3.1 Mode of Action of Enzymes

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Explain the Induced Fit Hypothesis which has superseded the previous model of enzyme action, the Lock and Key Hypothesis.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 of the induced fit hypothesis in Fig. 1.

Describe and 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 are only formed for a short amount of time.

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

Fig. 2

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]

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.

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]

Describe how the student could prepare and use chromatograms to compare the products of hydrolysis of the protein by the two different proteases.

Your method should be set out in a logical order and be detailed enough to allow another person to follow it.

Details of how the proteins were hydrolysed should not be included.

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.

Using an internet search the student found that:

• electrophoresis can also be used to separate the products of enzyme hydrolysis of proteins
• a combination of electrophoresis and chromatography has been used to study haemoglobin from people with sickle cell anaemia.

In an investigation, samples of haemoglobin from people with sickle cell anaemia and people without sickle cell anaemia were hydrolysed by an endoprotease. Each sample was placed centrally on one side of a piece of chromatography paper and separated by electrophoresis.

Negatively charged peptides moved to the anode (+) and positively charged peptides moved to the cathode (–).

After electrophoresis, the paper was turned through 90° and chromatography was used to further separate the peptides.

Fig. 3 shows the main stages of this experiment.

Fig. 3

Sickle cell haemoglobin and normal haemoglobin have a difference in amino acid sequence.

In Fig. 3, draw a circle around the spot in each chromatogram that shows that the two types of haemoglobin have different amino acid sequences.

[1]

(ii)

Explain your answer to part (i).

[1]

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

Fig. 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 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

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).