1.4 Proteins: Enzymes

Certain plants that reproduce sexually contain an enzyme called pyrophosphatase. This enzyme plays a role in ensuring self-incompatibility, which is a mechanism that prevents a plant from fertilising itself. The selective advantage of self-incompatibility is that more cross-breeding can occur within a species, which has long term benefits for evolution and for maintaining a wide pool of alleles.

Known volumes of pyrophosphatase and substrate can be mixed in a cuvette with a blue dye that starts as colourless and develops colour over time. The rate of colour development can be measured in a colorimeter by measuring the absorbance of light of wavelength 620 nm (red light). 

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

Figure 1

State why the wavelength of 620 nm is selected for this experimental measurement.

Use Figure 1 to calculate the rate of the reaction at 100 seconds. Give your answer in suitable units. 

Predict and explain whether your answer to 2 (b) would be higher, lower or the same if a higher concentration of enzyme at the beginning of the experiment.

As temperature increases, the rate at which pyrophosphatase works increases, then decreases.  Explain why these changes take place.  

Describe and explain the similarities and differences between competitive and non-competitive enzyme inhibition. 

Many products of multi-step cellular reactions act as inhibitors of the enzymes that catalyse the preceding steps in a metabolic pathway. For example, ATP acts as a non-competitive inhibitor of the enzyme pyruvate kinase, which catalyses the final step of glycolysis, the first stage of cellular respiration of glucose. 

Suggest how the inhibition of pyruvate kinase by ATP allows cells to prevent overproduction and wasting of cellular energy.

Figure 1 shows the effects of increasing substrate concentration on enzyme activity with and without two types of inhibitor, competitive and non-competitive. 

 Figure 1

Sketch a line on both graphs to indicate the effect of increasing inhibitor concentration in each case. Explain the position and shape of each line. 

Compare and contrast the features of a substrate and a competitive inhibitor

Define the term ‘specificity’ in the context of enzymes. 

In humans, the enzyme sucrase hydrolyses sucrose. This reaction occurs in the small intestine at 37°C. Explain why sucrase only hydrolyses sucrose, and why this reaction can take place at normal body temperature. 

A sucrose solution was split into two equal portions. One portion (A) was left untreated at room temperature, and the other (B) was treated with sucrase and left for 30 minutes at room temperature. Both solutions were tasted by human volunteers. Identify which solution would taste sweeter and explain your choice.

Suggest two sources of error that could arise from an experiment to measure the effect of temperature on the rate of an enzyme-controlled reaction. Assume that in this experiment, the dependent variable is measured as the volume of a gas produced.

A significant amount of research has been conducted on the enzyme composition of extremophile microorganisms, in order to discover new enzymes that can be used in the home in extreme conditions. One such organism, Planococcus halocryophilus, is a psychrophile (it grows at cold temperatures around 0°C). Trials with the enzymes of P. halocryophilus have discovered applications of these enzymes in the detergent industry. 

Suggest and explain how these trial results are encouraging for the laundry detergent industry.  

Many commercially-produced biological laundry detergents contain a range of different enzymes. Explain why a range of enzymes can improve the detergent’s performance in the home.

Papain is a proteolytic enzyme derived from papaya fruit. It has been used in contact lens cleaning solutions to remove denatured protein-containing deposits that accumulate on the surfaces of contact lenses during long periods of wear. The periodic removal of protein deposits increase wearer comfort and extends wearing time.

The principal protein component of tear film fluid is lysozyme. Suggest a reason for the presence of lysozyme in tear film fluid.  

Lysozyme and other proteins present in tear film fluid can denature rapidly when in contact with contact lens material. This denatured material loses its original function and forms deposits on the lens surface. Describe and explain the mode of action of papain against the denatured protein deposits on the surfaces of the contact lenses.

A protease is an enzyme that digests protein. A research scientist isolated protease C from a particular species of bacteria. They investigated the effect of temperature on the rate of hydrolysis of a protein by protease C.  After 4 minutes the mass ofprotein hydrolysed was measured at each temperature. The results can be seen in Table 1 below.

Table 1 

Fill in the missing details of Table 1.

Plot a graph of the results seen in Table 1.

A research assistant concluded from their graph of the data in Table 1 that the bacterium's habitat was likely around 25°C. Evaluate this conclusion.

Suggest how the research scientist controlled the pH throughout the experiment.

In their first few weeks of life puppies produce large amounts of the enzyme lactase so that they can break down their mothers milk. However as they grow older and are weaned onto solid dog food they produce less and less lactase. Milk can still be beneficial for an older puppy because of its high calcium content. 

Cow’s milk contains large amounts of the sugar lactose but it can be made suitable for older puppies via treatment with the enzyme lactase. This makes the cow’s milk lactose-free. Beads are coated with the enzyme lactase and placed in a glass cylinder, as shown in Figure 1 below. As the cow’s milk flows over the beads the lactose is hydrolysed.

Figure 1

Lactose is broken down into glucose and galactose by lactase. Galactose has a similar structure to a section of the lactose molecule. Explain how galactose acts as an inhibitor of lactase.

Attaching the enzyme to the beads is a more efficient method for using the enzyme, rather than directly adding it to cow’s milk. Suggest four reasons why it is a more efficient method.

The scientist varied the flow rate of the milk through the column. The effect of flow
rate on the concentration of glucose in Milk B is shown in Table 1 below.

 Table 1

Using the information from Table 1 suggest which flow rate should be used by a manufacturer wanting to produce large volumes of lactose free puppy milk. Justify your answer.

Many humans also struggle with digesting cow’s milk. Those who are lactoseintolerant have little or no lactase enzymes.

Fortunately, lactase tablets can be taken to help aid in the digestion of lactose heavy foods. These tablets don’t have to be stored at a lower temperature but other enzymes used in experiments can often require storage in a laboratory refrigerator.Use your knowledge of protein structure to explain why this is so.

An enzyme can only catalyse one reaction but a substrate can be hydrolysed by more than one enzyme. Explain why.

A lab technician was investigating the effect of temperature on the rate of an specific enzyme-controlled reaction.  Water baths were used to maintain temperatures of 30 and 50 degrees Celsius. For the reactions at both temperatures, the same concentration of substrate and enzyme was used. Figure 1 shows their results.

Figure 1

Sketch tangents to find the initial rates of reaction for both temperatures. Then use these values to calculate the ratio of the initial rates of reaction at 50°C : 30°C.
Show your working.

Ratio = _________:1

Explain the difference in the initial rates of reaction at 50 °C and 30 °C.

Explain the difference in the rates of reaction at 50 °C and 30 °C after 10 minutes.

Scientists investigated the effect of pH on the activity of enzyme A. Agar plates containing the enzyme substrate were used. The substrate caused the agar to go a grey colour. The scientists created four wells of equal size in the agar of each plate. A drop of enzyme A solution was added to each of the wells. The pH of the enzyme solution was different in each well. The agar plates were incubated for 5 hours at a constant temperature. 

Figure 1 below shows how the agar plates looked after they were incubated. 

Figure 1

Describe and explain the results seen in Figure 1.

While the scientists were investigating another enzyme, enzyme B, they found that a change in pH from 7.2 to 7.6 had a very large effect on the rate of reaction controlled by enzyme B. Using your knowledge of pH, explain why a small change in pH produces a large effect on the rate of reaction.

Research scientists have investigated the effects of both competitive and non-competitive inhibitors of enzyme A.  One of the inhibitors being investigated is represented  in Figure 2 below. State what type of inhibitor molecule 1 is and describe how it works.

Figure 2

Suggest how the inhibition of enzyme A by molecule 1 could be overcome.