juice with 1 cm^3 of decolourised orange juice.
In addition to the fruit juices mentioned in the procedure, two enzyme inhibitors, PHBA and phenylthiourea, were also investigated. The results of the investigation are shown below.
Test substance % transmission through solution after 5 minutes
Distilled water 27
Lemon juice 90
Orange juice 74
PHBA 99
Phenylthiourea 92
c) Phenylthiourea is structurally dissimilar to catechol and is known to bind to the oxidase enzyme at a copper atom, which is not located at the active site.
i) State the type of inhibition carried out by phenylthiourea. [1]
Q7 c)i) Non-competitive inhibition/allosteric inhibition; [1]
86
Past Paper Question - January 2012 Q7 c)ii) AS1 (Legacy Material)
Q7 An oxidase enzyme found in apples is responsible for the flesh of the fruit discolouring and turning brown when it is exposed to air. This is because the enzyme oxidises chemicals called phenols which occur in the apple, eventually forming dark coloured products. Many recipes involving cut or crushed apple suggest adding an acid fruit juice, like lemon juice, to the pieces of apple to stop them from going brown.
An investigation was carried out to determine the effectiveness of various substances in preventing discolouration. Apple extract was used as the source of the enzyme and catechol, one of the natural phenols in apple tissue, was used as the substrate. When a solution of catechol is oxidised, an orange colour develops. The more active the enzyme, the more intense the colour.
The method used was as follows:
1. Using measuring cylinders add 4 cm^3 of Apple extract and 1 cm^3 of distilled water to a clean test tube.
2. Add 10 cm^3 of 0.1 M catechol solution to the test tube, and shake thoroughly.
3. After 5 minutes decant some of the liquid into a clean cuvette.
4. Using a colorimeter, with a blue filter, measure the % transmission through the mixture.
5. Repeat steps 1-4 with fresh solutions, but replace the distilled water with 1 cm^3 of decolourised lemon juice.
6. Repeat step 5, but replace the lemon juice with 1 cm^3 of decolourised orange juice.
In addition to the fruit juices mentioned in the procedure, two enzyme inhibitors, PHBA and phenylthiourea, were also investigated. The results of the investigation are shown below.
Test substance % transmission through solution after 5 minutes
Distilled water 27
Lemon juice 90
Orange juice 74
PHBA 99
Phenylthiourea 92
c) Phenylthiourea is structurally dissimilar to catechol and is known to bind to the oxidase enzyme at a copper atom, which is not located at the active site.
ii) On the axes below, sketch a line to show how the level of inhibition by phenylthiourea is affected by substrate concentration. [1]
Graph has already been drawn, you must simply sketch a line.
X-axis = Substrate concentration
Y-axis = Level of inhibition
Q7 c)ii) Line rising to become horizontal at a low substrate concentration; [1]
87
Past Paper Question - June 2012 Q7 b)i) AS1 (Legacy Material)
Q7 b) Lipids are present in foods such as gravy and butter and can leave stains on clothes. Some biological washing powders may include lipase, which can help to remove such stains.
(i) Describe the mechanism of action of an enzyme such as lipase. [3]
Q7 b)i) Any three from
• the enzyme’s active site has a complementary fit to/is induced to fit the substrate shape
• forming an enzyme-substrate complex/enzyme and substrate molecules must collide
• the activation energy is lowered
• the change in shape of the enzyme puts a strain on bonds
(between glycerol and fatty acids)/glycerol and fatty acid (products) are released from the active site as they have a different shape to the substrate(s) [3]
88
Past Paper Question - June 2012 Q7 b)ii) AS1 (Legacy Material)
Q7 b) Lipids are present in foods such as gravy and butter and can leave stains on clothes. Some biological washing powders may include lipase, which can help to remove such stains.
ii) Biological washing powders are less effective stain removers at temperatures above 60°C. Explain precisely why this is the case. [2]
Q7 b)ii) At temperatures above 60 °C, the ionic bonds/hydrogen bonds are broken;
shape of active site is altered/binding sites are altered; [2]
89
Past Paper Question - June 2012 Q7 c)i)+ii) AS1 (Legacy Material)
Q7 c) Enzymes in biological washing powders are often immobilised in beads of silica. The graph below shows the activity of one enzyme in a free and in an immobilised state, over a range of pH values.
i) Describe the main differences in enzyme activity which result from immobilisation. [2]
ii) Suggest explanations for the differences shown. [2]
(Go do this past paper question)
Q7 c)i) Immobilisation decreases the maximum activity of the enzyme/activity at optimum pH (6.5);
though increases the activity over a wider pH range; [2]
(ii) Some enzymes when immobilised are inaccessible/may have a blocked active site;
immobilised enzyme has increased stability/lower breakage of (ionic) bonds; [2]
90
Past Paper Question - June 2011 Q6 a) AS1 (Legacy Material)
Q6 Enzymes can be immobilised for use in biotechnology.
a) Outline two methods used to immobilise enzymes.
1. ___________________________________________________________________
_____________________________________________________________________
2. ___________________________________________________________________
_____________________________________________________________________ [2]
Q6 a) Any two from
• adsorption on glass, alginate beads or matrix (enzyme is attached to outside of an inert material)
• entrapment (enzyme is trapped inside insoluble beads or microspheres, e.g. calcium (or sodium) alginate beads)
* cross-linkage (enzyme is bonded covalently to a matrix by a chemical reaction).
* encapsulation/enmeshment (inside a selectively permeable membrane e.g. nylon)
[Descriptions in brackets accepted only with detail included] [2]
91
Past Paper Question - June 2011 Q6 b)i)+ii)+iii) AS1 (Legacy Material)
Q6 Enzymes can be immobilised for use in biotechnology.
b) The graph below shows the effect of pH on the enzyme sucrase when in solution and when immobilised.
i) The graph shows that enzyme immobilisation reduces the activity of sucrase at the optimum pH. Calculate the percentage reduction in the rate of the activity for this immobilised sucrase. [3]
ii) Explain the reduction in the activity of the immobilised sucrase. [2]
iii) Using the information in the graph, describe and explain one other difference in the activity of the sucrase in solution and when immobilised. [2]
(Go do this past paper question)
Q6 b)i) Values from graph: 35 and 30;
division by maximum: 5 4 35;
percentage: (5 4 35) 3 100 5 14.3; [3]
ii) Fewer collisions between enzyme and substrate/fewer enzyme-substrate complexes formed;
enzyme not free to move around/some immobilized enzyme active sites not exposed/available/some of the enzyme denatured/substrate must diffuse through support medium to reach enzymes; [2]
iii) Immobilisation increases the range of pH over which the
enzyme remains active;
prevents enzyme structure being disrupted by pH/increases stability of the enzyme; [2]
[References to influence of temperature is NOT acceptable]
92
Past Paper Question - June 2011 Q7 d) AS1 (Legacy Material)
Q7 An experiment was carried out on the rate of absorption of two monosaccharide sugars by living intestinal cells and intestinal cells that have been poisoned with cyanide (a chemical which inhibits production of ATP in cells by inhibiting cell respiration). The results are shown in the table below.
Monosaccharide Rate of absorption/arbitrary units
Living intestinal cells Cyanide-treated
intestinal cells
Glucose 1.00 0.33
Arabinose 0.29 0.29
d) Cyanide, which was used in this investigation to inhibit cell respiration, can be described as a non-competitive enzyme inhibitor. Explain what is meant by the term 'non-competitive'. [2]
Q7 d) Any two from
• an enzyme inhibitor that bears no resemblance to the enzyme’s natural substrate/may attach to a part of the enzyme other than the active site/doesn’t compete with substrate for the active site
* alters the shape of the active site/permanently binds to (blocks) the active site
* inhibition does not depend on the relative concentration of the inhibitor [2]
93
Past Paper Question - January 2011 Q7 b)i)+ii) AS1 (Legacy Material)
Q7 This question is about the digestion of jelly by protein-digesting enzymes.
b) The procedure used for an investigation of the effect of pH on the activity of two protein-digesting enzymes is outlined below.
1. Five buffer solutions were prepared at pH 4, 6.4, 7.4, 8, and 9.
2. 10 strips of jelly were cut from a jelly block. Each strip was
approximately 1 to 2 mm in thickness.
3. Each strip was trimmed to a rectangle of 10 by 20 mm and the
surplus jelly discarded. Trimmed strips were placed in separate
Petri dishes.
4. 10 cm3 of each of the five buffer solutions was added to two of
the Petri dishes, so that two Petri dishes had pH 4, two had pH 6.4, etc. The Petri dishes were divided into two sets, each set having one dish at each pH.
5. 10 cm3 of enzyme A was added to one set of the Petri dishes and 10 cm3 of enzyme B was added to the second set of Petri dishes.
6. All Petri dishes were left at room temperature for 90 minutes.
7. The area of the jelly remaining was measured and the results are
shown in the table below.
Area of jelly remaining/mm^2
pH Enzyme A Enzyme B
4 0 200
6.4 100 190
7.4 171 98
8 190 48
9 200 0
(i) Plot the above results, using an appropriate graphical technique. [5]
(ii) Describe the trends evident in the graph. [3]
Q7 b)i) Caption (e.g. The effect of pH on the activity of two enzymes);
pH as the independent variable along the shared x-axis;
labels and units of measurement shown, with a key to identify enzymes A and B;
points accurately plotted in a well-scaled graph (using the graph paper to maximal effect);
points joined with short straight lines; [5]
ii) Any three from
• for enzyme A, as pH increases the area of jelly remaining increases/enzyme A is more effective in acidic conditions (pH4/low pH)
• for enzyme B, as pH increases the area of jelly remaining decreases/enzyme B is more effective in alkaline conditions (pH7.4, 8 and 9/high pH)
• the more jelly remaining, the less digestion has taken place (allow converse)/the lower the activity of the enzyme (allow converse)
• enzymes A and B have different optimum pHs
• enzymes A and B have the same activity/amount of jelly
remaining at pH7 (approximately) [3]
94
Past Paper Question - January 2011 Q7 c) AS1 (Legacy Material)
Q7 This question is about the digestion of jelly by protein-digesting enzymes.
The procedure used for an investigation of the effect of pH on the activity of two protein-digesting enzymes is outlined below.
1. Five buffer solutions were prepared at pH 4, 6.4, 7.4, 8, and 9.
2. 10 strips of jelly were cut from a jelly block. Each strip was
approximately 1 to 2 mm in thickness.
3. Each strip was trimmed to a rectangle of 10 by 20 mm and the
surplus jelly discarded. Trimmed strips were placed in separate
Petri dishes.
4. 10 cm3 of each of the five buffer solutions was added to two of
the Petri dishes, so that two Petri dishes had pH 4, two had pH 6.4, etc. The Petri dishes were divided into two sets, each set having one dish at each pH.
5. 10 cm3 of enzyme A was added to one set of the Petri dishes and 10 cm3 of enzyme B was added to the second set of Petri dishes.
6. All Petri dishes were left at room temperature for 90 minutes.
7. The area of the jelly remaining was measured and the results are
shown in the table below.
Area of jelly remaining/mm^2
pH Enzyme A Enzyme B
4 0 200
6.4 100 190
7.4 171 98
8 190 48
9 200 0
c) Explain why pH influences the activity of an enzyme. [2]
Q7 c) Ionic bonds in the tertiary structure may be disrupted (and result in coagulation of the protein);
disrupting the shape of the active site/therefore fewer enzyme- substrate complexes are formed;
Insist on reference to terms such as tertiary/globular structure,
active site. [2]
95
Past Paper Question - January 2011 Q7 d) AS1 (Legacy Material)
Q7 This question is about the digestion of jelly by protein-digesting enzymes.
The procedure used for an investigation of the effect of pH on the activity of two protein-digesting enzymes is outlined below.
1. Five buffer solutions were prepared at pH 4, 6.4, 7.4, 8, and 9.
2. 10 strips of jelly were cut from a jelly block. Each strip was
approximately 1 to 2 mm in thickness.
3. Each strip was trimmed to a rectangle of 10 by 20 mm and the
surplus jelly discarded. Trimmed strips were placed in separate
Petri dishes.
4. 10 cm3 of each of the five buffer solutions was added to two of
the Petri dishes, so that two Petri dishes had pH 4, two had pH 6.4, etc. The Petri dishes were divided into two sets, each set having one dish at each pH.
5. 10 cm3 of enzyme A was added to one set of the Petri dishes and 10 cm3 of enzyme B was added to the second set of Petri dishes.
6. All Petri dishes were left at room temperature for 90 minutes.
7. The area of the jelly remaining was measured and the results are
shown in the table below.
Area of jelly remaining/mm^2
pH Enzyme A Enzyme B
4 0 200
6.4 100 190
7.4 171 98
8 190 48
9 200 0
d) There are several potential problems with the design of this experiment. Suggest four possible problems in the procedure used. [4]
Q7 d) Any four from
• the strips of gelatine were not of uniform thickness
• there was no control experiment (to check that the jelly did not
dissolve in water or the different buffer solutions)
• temperature was not controlled
• the range of the pH buffers was not sufficient
• a shorter time period or measurements at intervals should be used
as all the jelly was digested at 90 minutes
• the procedure was not replicated/two or more strips of jelly could
have been included in each Petri dish
• the enzyme should be mixed with the buffer before adding to the
jelly
• mass remaining would be a better measure of enzyme activity
(dependent variable)
• area digested should have been determined as initial area minus
the area remaining
• other appropriate suggestion [4]
96
Past Paper Question - June 2013 Q7 a)i) AS1 (Legacy Material)
Q7 a) Enzymes are biological catalysts which work by lowering the activation energy of a reaction. Two models of enzyme action have been proposed: the lock and key hypothesis and the induced fit model.
i) Explain the term 'activation energy'. [1]
Q7 a)i) The energy needed to make a reaction occur; [1]
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Past Paper Question - June 2013 Q7 a)ii) AS1 (Legacy Material)
Q7 a) Enzymes are biological catalysts which work by lowering the activation energy of a reaction. Two models of enzyme action have been proposed: the lock and key hypothesis and the induced fit model.
ii) Describe one similarity and one difference between the lock and key hypothesis and the induced fit model of enzyme action. [2]
Q7 a)ii) Similarity: both involve shape complementarity between substrate and active site of the enzyme/complementary binding sites on enzyme and substrate/both involve formation of ES complex/both involve an active site on the enzyme/other appropriate similarity;
Difference: in the induced fit model, full complementarity only occurs once binding has occurred between enzyme and substrate, whereas lock and key hypothesis states that they are complementary from the outset; [2]
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Past Paper Question - June 2013 Q7 b)i) AS1 (Legacy Material)
Q7 b) The cells of many fruits and vegetables including apples, bananas and lettuce contain the enzyme catechol oxidase. The enzyme is found in the cytoplasm, whereas its substrate catechol is usually confined to the vacuole. The reaction is a major cause of browning in fruits and vegetables. The enzyme's action is summarised below.
Catechol Non-enzymatic
oxidase oxidation
Catechol ----------> Benzoquinone ---------> Melanin
(brown/black)
i) Explain how dropping a banana or apple leads to the development of brown/black 'bruised' areas on the fruit. [2]
Q7 b)i) Any two from
• vacuole membrane/tonoplast is ruptured (as a result of mechanical damage)
• allowing enzyme and substrate to come together/ enzyme–substrate complex forms
• the production of melanin results in brown/black colour/
bruised areas [2]
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Past Paper Question - June 2013 Q7 b)ii) AS1 (Legacy Material)
Q7 b) The cells of many fruits and vegetables including apples, bananas and lettuce contain the enzyme catechol oxidase. The enzyme is found in the cytoplasm, whereas its substrate catechol is usually confined to the vacuole. The reaction is a major cause of browning in fruits and vegetables. The enzyme's action is summarised below.
Catechol Non-enzymatic
oxidase oxidation
Catechol ----------> Benzoquinone ---------> Melanin
(brown/black)
ii) Suggest an explanation for the fact that lettuce leaves will brown faster if they are cut with a knife, rather than torn by hand. [2]
Q7 b)ii) Cutting results in destruction of cells, so that more enzyme is released; whereas tearing leaves cells intact/breaks the tissue along cell walls; [2]
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Past Paper Question - June 2013 Q7 c) AS1 (Legacy Material)
Q7 The cells of many fruits and vegetables including apples, bananas and lettuce contain the enzyme catechol oxidase. The enzyme is found in the cytoplasm, whereas its substrate catechol is usually confined to the vacuole. The reaction is a major cause of browning in fruits and vegetables. The enzyme's action is summarised below.
Catechol Non-enzymatic
oxidase oxidation
Catechol ----------> Benzoquinone ---------> Melanin
(brown/black)
c) The catechol oxidase molecules consist of four polypeptide chains, each of which is attached to a copper atom. The enzyme is not functional without the copper atoms. State the terms used to describe the role of copper in the action of the enzyme. [1]
Q7 c) Cofactor; [1]
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Past Paper Question - June 2013 Q7 d) AS1 (Legacy Material)
Q7 The cells of many fruits and vegetables including apples, bananas and lettuce contain the enzyme catechol oxidase. The enzyme is found in the cytoplasm, whereas its substrate catechol is usually confined to the vacuole. The reaction is a major cause of browning in fruits and vegetables. The enzyme's action is summarised below.
Catechol Non-enzymatic
oxidase oxidation
Catechol ----------> Benzoquinone ---------> Melanin
(brown/black)
d) The following are methods for decreasing the activity of catechol oxidase and thus preventing browning in fruits and vegetables:
1. Cut fruit is coated with an antioxidant such as ascorbic acid, which prevents oxygen from reaching the cells of the fruit.
2. Vegetables are blanched(immersed in boiling water for one minute) prior to preservation.
3. Compounds such as cinnamic acid are added to fruit juice. Cinnamic acid is structurally similar to catechol.
Select the treatment 1, 2 or 3 which
* Represents an example of competitive inhibition
* Permanently changes the shape of the enzyme [2]
Q7 d) 3;
| 2; [2]
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Past Paper Question - June 2013 Q7 e)i)+ii)+iii) AS1 (Legacy Material)
Q7 The cells of many fruits and vegetables including apples, bananas and lettuce contain the enzyme catechol oxidase. The enzyme is found in the cytoplasm, whereas its substrate catechol is usually confined to the vacuole. The reaction is a major cause of browning in fruits and vegetables. The enzyme's action is summarised below.
Catechol Non-enzymatic
oxidase oxidation
Catechol ----------> Benzoquinone ---------> Melanin
(brown/black)
The following are methods for decreasing the activity of catechol oxidase and thus preventing browning in fruits and vegetables:
1. Cut fruit is coated with an antioxidant such as ascorbic acid, which prevents oxygen from reaching the cells of the fruit.
2. Vegetables are blanched(immersed in boiling water for one minute) prior to preservation.
3. Compounds such as cinnamic acid are added to fruit juice. Cinnamic acid is structurally similar to catechol.
e) Spoilage of fruit juice can also be prevented by removing the catechol oxidase enzyme. One way of doing this is to pour the juice through a column containing a protease enzyme immobilised on glass beads.
An experiment was carried out to determine the optimum flow rate for the juice through the column. A tap was used to adjust the flow rate. Once the juice had been passed through the column, it was tested to determine the concentration of catechol oxidase remaining. The results of the investigation are shown in the following table.
Flow rate/ mm^3 min^-1 Concentration of catechol oxidase
remaining in treated juice/ arbitrary units
10 0
20 0
30 5
40 45
50 85
60 125
70 140
i) Plot the results using a suitable graphical technique. [4]
ii) It was concluded that the optimum flow rate for the juice through the column was 20 mm^3 min^-1. Explain how this conclusion was reached. [2]
iii) Describe and explain the trend shown by the results for flow rates of 40-70 mm^3 min^-1. [2]
Q7 e)i) Appropriate caption (e.g. Concentration of catechol oxidase remaining in fruit juice as flow rate through an immobilised enzyme column is changed), including concentration of catechol oxidase and flow rate;
axes the correct way round (flow rate on x-axis);
selection of appropriate scale and axes labelled including units;
points plotted accurately, and joined by short straight lines; [4]
[X-axis = Flow rate/ mm3 min-1, Y-axis = Concentration of catechol oxidase remaining in treated juice/ arbitrary units]
ii) At flow rate of 20 mm3 min–1, no catechol oxidase remains in the fruit juice;
20 mm3 min–1 represents a more efficient/more cost effective process than at 10mm3 min–1 [2]
iii) As flow rate increases, the amount of enzyme remaining in the juice increases/the amount of enzyme (catechol oxidase) removed decreases; at higher flow rates, there is insufficient time for substrate molecules
to be catalysed by the enzyme/for substrates to bind to the active sites of the protease molecules; [QWC: must be clear which enzyme is being referred to. Protease is the catalytic enzyme, catechol oxidase is the (protein) substrate broken down.] [2]
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Past Paper Question - June 2018 Q7 a) AS1 (Revised Spec.)
Q7 Hypertension (high blood pressure) is a condition which often goes undetected as it has very few obvious symptoms. If left untreated, it increases the risk of heart attack and stroke.
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Adults with hypertension often have increased blood levels of a molecule called angiotensin 2. An enzyme Angiotensin Converting Enzyme (ACE) converts the molecule angiotensin 1 to angiotensin 2.
ACE
Angiotensin 1 ------> Angiotensin 2
Angiotensin 2 narrows blood vessels, which in turn contributes to increased blood pressure.
a) With reference to the mechanism of enzyme action, describe fully how ACE converts angiotensin 1 to angiotensin 2. [3]
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Q7 a) Angiotensin 1 fits exactly into/complimentary fit with the active site of the ACE/ACE active site mounds to fit around angiotensin 1;
Bonds are made/broken within angiotensin 1 which changes its shape;
Angiotensin 2 (product) no longer fits the active site so is released; [3]
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Past Paper Question - June 2018 Q7 b) AS1 (Revised Spec.)
Q7 Hypertension (high blood pressure) is a condition which often goes undetected as it has very few obvious symptoms. If left untreated, it increases the risk of heart attack and stroke.
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Adults with hypertension often have increased blood levels of a molecule called angiotensin 2. An enzyme Angiotensin Converting Enzyme (ACE) converts the molecule angiotensin 1 to angiotensin 2.
ACE
Angiotensin 1 ------> Angiotensin 2
Angiotensin 2 narrows blood vessels, which in turn contributes to increased blood pressure.
Enzyme inhibition may be used to treat hypertension. ACE inhibitors are medicines which prevent angiotensin 1 being converted into angiotensin 2.
b) Describe and explain two ways in which these inhibitors may inhibit the ACE enzyme. [3]
Q7 b) Competitive and non-competitive inhibition;
Inhibitor may occupy the active site so that angiotensin 1 cannot bond/inhibitor has a similar shape to angiotensin 1;
Inhibitor may interact with another site on the enzyme changing the shape of the active site; [3]
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Past Paper Question - June 2018 Q7 c)i)+ii) AS1 (Revised Spec.)
Q7 Hypertension (high blood pressure) is a condition which often goes undetected as it has very few obvious symptoms. If left untreated, it increases the risk of heart attack and stroke.
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Adults with hypertension often have increased blood levels of a molecule called angiotensin 2. An enzyme Angiotensin Converting Enzyme (ACE) converts the molecule angiotensin 1 to angiotensin 2.
ACE
Angiotensin 1 ------> Angiotensin 2
Angiotensin 2 narrows blood vessels, which in turn contributes to increased blood pressure.
Enzyme inhibition may be used to treat hypertension. ACE inhibitors are medicines which prevent angiotensin 1 being converted into angiotensin 2.
While ACE is an enzyme which can contribute to heart disease, other enzymes can act as biomarkers and are useful as indicators of health.
Two examples of biomarkers which can be used to assess heart health are the enzyme creatine kinase and another molecule called troponin. Both of these molecules are released from damaged heart muscle.
The graph below shows the levels of both of these biomarkers over a 100-hour period (96 hours = 4 days) in the blood of an individual who has suffered from chest pain.
[The graph is present on page 13 of the S3 Biology Christmas Test (Assessment 2)]
c) i) Describe the differences between the trends shown in the graph for creatine kinase and troponin. [2]
ii) Using the graph, suggest two reasons why doctors use the troponin blood test (rather than creatine kinase) to help diagnose a heart attack. [2]
Q7 c)i) Any two from:
• Troponin increases to a much higher level than CK.
• CK levels start to decrease sooner (after 27 hours) than troponin (which reaches its peak at 35 hours).
• Troponin remains in the blood in high levels for longer/no creatine kinase after 50 hours.
• Troponin decreases at a slower rate than CK. [2]
ii) If blood is not tested within a couple of days then the rise in CK may be missed;
CK levels may not increase enough to be detected; [2]
