Question

Amyloid precursor protein (APP) is an integral membrane protein with important functions at synapses between nerve cells. It is cleaved within the membrane to release an extracellular peptide fragment called Ab. When Ab peptides accumulate, they can aggregate to form amyloid fibrils associated with Alzheimer’s disease

The cleavage of APP to Ab is catalyzed by Presenilin-1. Presenilin-1 is inhibited by transition state analogues specific for aspartyl proteases.

A. In one sentence, describe why transition state analogues are excellent competitive inhibitors of enzymatic activity.

B. Presenilin-1 has two important catalytic residues, Asp257 and Asp385, which cleave APP to Ab within the hydrophobic membrane. Describe TWO reasons why these catalytic aspartates have elevated pKa values.

C. During catalysis, there is a buildup of negative charge on the oxygen atom of the scissile amide bond. To stabilize the transition state, Asp385 donates a proton to the oxyanion that forms. To what amino acid would you mutate Asp385 to prevent proton transfer whilst having minimal impact on the rest of the protein? Will this amino acid still be able to stabilize the transition state via hydrogen bonding?

D. Aspartyl proteases directly activate water for hydrolysis without forming a covalent enzyme-substrate intermediate. Draw a mechanism for peptide hydrolysis. Clearly show Asp385 stabilizing the oxyanion transition state. Clearly show what role Asp257 may have.

Answer 1