Question 1 In a directed evolution experiment, the substitution of an asparagine

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Question 1 In a directed evolution experiment, the substitution of an asparagine for a lysine in the active site of a protein enhanced the binding of a negatively charged fatty acid substrate to a human fatty acid hydroxylase enzyme. The same mutation was introduced into a homologous hydroxylase enzyme from the model organism, Danio rerio (Zebrafish), which was proposed (but not proven) to act on fatty acids and fatty acid amides. This mutation, however, resulted in a misfolded protein that failed to bind any fatty acid or fatty amide substrates at all. Modelling of the two enzymes suggested that the active site of the Zebrafish enzyme contained mostly similar residues to the human form, with the exception of an arginine residue positioned in the substrate-binding pocket in close proximity to the introduced mutation. In the human enzyme, there is a serine residue at this position. Potential Question 1.2) What are the pros/cons of the directed evolution approach Potential Question 1.3) After examining the original system, what mutations would you make and how Potential Question 1.4) Outline a strategy by which you would attempt to engineer a catalyst of this reaction that was stable and had the required catalytic activity towards the protein of interest Question 1.5 How would you screen the activity of the protein Question 1.6 What conditions should it be used under Question 1.7 How might we design a direct evolution or rational design strategy to improve the protein

Explanation / Answer

ans-1.2 pros of direct evolution include enhancement of process while its cons inculde the complete inhibition of the process due to error in folding of proteins.

ans-1.3 the mutation which can be carried out after examining the original system is that the arginine and serine can be interchanged at the specific positions.

ans-1.4 the catalyst can be synthesized by first changing the arginine with the serine and then changing asparagine with lysine.

ans-1.5 the activiy of the protein can be observed by checking the binding of negatively charged fatty acids to the proteins.

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