OC2 - chemical methods for locating the amino acid residues in binding sites

Question 1

chemical modification

Answer 1

amino acids thought to be crucial to the proteins function are selected and chemically modified,
if this modification alters the activity of the protein, the amino acid modified is crucial in the binding site.
e.g. phosphorylation
e.g. chymotrypsin

Question 2

chymotrypsin

Answer 2

a protease enzyme that employs reactive serine in its active site (serine protease)
treatment with DIPF, which covalently bonds to serine (ser-195), inactivates chymotrypsin, in an irreversible modification, this demonstrates that ser-195 residues is of critical importance in the binding site of chymotrypsin

Question 3

pH dependence of activity

Answer 3

an alteration in the pH will affect the acidic and basic residues on the binding sites leading to a shift in the equilibrium point of ionisation, so this can be used to investigate the roles of particular amino acid residues in enzyme activity.
a comparison of activity before and after alteration of pH can reveal if the amino acid residue was critical to the proteins function.