Mode of Action of Enzymes (Cambridge (CIE) AS Biology)

Lysozyme is an enzyme found in many secretions of the body, such as tears, saliva and milk. It is able to break down the peptidoglycan cell walls of bacteria and form part of the body's chemical defence system against pathogens.

Fig.1 shows a molecular model of lysozyme.

Fig. 1

With reference to Fig.1 and the parts labelled A and B, explain the term 'secondary structure'.

Enzymes, such as lysozyme, are described as being 'biological catalysts'.

Outline what is meant by the term 'biological catalyst'.

State whether lysozyme can be classified as an intracellular or extracellular enzyme and explain your answer.

Lysozyme molecules will eventually stop working and are broken down.

Outline how cells replace the enzymes that are broken down.

Fig.1 represents the change in the amount of energy needed for two reactions (P and Q) to occur over time.

Fig. 1

(i) Identify the reaction in Fig.1 that is catalysed by an enzyme.

[1]

(ii) Explain your answer in part i).

[1]

The enzyme lactase catalyses the breakdown of the glycosidic bond in lactose.

A student investigated the effect of increasing the concentration of lactose on the rate of activity of lactase.

Five test tubes were set up with each containing 3 cm³ of different concentrations of a lactose solution. The test tubes were placed in a water bath at 38°C for ten minutes. A container with a lactase solution was also put into the water bath.

After ten minutes, 1 cm³ of the lactase solution was added to each test tube. The test tubes were returned to the water bath and kept at 38°C for another ten minutes.

Thereafter, the temperature of the water bath was raised to boiling point before Benedict's solution was added to each test tube. The time taken for a colour change was recorded and used to calculate the rate of enzyme activity.

The results are shown in Fig. 2

Fig. 2

Explain why the action of lactase can be described as 'catabolic'.

State what happened to lactase when the temperature in this investigation was raised to boiling point.

Describe and explain the results shown in Fig. 2

Fig. 1 shows two ways in which enzymes interact with their substrates.

Fig. 1

Explain the difference between the two ways in which enzymes interact with their substrates as shown in Fig. 1

The amount of substrate available can have an impact on the rate of an enzyme catalysed reaction. A group of students carried out an investigation into the effects of substrate concentration on the rate of the reaction.

They had ten test tubes for each of the different substrate concentrations to which they added 2 cm³ of an enzyme solution. The test tubes were incubated at 40°C for 20 minutes, during which time the mass of the product that formed was measured as an indication of the rate of the reaction.

Their results are shown in Table 1.

Table 1

Calculate the percentage increase in the amount of product formed during the duration of this investigation.

Show your working.

Explain why it was important that the students kept the temperature constant during the investigation described in part b).

State one other factor, besides temperature, that could affect the structure of the enzyme molecules during the investigation mentioned in part b).

A student wanted to investigate the effect of enzyme concentration on the rate of a reaction. The student made a serial dilution of a 10% amylase solution. He had three test tubes for each of the different concentrations of amylase that contained 2 cm³ of starch solution.

After incubating both amylase and starch solutions for 10 minutes at 40°C, he mixed 1 cm³ of the different amylase solutions with the starch solution in the relevant test tubes. The student removed samples from the mixtures at 1 minute intervals and tested for the presence of starch using iodine solution. He recorded the time taken for all the starch to be digested by the amylase.

His results are presented in Table 1

Table 1

(i)State the conclusion that can be drawn from these results.

[1]

(ii) Explain the importance of having three test tubes for each of the different concentrations of amylase.

[1]

The student judged the colour change of the iodine solution by eye.

Describe a more accurate method that the student could use to determine when all the starch had been digested.

The student repeated the investigation described in part a) but incubated the starch and amylase solution at 60°C.

Suggest and explain the possible outcome of this investigation.

Identify two variables that must be controlled during the investigation described in part a).

Explain how the levels of protein structure enable enzyme specificity. 

Carbonic anhydrase is an enzyme that has a Zn²⁺ ion within its active site.

State the name given to this kind of structure in a protein and identify the level of protein structure that this affects. 

Carbonic anhydrase catalyses the conversion of carbon dioxide and water into carbonic acid, as well as the reverse of this reaction as follows:

CO₂ + H₂O ⟶ H₂CO₃

H₂CO₃ ⟶ CO₂ + H₂O

Suggest how one enzyme is able to catalyse two different reactions.

When it is not catalysed by an enzyme, the rate at which carbon dioxide combines with water to produce carbonic acid is 0.067 reactions per second. With the addition of carbonic anhydrase, and in optimal conditions, the same reaction is 10⁷ times faster.

Calculate the rate at which carbonic acid is produced when the reaction is catalysed by carbonic anhydrase. Give your answer in standard form. 

Some species of sexually-reproducing plants produce an enzyme called pyrophosphatase. This enzyme plays a role in ensuring self-incompatibility, preventing a plant from fertilising itself. The advantage of self-incompatibility is that more cross-breeding can occur within a species; this maintains a large gene pool and so ensures that genetic variation is present.

Known volumes of pyrophosphatase and substrate can be mixed in a cuvette with a dye that starts as colourless and turns blue over time as the reaction progresses. The rate of colour development can be measured in a colorimeter by measuring the absorbance of light by the sample in the cuvette over time. A red filter was used in the colorimeter, meaning that a beam of red light (at a wavelength of 620 nm) was used to assess the absorbance of the solution.

Fig. 1 shows the mean rate of reaction of pyrophosphatase measured over five repeats at 20°C.

Fig. 1

Use your knowledge of colorimetry to suggest why a red filter was selected for the colorimeter.

Use Fig. 1 from part (a) to calculate the rate of the reaction at 100 seconds.

Give your answer in suitable units. 

(i) Predict the effect that a higher enzyme concentration at the start of the experiment would have on the results calculated in part (b).

[1]

(ii) Explain your answer in part (i).

[1]

As the temperature increases, the rate at which pyrophosphatase works also increases up to a point, before decreasing. 

Explain why these changes in the reaction rate take place.  

Enzymes are known to reduce the activation energy of biochemical reactions.

Suggest and explain two mechanisms by which activation energy might be lowered.

Use the context of an anabolic reaction.

Explain the induced fit hypothesis.

The first step of cellular respiration is the phosphorylation of glucose in the cell cytoplasm.

This is catalysed by the enzyme hexokinase:

glucose + ATP → glucose 6-phosphate + ADP

Fig. 1 shows the images obtained of the enzyme and enzyme-substrate complex. X-ray crystallography was used to obtain these images.

Fig. 1

Identify the evidence for the induced fit hypothesis in Fig. 1.

Explain two ways in which an enzyme can become denatured.

A researcher investigated the effect of pH on the activity of the stomach enzyme pepsin. 

Their results are shown in Fig.1 below. 

Fig. 1

The rate of reaction can be calculated by using the following formula:

Calculate the rate of reaction at 20 seconds at pH 2. Give your answer in suitable units.

Explain the differences between the curves at pH 2 and pH 4.

Explain why enzyme-substrate complexes only form for a short time period.

A copy of Fig. 1 is reproduced below as Fig. 2.

Fig. 2

(i) Sketch a curve on Fig. 2 to predict the outcome if the temperature were decreased from 37°C to 25°C at pH 2.

[1]

(ii) Explain the features of the curve you have drawn in part (i).

[2]

A student was provided with two proteases that hydrolyse the amino acid chains of protein in different ways, producing mixtures of single amino acids and peptides of varying lengths:

• An endoprotease that hydrolyses peptide bonds between specific amino acids within the protein molecule. The enzyme only functions if there is a minimum of two amino acids on each side of the hydrolysis site.

• An exoprotease that hydrolyses protein molecules by removing amino acids one at a time from the terminal carboxyl (–COOH) end. This enzyme only functions if the substrate molecule has a minimum of three amino acids.

The student used these enzymes to hydrolyse a protein formed by the linking of two polypeptides.

Fig. 1 shows the hydrolysis sites of these two enzymes on this protein.

Fig. 1

The student investigated the effect of these two enzymes on the hydrolysis of this protein by incubating the protein separately with each of the enzymes.

• Each mixture of enzyme and protein was incubated at 35 °C and a pH of 7.6.

• At intervals of 5 minutes, a sample of each mixture was removed using a capillary tube.

• The products of hydrolysis within each sample were separated by chromatography using the same solvent.

• The products of hydrolysis were located by spraying the chromatogram with a specific dye that stains proteins, peptides and amino acids.

• The student continued sampling the mixtures every 5 minutes and running chromatograms for each sample until hydrolysis of the protein was completed by each enzyme.

(i) Identify the independent variable in this investigation. 

[1]

(ii) Identify three variables, other than the chromatography solvent and the specific dye, that the student standardised in this investigation.

[1]

(iii) Describe how the student could have standardised two of the variables you described in (a) (ii).

[2]

(iv) Suggest how the student could have determined when hydrolysis of the protein was complete.

[1]

Fig. 2 shows the chromatograms produced when complete hydrolysis of the protein by each protease had occurred. Fig. 2 also shows the time taken for complete hydrolysis.

Fig. 2

The student concluded that:

1. The endoprotease worked faster than the exoprotease because fewer bonds needed to be hydrolysed.

2. The products of hydrolysis by the exoprotease were all single amino acids giving more spots on the chromatogram.

3. Hydrolysing the protein with a mixture of endoprotease and exoprotease would give the same results as for the exoprotease alone but more quickly.

State and explain whether each of these conclusions is supported or not supported by all of the information provided about these two enzymes, including the evidence in Fig. 2.

Trypsin is a protease enzyme that digests a milk protein called casein. Casein causes milk to have an opaque white appearance. When it is digested by trypsin the white colour changes to colourless. 

A student investigated the effect of trypsin concentration on the rate of casein digestion. In preparation for this they needed to prepare 10 cm³ of 6 different trypsin test solutions. They started with a large container of 1% trypsin stock solution and some distilled water.

Complete the table below to show how they would make up the test solutions required for the experiment.

The student used a colorimeter to measure the rate of reaction at different trypsin concentrations. 

Suggest why a colorimeter would be a suitable piece of equipment to use for this investigation.

Suggest an experimental method that could be used by the student to investigate the effect of trypsin concentration on the rate of casein digestion.

Your answer should include the use of a colorimeter. 

Some example data from this experiment is shown below:

(i) Describe the relationship between trypsin concentration and rate of casein digestion. 

[2]

(ii) Explain the relationship between trypsin concentration and rate of casein digestion. 

[1]