9700_w04_2 question 5

5 Enzymes catalyse reactions in which substrate molecules are converted to products.

(a) There are two main approaches to investigation of the activity of an enzyme. State the
two ways in which the activity of an enzyme can be found.

1 .......................................................................................................................................

..........................................................................................................................................

2 .......................................................................................................................................

......................................................................................................................................[2]

9700_s05_2 question 5
Collagen is continuously broken down in the extracellular matrix by the enzyme collagenase,
which catalyses the hydrolysis of the peptide bond between the amino acids glycine and
isoleucine.

(b) Suggest how collagenase is only able to act on the peptide bond between glycine and
isoleucine and not on peptide bonds between any other amino acids.

..........................................................................................................................................

..........................................................................................................................................

..........................................................................................................................................

..................................................................................................................................... [2]

9700_s07_2 question 2

Starch phosphorylase is an enzyme found in plant cells. In potato tuber cells, the enzyme
takes part in the breakdown of starch when the tuber begins to grow.

starch phosphorylase
starch + phosphate ions glucose 1-phosphate

A student investigated the effect of pH on this reaction using two buffer solutions.

The student prepared four test-tubes, A to D, as shown in Table 2.1 and described
below.

The student made an extract of potato tissue that contained the enzyme. Some of this
extract was boiled.

A solution of potassium dihydrogen phosphate was added to some tubes as a source of

phosphate ions.

The test-tubes were left for ten minutes in a water bath at 30 °C and then samples were
tested with iodine solution.
 test- contents
tube
volume volume of volume of pH of buffer enzyme results with
of starch glucose potassium solution extract iodine solution
solution / 1-phosphate dihydrogen after ten
cm3 solution / cm3 phosphate minutes
solution / cm3
A 2 0.5 6.5 unboiled negative

B 2 0.5 2.0 unboiled positive

C 2 0.5 6.5 boiled positive

D 2 6.5 boiled negative

(b) (i) State what the student would conclude from a positive result with iodine solution.

..............................................................................................................................[1]

(ii) Explain why the student boiled some of the extract in this investigation.

..................................................................................................................................

..................................................................................................................................

..................................................................................................................................

..............................................................................................................................[2]

9700_w10_23 question 3
3 The enzyme sucrase is used by many organisms for the hydrolysis of sucrose. Fig. 3.1 shows
a diagram of the enzyme and its substrate.

sucrose

sucrase

(iii) Copper ions (Cu2+) will inhibit the enzyme sucrase.

Suggest which type of inhibition occurs.

.............................................................................................................................. [1]
9700_w05_2 question 2

The enzyme lipase catalyses the hydrolysis of ester bonds in triglycerides. As the reaction
proceeds there is a decrease in pH. The progress of the reaction may be followed by using a
pH meter.

A solution containing tristearin was placed in a water bath at 25 °C. When the solution had
reached this temperature, lipase was added and the mixture stirred. The pH of the reaction
mixture was recorded every minute for 20 minutes. The results are shown in Fig. 2.2.

11

10

8
pH
7

4
0 5 10 15 20
time / min

Fig. 2.2

(d) Using the data in Fig. 2.2, state the time when

(i) lipase was added;

...............................................................................................................................[1]

(ii) the reaction ended.

...............................................................................................................................[1]

(e) Explain why the pH decreases during this reaction.

..........................................................................................................................................

......................................................................................................................................[1]
9700_w09_22 question 2

2 Sucrase is the enzyme that catalyses the hydrolysis of sucrose. A student investigated the
effect of substrate concentration on the activity of this enzyme.

Six test-tubes were set up each containing 10 cm3 of different concentrations of sucrose
solutions. The test-tubes were left in a water bath at 30 °C for ten minutes.

After ten minutes, 5 cm3 of a sucrase solution at 30 °C was added to each test-tube and the
reaction mixtures were stirred.

After a further five minutes, the temperature of the water-bath was raised to above 85 °C
and the same volume of Benedict’s solution added to each test-tube in turn. The student
recorded the time when a green colour first became visible in each test-tube.

The concentrations used and the student’s results are shown in Table 2.1.

Table 2.1

concentration of time taken for green

sucrose / g dm–3 colour to appear / s
5 278
10 145
15 95
20 75
50 47
100 45

(a) Explain why the temperature of the water-bath was raised to above 85 °C.

..........................................................................................................................................

..........................................................................................................................................

..........................................................................................................................................

...................................................................................................................................... [2]
9700_s10_23 question 5
(c) The enzyme urease is known to be affected by competitive inhibitors. A student carried
out an investigation to determine the percentage of urea hydrolysed by urease at various
time intervals
• without any inhibitor
• with a competitive inhibitor.

The experiment was carried out in test tubes set up as follows:

Tube A – 1 cm3 of urease solution, 10 cm3 pH 7.5 buffer solution, 1 cm3 urea solution.
Tube B – 1 cm3 urease solution, 9 cm3 pH 7.5 buffer solution, 1 cm3 inhibitor, 1 cm3 urea
solution.
Tube C – 1 cm3 water, 10 cm3 pH 7.5 buffer solution, 1 cm3 urea solution.

The results are shown in the table below.

percentage of urea remaining

time / min
Tube A Tube B Tube C

0 100 100 100

5 55 99 100

10 29 98 100

15 14 96 100

20 8 95 100

25 5 92 100

30 3 90 100

(i) State how Tube C acts as a control for this investigation.

..................................................................................................................................

..............................................................................................................................[1]

9700_s13_22 question 4

(d) Freezing temperatures can also completely stop enzyme activity by causing
the molecules to undergo ‘cold denaturation’. Enzyme activity is not recovered
when temperatures are increased to a normal working temperature range.
(ii) Suggest how the molecular structure of the enzyme changes during ‘cold
denaturation’.

..................................................................................................................................

..................................................................................................................................

..................................................................................................................................

............................................................................................................................. [2]
9700_s13_23 question 4
4 The enzyme, catechol oxidase, causes a brown colour to develop when slices of many fruits,
such as apples, are exposed to air.

The enzyme catalyses the following reaction:

catechol oxidase
catechol + oxygen quinone + water

Quinone is then immediately further oxidized in air to a brown-coloured substance. Catechol

and quinone are colourless.

A student investigated the rate of this reaction under different conditions.

(a) State how the student could follow the progress of this reaction.

..........................................................................................................................................

..................................................................................................................................... [1]

(d) Lemon juice contains citric acid. Adding even a small amount of diluted lemon juice to
apple slices slows the appearance of the brown colour.

Suggest an explanation for this observation.

..........................................................................................................................................

..........................................................................................................................................

..........................................................................................................................................

..................................................................................................................................... [2]
9700_w14_23 question 4

(b) Suggest how enzymes which use the induced fit mechanism can be less affected by
competitive inhibitors than those which use the lock and key mechanism.

..................................................................................................................................................

..................................................................................................................................................

..................................................................................................................................................

..................................................................................................................................................

.............................................................................................................................................. [2]
9700_s15_22 question 4

4 Many microorganisms can digest cellulose by using a group of enzymes collectively known as
cellulases. Cellobiose is the disaccharide produced during cellulose digestion.

The cellulase known as β-glucosidase completes the digestion of cellulose by hydrolysing the
cellobiose molecule to produce two β-glucose molecules.
(b) β-glucosidase was extracted from two different bacteria, Agrobacterium tumefaciens and
Thermotoga maritima.

Fig. 4.1 shows the results of an investigation into the effect of temperature between 0 °C and
100 °C, on the activity of each enzyme.

• L represents the lowest temperature at which activity of each enzyme was detected.
• H represents the highest temperature at which activity of each enzyme was detected.

100

80
relative
activity /
% of
maximum 60 H
activity

40
L

20

L
H
0
0 20 40 60 80 100
temperature / °C
Key
enzyme A (extracted from A. tumefaciens)
enzyme T (extracted from T. maritima)

Fig. 4.1
 (ii) Both enzyme A and enzyme T act on cellobiose. They have a similar, but not identical,
primary structure.

Suggest how similarities and differences in the primary structure of the two enzymes
could help to explain the results obtained in the investigation.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

.......................................................................................................................................[4]

9700_w15_21 question 5
(d) The measles pathogen must carry out RNA replication to make new RNA molecules for the
new pathogens. This happens inside the infected cell.

The pathogen carries its own enzyme for RNA replication, but no other enzymes.

Explain why the measles pathogen cannot use an enzyme from the cell to carry out RNA
replication.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

.............................................................................................................................................. [2]
 9700_w15_23 question 3

3 The enzyme catalase is found in many plant and animal tissues.

The enzyme catalyses the decomposition of hydrogen peroxide, which is a toxic product of
metabolism. The reaction is:

catalase
2H2O2 O2 + 2H2O

A research team investigated the activity of two forms of catalase, P and Q, extracted from
Anopheles gambiae, an important vector of malaria. The team investigated the effect of increasing
concentrations of hydrogen peroxide on the activity of these two forms of catalase.

The results are shown in Fig. 3.1.

140
catalase P
120

100
catalase Q
80
activity of catalase
/ arbitrary units 60

40

20

0
0 50 100 150 200 250 300
concentration of hydrogen peroxide / mM

Fig. 3.1

(b) Each molecule of catalase consists of four identical polypeptides. The two forms of catalase in
A. gambiae differ by only one amino acid at position 2 in the amino acid sequence. Catalase P
has serine and catalase Q has tryptophan.

Suggest how the difference in one amino acid is responsible for the lower activity of catalase Q
compared with catalase P.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

.............................................................................................................................................. [2]
(c) Female mosquitoes feed on blood in order to produce their eggs. After feeding, the metabolic
rate increases for egg production.

The researchers allowed female mosquitoes to feed on blood. They found that female
mosquitoes with only catalase P produced more eggs than those with only catalase Q.

Suggest why there is a difference in egg production between the two types of A. gambiae.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

.............................................................................................................................................. [2]
9700_s16_22 question 6a
(iii) Glycogen branching enzyme is another enzyme that is required for glycogen synthesis.

Suggest why glycogen branching enzyme is needed in addition to glycogen synthase.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

.......................................................................................................................................[1]
9700_s16_23 question 5c
(ii) M. tuberculosis and humans both use RNA polymerase for transcription.

Suggest why rifampicin does not affect transcription in human cells.

...........................................................................................................................................

.......................................................................................................................................[1]
9700_w16_22 question 3
(b) The glucose isomerase used in the production of high fructose corn syrup is extracted from a
strain of a bacterium, Thermus thermophilus, which is found in hot springs. The enzyme has
an optimum temperature of 95 °C.

Suggest and explain the advantages of using glucose isomerase from T. thermophilus to
produce high fructose corn syrup, rather than using glucose isomerase that has an optimum
temperature of 37 °C.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...............................................................................................................................................[3]
9700_w16_23 question 2

2 (a) Explain how enzymes lower the activation energy needed to allow reactions to proceed.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...............................................................................................................................................[2]

9700_s17_22 question 2

(d) A separate investigation into the effect of pH on the same bacterial lipase compared the
enzyme free in solution with the enzyme immobilised by physical attachment to a stable
polymer.

At a temperature of 37 °C, the optimum pH of the enzyme free in solution was the same as
that shown in Fig. 2.2. The optimum pH of the immobilised enzyme was measured as pH 4.

(i) Suggest one reason to explain why the enzyme free in solution has a different optimum
pH compared to the immobilised enzyme.

...........................................................................................................................................

...........................................................................................................................................

.......................................................................................................................................[1]

9700_m18_22 question 3

(b) On a commercial scale, immobilised lactase can be used to produce lactose-free milk.

One of the products of the reaction shown in Fig. 3.1 acts as an inhibitor of lactase. This is an
example of product inhibition.

(i) Suggest why product inhibition is useful in K. lactis when lactase is acting as an
intracellular enzyme, but can be a disadvantage when extracted lactase is used free in
solution for the production of lactose-free milk.

...........................................................................................................................................

...........................................................................................................................................

...................................................................................................................................... [2]

(ii) Suggest how using immobilised lactase in a commercial application helps to reduce the
problem of product inhibition.

...........................................................................................................................................

...................................................................................................................................... [1]

(iii) The first large-scale production of lactose-free milk with an immobilised enzyme used
lactase trapped in cellulose triacetate fibres.
Suggest one feature of cellulose triacetate that makes it useful as an immobilising
material.

...........................................................................................................................................
9700_w18_21 question 2
(c) Enzymes, such as lactase, are often immobilised for use in the food industry.

A scientist carried out an investigation to determine the effects of temperature on the activity
of lactase when it was immobilised and when it was free in solution.

The scientist produced alginate beads containing lactase for use in this investigation. The
beads varied in size. The scientist selected small beads for the investigation and put them
into a glass column.

(i) Suggest the advantage of using small beads rather than large beads.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...................................................................................................................................... [2]

9700_w19_23 question 3

(b) Neuraminidase removes parts of the host cell receptors that bind to haemagglutinin. This
helps newly-formed viruses to leave host cells.

Drugs have been developed to act on neuraminidase. These drugs prevent viruses from
leaving host cells.

Suggest and explain how these drugs act to prevent viruses leaving cells.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

............................................................................................................................................. [3]
9700_s19_21 question 3

3 Neutrase® is an enzyme that is used to hydrolyse proteins in solution. When the enzyme is mixed
with a 2% protein solution the reaction mixture changes from white to colourless.

A student carried out an experiment to find the effect of copper sulfate and potassium sulfate on
the activity of Neutrase®.

The student made four reaction mixtures in test-tubes A to D. Test-tubes A to C contained equal
volumes of protein solution and 0.1 cm3 of solutions of copper sulfate or potassium sulfate.
Test-tube D contained the same volume of protein solution and 0.1 cm3 of water.

0.5 cm3 of a 1% Neutrase® solution was added to test-tube A and immediately placed into a
colorimeter. The colorimeter was used to measure the intensity of light that is absorbed by the
solution (absorbance) over 100 seconds. The procedure was repeated with the other reaction
mixtures, B, C and D.

The results are shown in Fig. 3.1.

1.4
A 0.05 mol dm–3 copper sulfate

1.2
B 0.01 mol dm–3 copper sulfate

1.0

0.8
absorbance C 0.01 mol dm–3 potassium sulfate
0.6
D water
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90 100
time / s

Fig. 3.1

(a) (i) Suggest and explain why measuring the absorbance of the reaction mixture over 100 s
is a suitable method for determining the activity of Neutrase®.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

..................................................................................................................................... [2]
(ii) With reference to Fig. 3.1:

• describe the effects of copper sulfate solution and potassium sulfate solution on the
activity of Neutrase®
• suggest explanations for the effects that you have described.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

..................................................................................................................................... [5]
9700_s20_21 question 3

3 The enzyme glucose 6-phosphate dehydrogenase (G6PD) is composed of two identical polypeptide
chains.

(a) Students investigated the activity of two forms of G6PD, J and K, at different concentrations
of substrate. K is a form of the enzyme that results from a mutation that changes one amino
acid in the polypeptide.

The results are shown in Fig. 3.1.

70

J
60

K
50
rate of reaction
/ μmol min–1

40

30

20

10

0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
concentration of substrate / mmol dm–3

Fig. 3.1
 (ii) With reference to Fig. 3.1 and Table 3.1, describe the effect of the mutation on the activity of
G6PD and suggest an explanation for this effect.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

..................................................................................................................................... [4]

9700_w21_22 question 3

3 Baculovirus is a virus that kills some of the insect pests of major crops. When the virus is released
to the outside of the insect body, it is contained within stable, protective structures known as
polyhedrons.

The main component of the polyhedron is a protein molecule, polyhedrin.

Polyhedrons can be sprayed onto plants as a bioinsecticide. They are ingested by feeding insect
larvae and once inside the insect gut they break down to release the virus.

(b) Suggest and explain the conditions present in the insect gut that can cause the breakdown of
polyhedrons.

...................................................................................................................................................

...................................................................................................................................................

...................................................................................................................................................

............................................................................................................................................. [2]
9700_s21_22 question 5

(d) Ultrasound is one possible method that can be used to destroy microorganisms that
contaminate fruit juices. Ultrasound is the term given to sound waves that are out of the range
of human hearing.

An investigation was carried out into the effect of ultrasound on the activity of cellulase,
pectinase and xylanase used in fruit juice manufacture.

For each enzyme, the effect of ultrasound was compared with no ultrasound on the:
• maximum rate of reaction (Vmax)
• Michaelis‑Menten constant (Km)
• catalytic efficiency (Vmax / Km)

Table 5.1 summarises the results. A higher Vmax / Km indicates a higher catalytic efficiency.

Table 5.1

comparison comparison Vmax / Km

enzyme method
of Vmax of Km / min–1
ultrasound higher higher 34
cellulase
no ultrasound lower lower 29
ultrasound same lower 945
pectinase
no ultrasound same higher 759
ultrasound higher same 146
xylanase
no ultrasound lower same 125

(i) In terms of changes in the interaction between enzyme and substrate when ultrasound
is used, suggest explanations for the lower Km for pectinase and the higher Vmax for
xylanase, as shown in Table 5.1.

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

...........................................................................................................................................

..................................................................................................................................... [3]

(ii) Explain whether the data shown in Table 5.1 supports the recommendation that
ultrasound can be used in the manufacture of fruit juices.

...........................................................................................................................................

...........................................................................................................................................

..................................................................................................................................... [1]
9700_w21_22 question 6
6 Amylase is an enzyme that catalyses the hydrolysis of starch into reducing sugars.

A student carried out an experiment to investigate the hydrolysis of starch using immobilised
amylase. Fig. 6.1 is a diagram of the apparatus that was used in the investigation.

starch
solution

tap

immobilised
amylase
in alginate beads

tap

beaker for
collecting
product

Fig. 6.1

The alginate beads were all the same size.

Both taps were opened to allow the starch solution to flow down the column and for the product to
be collected.

The product was tested for the presence of reducing sugar and starch.

(b) One standardised variable in the investigation is the size of the alginate beads.

Suggest one reason why using larger or smaller alginate beads in the column would affect
the results obtained.

...................................................................................................................................................

...................................................................................................................................................

............................................................................................................................................. [1]