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Amyloid precursor protein (APP) is an integral membrane proteinwith important functions at synapses between nerve cells. It iscleaved within the membrane to release an extracellular peptidefragment called Ab. When Ab peptides accumulate, they can aggregateto 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 specificfor aspartyl proteases.

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

B. Presenilin-1 has two important catalytic residues, Asp257 andAsp385, which cleave APP to Ab within the hydrophobic membrane.Describe TWO reasons why these catalytic aspartates have elevatedpKa values.

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

D. Aspartyl proteases directly activate water for hydrolysiswithout forming a covalent enzyme-substrate intermediate. Draw amechanism for peptide hydrolysis. Clearly show Asp385 stabilizingthe oxyanion transition state. Clearly show what role Asp257 mayhave.