Abstract
Undertaking to modulate the catalytic properties of trypsin, highly conserved G187, K188 and D189 were replaced with aromatic amino acid residues in order to perturb the electrostatics and to amplify hydrophobic interactions of the substrate binding site. The kinetic parameters of the wild-type trypsin and G187W/K188F/D189Y mutant were determined with synthetic tetrapeptide substrates and β-casein at different pHs. Compared with trypsin, the mutant G187W/K188F/D189Y exhibits 1.3-fold increase in Km values for the substrates containing arginine and lysine. This mutant shows a 30- to 40-fold decrease of its kcat and its second-order rate constant kcm/km decreases = 40- and 55-fold for substrates containing arginine and lysine, respectively. The kinetic analysis reveals that the mutant conserves the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. Surprisingly, as demonstrated only by proteolysis of a natural substrate (β-casein), the mutant cleaves also peptide bonds involving asparagine and glutamine.
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