Reference no: EM132657953

CHT353 Drug Targets - Cardiff University

Enzyme Inhibition

Question 1. The reaction of carbon dioxide with water (shown below) is catalyzed in the eye by the enzyme carbonic anhydrase II (HCA II). This is an important reaction because it is involved in maintaining the shape of the eyeball. Unfortunately, too much HCA II activity can lead to high pressures in the eye and glaucoma.

Because water is in large excess, the enzyme-catalyzed rate of carbonic acid (H₂CO₃) as a function of [CO₂] follows Michaelis-Menten steady-state kinetics. Write down the Michaelis- Menten equation for HCA II and hence derive the following expression:

v_o = V_max - K_M.vo/[CO₂]

Where v_o is the initial rate, V_max is the maximum rate, and K_M is the CO₂ concentration at which v_o = 0.5V_max.

Question 2. Calculate the minimum time taken for CO₂ to react with water in the presence of HCA II given that 10^-6 moles of the enzyme produces 0.6 moles of carbonic acid (H₂CO₃) per second (V_max).

Question 3. Competitive inhibitors of HCA II, such as Trusopt (shown below), are used to reduce high internal pressures of CO₂ in the eye. Draw a plot showing how v_o versus v_o/[S] changes for the HCA II-catalysed production of H₂CO₃ in the presence of increasing concentrations of Trusopt.

Hint: Use a suitably modified form of the expression you derived above.

Question 4. Briefly explain why uncompetitive inhibitors are more effective at stopping enzyme-catalyzed reactions in vivo (in a living cell) than competitive inhibitors, and hence explain why most drugs are deliberately chosen to be competitive inhibitors.

Ligand Binding

Question 5. When water molecules surround methane (CH₄) the enthalpy of the system decreases. This is due to the formation of dispersion interactions and strong hydrogen bonds between water molecules on the surface of the methane molecule. Yet methane is almost completely insoluble in water, the free energy change (ΔG_sol) associated with moving methane from the gas phase into aqueous solution being ΔG_sol = +26.4 kJ/mol. Briefly explain why ΔG_sol is so unfavorable.

Question 6. Somewhat counter-intuitively, most of the interaction energy needed to form molecular complexes in water is provided by dispersion interactions between hydrophobic groups located on separate molecules. Provide evidence to support this assertion by calculating the free energy for the dimerization of two CH₄ molecules in water (ΔG_dim) given the following information:

Question 7. Again counter-intuitively, the contribution of hydrogen bonds to ligand binding affinity is also less than might be expected based on their energies in the gas-phase. For example, studies of intermediate binding to the enzyme tyrosyl-tRNA synthetase showed that removing a hydrogen bond to an uncharged group on the substrate by changing Tyr-34 to a phenylalanine residue (see figure) decreased the binding energy by only 2 kJ/mol. This is surprising given that similar hydrogen bonds in the gas-phase have an energy of approximately 20 kJ/mol. Briefly explain this experimental observation.