Influence of G187W/K188F/D189Y mutation in the substrate binding pocket oftrypsin on beta-casein processing

Undertaking to modulate the catalytic properties of trypsin, highly conserv ed G187, K188 and D189 were replaced with aromatic amino acid residues in o rder to perturb the electrostatics and to amplify hydrophobic interactions of the substrate binding site. The kinetic parameters of the wild-type tryp sin and G187W/K188F/D189Y mutant were determined with synthetic tetrapeptid e substrates and beta-casein at different pHs. Compared with trypsin, the m utant G187W/K188F/D189Y exhibits 1.3-fold increase in K-m values for the su bstrates containing arginine and lysine. This mutant shows a 30- to 40-fold decrease of its k(cat) and its second-order rate constant k(cat)/K-m decre ases approximate to 40- and 55-fold for substrates containing arginine and lysine, respectively. The kinetic analysis reveals that the mutant conserve s the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. Surprisingly, as demonstrated only by proteolysis of a natural substrate (beta-casein), the mutant cleaves also peptide bonds inv olving asparagine and glutamine.