1. 1
   A dairy produces lactose-free milk by passing milk through a column packed with alginate beads in which the enzyme lactase is immobilised. Lactase hydrolyses lactose into glucose and galactose. In trials at 35 °C with a fixed flow rate, the rate of lactose breakdown rose steeply as the lactose concentration of the milk was increased from 10 to 40 mmol dm⁻³, rose only slightly between 40 and 80 mmol dm⁻³, and did not change between 80 and 120 mmol dm⁻³.

   (a)

   Why did the rate of lactose breakdown stop increasing above 80 mmol dm⁻³?

   [1 mark]

   • AThe lactase had been denatured by the high lactose concentration
   • BThe products glucose and galactose had been used up
   • CAll the active sites of the lactase were occupied, so enzyme concentration had become the limiting factor
   • DLactose molecules could no longer move at high concentration

   (b)

   The lactase in the beads cannot move. How do enzyme–substrate collisions still occur?

   [1 mark]

   • ALactose molecules move by random motion in the milk and diffuse into the beads to reach the active sites
   • BThe alginate actively pumps lactose into the active sites
   • CLactase molecules leave the beads to find lactose and then return
   • DCollisions are not needed because the enzyme is immobilised

   (c)

   State, with a reason, whether the reaction catalysed by lactase is anabolic or catabolic.

   [2 marks]

   Total for question 1: 4 marks

2. 2
   In the bacterium Escherichia coli, the amino acid isoleucine is made from threonine by a pathway of five enzyme-catalysed steps. The first enzyme, threonine deaminase, has a binding site for isoleucine that is separate from its active site. When isoleucine accumulates in the cell, the rate of the first step falls; when isoleucine is used up in protein synthesis, the rate rises again. A mutant strain whose threonine deaminase cannot bind isoleucine produces and excretes large amounts of isoleucine.

   (a)

   What is the name of the site to which isoleucine binds on threonine deaminase?

   [1 mark]

   • AActive site
   • BAllosteric site
   • CTranspeptidase site
   • DCatalytic site

   (b)

   Why is it an advantage for isoleucine to inhibit the first enzyme of the pathway rather than the last?

   [1 mark]

   • AIt stops protein synthesis in the cell
   • BIt makes the reaction irreversible
   • CIt increases the rate at which isoleucine is produced
   • DIt prevents the intermediates of the pathway from accumulating and saves threonine for other uses

   (c)

   Explain how the binding of isoleucine reduces the activity of threonine deaminase and allows the rate of isoleucine production to recover.

   [2 marks]

   Total for question 2: 4 marks

3. 3
   HMG-CoA reductase catalyses a step in the synthesis of cholesterol in liver cells. Researchers measured its activity in arbitrary units at a low and a high concentration of its substrate. With no inhibitor, activity was 40 at low and 100 at high substrate concentration. With a statin drug, activity was 12 at low and 95 at high substrate concentration. With an experimental compound Q, activity was 20 at low and 50 at high substrate concentration.

   (a)

   Using the data, deduce which type of inhibitor the statin and compound Q are.

   [3 marks]

   (b)

   Explain the difference between competitive and non-competitive inhibition in terms of the interactions between inhibitor, substrate and enzyme.

   [4 marks]

   Total for question 3: 7 marks

4. 4
   Penicillin was first used widely in the 1940s. Its molecules have a shape similar to part of the substrate of transpeptidases, enzymes that form cross-links between the peptidoglycan chains of bacterial cell walls. Penicillin binds permanently to the active site of transpeptidase, where it forms a covalent bond with an amino acid. Bacteria exposed to penicillin while growing burst. By the 1960s, strains of Staphylococcus aureus had appeared that carry a gene coding for a modified transpeptidase, and these strains grow normally in the presence of penicillin.

   (a)

   Explain how penicillin kills growing bacteria and how the change in transpeptidase confers resistance to penicillin.

   [6 marks]

   (b)

   Discuss the relationship between the structure of an enzyme's active site, its specificity and denaturation, using transpeptidase and penicillin as examples.

   [6 marks]

   Total for question 4: 12 marks

End of questions