03.13 End of Chapter Questions
Questions
1 The diagram below shows an enzyme and two inhibitors of the enzyme, X and Y. Which of the following describes the two inhibitors? [1]
A X and Y are competitive inhibitors.
B X and Y are non-competitive inhibitors.
C X is a competitive inhibitor and Y is a non-competitive inhibitor.
D X is a non-competitive inhibitor and Y is a competitive inhibitor.
B;
2 In a reaction controlled by an enzyme, which of the following graphs shows the effect of substrate concentration on the rate of the reaction? [1]
D;
3 The graph shows the progress of the digestion of starch by the enzyme salivary amylase. Why does the reaction slow down?
A End-product inhibition by maltose.
B The salivary amylase is becoming denatured.
C The salivary amylase is gradually becoming saturated with starch.
D There are fewer and fewer substrate molecules left to bind with the salivary amylase.
[1]
D;
4 If methylene blue dye is added to a suspension of yeast cells, living cells do not take up the stain, and they remain colourless. However, dead cells are stained blue. This fact was used to carry out an investigation into the rate at which yeast cells were killed at two diff erent temperatures (at high temperatures the yeast enzymes will be denatured). The results are shown in the diagram below.
Which of the following is correct?
| The higher temperature is | The vertical axis (y-axis) should be labelled | |
| A | X | % coloured cells |
| B | Y | % colourless cells |
| C | X | % colourless cells |
| D | Y | % coloured cells |
[1]
C;
5 Copy the graph in question 3 and draw a line from which the initial rate of reaction could be calculated. [1]
straight line drawn from origin at zero to show steepest gradient of curve;
6 The graph shows the eff ect of changes in pH on the activity of the enzyme lysozyme.
a Describe the eff ect of pH on this enzyme. [2]
maximum activity / optimum pH, is pH 5;
activity gradually increases between pH 2 and pH 5, and decreases from pH 5 to pH 10;
activity very low at pH 2 and pH 10; AW
b Explain why pH aff ects the activity of the enzyme. [4]
pH is a measure of the hydrogen ion concentration;
hydrogen ions are positively charged;
hydrogen ions can interact with the R groups of amino acids;
affects ionic bonding / affects ionisation of R groups;
affects tertiary structure / affects 3D shape of enzyme;
therefore substrate may not fit active site (as precisely);
[Total: 6]
7 The graph below shows the eff ect of temperature on the rate of reaction of an enzyme.
a What is indicated by X? [1]
optimum temperature;
b What temperature would X be for a mammalian enzyme? [1]
37 °C; accept 40 °C
c Explain what is happening in region A. [3]
as temperature increases the kinetic energy of the molecules increases;
the rate of collision between substrate and, enzyme / active site, increases;
rate of reaction increases;
d Explain what is happening in region B. [3]
the enzyme is gradually being denatured;
when the rate is zero the enzyme is completely denatured;
ORA enzyme loses tertiary structure;
substrate no longer fits into active site / active site loses its (specific) shape so substrate does not fit;
AVP e.g.hydrogen bonds broken / increased vibration of enzyme molecule;
e Enzymes are eff ective because they lower the activation energy of the reactions they catalyse.
Explain what is meant by ‘activation energy’. [2]
the extra energy which must be given to the substrate;
before it can be converted into the product;
[Total: 10]
8 The reaction below occurs during aerobic respiration. The reaction is catalysed by the enzyme
succinate dehydrogenase.
a Name the substrate in this reaction. [1]
succinic acid;
b The molecule malonic acid, which is shown here, inhibits this reaction.
It does not bind permanently to the enzyme. Describe how malonic acid inhibits the enzyme succinate dehydrogenase. [3]
malonic acid acts as a competitive inhibitor;
it has a similar shape / structure to succinic acid;
it therefore competes with succinic acid for a place in the active site of the enzyme;
c Heavy metals such as lead and mercury bind permanently to –SH groups of amino acids present in enzymes. These –SH groups could be in the active site or elsewhere in the enzyme.
i Name the amino acid which contains –SH groups. [1]
cysteine;
ii Explain the function of –SH groups in proteins and why binding of heavy metals to these groups
would inhibit the activity of an enzyme. [4]
–SH groups form disulfide bridges;
used to determine tertiary structure;
heavy metal would prevent formation of disulfide bridges;
could change shape of active site;
heavy metal could affect shape either by binding directly in the active site, or by binding at another site which then results in change in shape of the active site;
substrate would not be able to fit into active site;
iii What type of inhibition would be caused by the heavy metals? [1]
(non-competitive) irreversible;
[Total: 10]
9 You are provided with three solutions: A, B and C. One solution contains the enzyme amylase, one contains starch and one contains glucose. Starch is the substrate of the enzyme. The product is the sugar maltose. You are provided with only one reagent, Benedict’s solution, and the usual laboratory apparatus.
a Outline the procedure you would follow to identify the three solutions. [6]
carry out Benedict’s test on solutions A, B and C;
a positive result / brick-red precipitate will be seen, with the glucose solution;
heat separate samples of the two remaining solutions, in boiling water bath / to high
temperature (e.g. 80 °C), for suitable time / at least two minutes (enzyme will be denatured);
for each heated solution, mix it with an unheated sample of the other solution;
leave several minutes / suitable time (for reaction to take place);
carry out Benedict’s test on the two tubes;
only one will give a positive result (due to presence of maltose) and this will be the one
which contained the unheated enzyme;
Accept alternative wording for all steps in the procedure, provided the same logical
sequence is described
b What type of reaction is catalysed by the enzyme? [1]
hydrolysis;
[Total: 7]
10 The activity of the enzyme amylase can be measured at a particular temperature by placing a sample into a Petri dish containing starch-agar (‘a starch-agar plate’). Starch-agar is a jelly containing starch. One or more ‘wells’ (small holes) are cut in the agar jelly with a cork borer, and a sample of the enzyme is placed in each well.
The enzyme molecules then diff use through the agar and gradually digest any starch in their path. At the end of the experiment, iodine in potassium iodide solution is poured over the plate. Most of the plate will turn blue-black as iodine reacts with starch, but a clear ‘halo’ (circle) will be seen around the well where starch has been digested. Measuring the size of the halo can give an indication of the activity of the enzyme.
A student decided to investigate the rate at which a mammalian amylase is denatured at 60 °C. She heated different samples of the enzyme in a water bath at 60 °C for 0, 1, 5, 10 and 30 minutes. She then allowed the samples to cool down to room temperature and placed samples of equal volume in the wells of five starch-agar plates, one plate for each heating period. She then incubated the plates in an oven at 40 °C for 24 hours.
The results of the student’s experiment are shown on the next page. A diagram of one dish is shown, and the real size of one halo from each dish is also shown.
a Why did the student cut four wells in each dish rather than just one? [1]
replication increases reliability; AW
b One dish contained samples from amylase which was not heated (time zero). This is a control dish. Explain the purpose of this control. [1]
to act as a reference to show what happens if there is no denaturation; AW
c Explain why the starch-agar plates were incubated at 40°C and not room temperature. [1]
40°C is the optimum temperature for a mammalian enzyme;
d Describe what was happening in the dishes during the 24 hours of incubation. [4]
enzyme / amylase (molecules) diffuse(s) from wells into the agar;
enzyme / amylase digests the starch;
to maltose;
forms rings / halos, of digested starch around the wells;
amount of digestion / rate of digestion, is related to degree of denaturation of enzyme /
amylase;
e Why was it important to add the same volume of amylase solution to each well? [1]
the more enzyme / amylase added, the greater the amount of digestion of starch
or
want results to be due to differences in preheating times, not to differences in amount of amylase / enzyme; AW
f Measure the diameter in mm of the representative halo from each dish. Record the results in a suitable table. [4]
| Time (heated) at 60 °C / min | Diameter of halo / mm |
| 0 | 24 |
| 1 | 19 |
| 5 | 10 |
| 10 | 6 |
| 30 | 0 |
table drawn with ruled lines for border and to separate columns and headings (ideally
ruled lines between rows, but not essential for mark);
correct headings to columns with units;
first column is independent variable (Time heated at 60 °C);
correct measurements of halos;
g Only one halo from each dish is shown in the diagrams. In practice there was some variation in the diameters of the four halos in each dish. How would you allow for this when processing your data? [1]
measure the four halos and calculate the mean;
(any anomalous results should be ignored)
h Plot a graph to show the eff ect of length of time at 60°C on the activity of the enzyme. [5]
x-axis (horizontal axis) is labelled ‘Time (heated) at 60 °C’, y-axis (vertical axis) is
labelled ‘Diameter’;
units given on axes, min / minutes and mm;
regular intervals on both axes (check that 0, 1, 5, 10, 30 are not regularly spaced on
x-axis);
points plotted accurately;
points joined with straight lines or smooth curve;
i Describe and explain your results. [4]
enzyme was completely denatured after 30 minutes;
rate of denaturation was rapid at first and then gradually slowed down;
data quoted;
enzyme loses tertiary structure;
substrate no longer fits into active site / active site loses its (specific) shape so substrate does not fit;
AVP e.g. hydrogen bonds broken / increased vibration of enzyme molecule;
j Another student discovered that amylases from fungi and bacteria are more resistant to high temperatures than mammalian amylases. Using starch-agar plates as a method for measuring the activity of an amylase at 40°C, outline an experiment that the student could perform to discover which amylase is most resistant to heat. Note that temperatures up to 120°C can be obtained by using an autoclave (pressure cooker). [5]
heat samples of mammalian, fungal and bacterial amylases at different temperatures;
suitable range, e.g. between 40 °C and 120 °C;
40 °C is a control (for reference to find out size of halo with no denaturation);
at least five temperatures, e.g. 40, 60, 80, 100, 120 °C;
heat for suitable length of time (e.g. one hour, at least ten min);
cool to room temp / 40 °C, add equal volumes to wells in starch–agar plates, replicate wells in each plate (e.g. four), leave 24 hours, test for starch, measure diameters of halos;
Background information: amylase enzymes from the bacterium Bacillus licheniformis and
the fungus Aspergillus have been developed by biotechnology companies for use in industrial processes. For example, a bacterial amylase that functions in the range 90–110 °C has been developed and is used in beer brewing and other processes, and a fungal amylase that operates in the range 50–60 °C is used for pastry baking and maltose syrup production.
k Enzymes are used in many industrial processes where resistance to high temperatures is an advantage. State three other variables apart from temperature which should be controlled in an industrial process involving enzymes. [3]
pH;
substrate concentration;
enzyme concentration;
[Total: 30]
11 Two inhibitors of the same enzyme, inhibitor A and inhibitor B, were investigated to discover if they were competitive or non-competitive. In order to do this, the rate of reaction of the enzyme was measured at diff erent concentrations of substrate without inhibitor, with inhibitor A and with inhibitor B. Graphs of the data were plotted as shown on the next page. The graphs showed that one inhibitor was competitive and the other non-competitive.
Copy the graphs.
a Label the graph for ‘no inhibitor’ to show the position of Vmax, ½Vmax and Km. [3]
see Figure 3.14b. Award 1 mark for each correct label;;;
b State the eff ect that inhibitor A had on V max and Km of the enzyme. [2]
inhibitor A had no effect on Vmax;
and increased Km;
c State the effect that inhibitor B had on Vmax and Km of the enzyme. [2]
inhibitor B decreased Vmax;
and had no effect on Km;
d Which inhibitor is competitive and which is non-competitive? Explain your answer. [4]
inhibitor A is competitive, B is noncompetitive;
A is competitive because:
it increased Km / did not affect Vmax;
decreased the affinity of the enzyme for its substrate;
the substrate is competing with the inhibitor for the active site;
the inhibition is overcome by increasing substrate concentration;
Or
Alternative ways of explaining the same marking points;
B is non-competitive because:
it did not affect Km/decreased Vmax;
did not affect the affinity of the enzyme for its substrate;
the substrate is not competing with the inhibitor for the active site;
the inhibition cannot be overcome by increasing substrate concentration;
Double-reciprocal plots of the data obtained produced the following graphs.
e Identify which line, X, Y or Z, corresponds to each of the following experiments:
i inhibitor absent
ii competitive inhibitor present
iii non-competitive inhibitor present
Briefly explain your answers to ii and iii. [5]
i Z; [1]
ii Y; [1]
iii X; [1]
Reasons:
Accept any valid points up to a maximum of 2 marks for each inhibitor, for example:
ii the lines Y and Z cross the y-axis at the same point, which is 1/Vmax;
therefore Vmax is the same for both;
line Y meets the x-axis at a less negative value than line Z;
therefore Km is increased; [max. 2]
iii the lines X and Z cross the y-axis at the same point, (which is –1/Km );
therefore both have the same Km;
line X crosses the y-axis higher than line Z, so 1/Vmax has a higher value;
therefore Vmax has a lower value; [max. 2]
[Total: 16]