Bio 2960 PKU Project
Assignment 3 Questions:
1. What, if any, secondary structure is present in the immediate area of the E280K mutation in the PAH protein (see figure 1)?
There are no secondary structures present in the immediate area of the E280K mutation in the PAH protein because the figure does not show a helix representing an alpha helix or an arrow representing a beta strand at position 280, amino acid K.
2. What domain of the protein is amino acid 280 in (see figure 2)?
It is the catalytic domain.
3. What percentage of PAH enzyme activity does the E280K mutant have?
The paper states that expression of E280K human PAH in E.coli results in an enzyme with only about 1%of the activity of the wild-type PAH.
4. Explain the likely mechanism behind the loss of enzyme activity for the E280K mutant PAH enzyme. Be as specific as possible.
In the active site of PAH, there are only 2 free, charged groups that happen to be glutamic acids. The substitution of Glu280 to lysine changes the electrostatic potential of the active site. What normally happens (without the mutation) is that Glu280 is able to hydrogen bond to His146 and can also form a salt bridge with Arg158. But this is not possible when Glu280 is mutated to K280. If we think about it, this makes sense since Glutamic acid is a negatively charged side chain while lysine is positively charged so it wouldn’t be able to have the same interactions as glutamic acid.