Question

The following question refers to the enzyme chymotrypsin.

For an experiment, you are asked to modify the sequence of chymotrypsin so that, it instead of cleaving C-terminal to a Phe/Tyr residue, it cleaves after Lys. Please describe the strategy you would take in your design, considering what you have learned. Please explain with clarity and detail.

Answer 1

Endopeptidases are enzymes which cleaves peptide bond in the protein. They can be divided into two types

Trypsin type and chymotrypsin type.

Among this chymotrypsin type cause the cleavage of only large sized hydrophobic amino Acids such as phenylalanine, tryptophan and tyrosine. In the active site of chymotrypsin serine is present.

The specificity of endopeptidases to hydrophobic or hydrophilic aminoacid depends on the residue which is present in the base of the enzymes S1 pocket. If in this position negatively charged aminoacid such as Aspartic acid is present it will cause the breakage of positively charged hydrophilic aminoacid such as lysine or arginine. Trypsin is the endopeptidase which cleaves lysine.

So, inorder to change the specificity of chymotrypsin to cleave lysine it has to be converted to the sequence of trypsin . So serine in the active site of chymotrypsin has to be changed to Aspartic acid then it will specifically cleave lysine.