Question

Three amino acids are crucial to the activity of Trypsin: Asp 102, His57, and Ser195.

a) select one of these residues and describe how replacing that residue with a glycine would affect the catalytic mechanism of Trypsin

b) Which kinetic parameter (K_m, V_max, K_cat...) would be altered due to the change that u made in part a? Explain.   

Answer 1

Trypsin is a serine protease and threonine is a precursor of glycine. Serine residue has a hydroxyl group at position 195 which acts as a nucleophile in the catalytic mechanism of Trypsin. If Ser195 is replaced with threonine (glycine) then trypsin becomes inactive because of the negative steric interaction which takes place between the methyl group on the beta carbon of threonine 195 and the disulfide bridge formed by cysteine 42 and cysteine 58. As the catalytic power of trypsin is reduced therefore the Kcat substantially gets reduced but the value of Km remains unchanged because of the substrate-binding not being changed significantly. Kcat/Km measures the enzyme specificity for different substrates