HOW THE SUBSTITUTION OF K188 OF TRYPSIN BINDING-SITE BY AROMATIC-AMINO-ACIDS CAN INFLUENCE THE PROCESSING OF BETA-CASEIN

Aspartyl 189 residue of trypsin is known to be essential for specific lysis of Arg-X and Lys-X bonds. Undertaking to modulate the catalytic properties of this protease, otherwise highly conserved K188 was repla ced with aromatic amino acid residues aiming the perturbation of the e lectrostatics and the amplifying of hydrophobic interactions of the su bstrate binding site. The catalytic properties of the mutants K188F, K 188Y, and K188W were measured at pH 7, 8, 9, and 10 using a pair of sy nthetic tetrapeptide p-nitroanilide substrates and beta-casein. The ki netic analysis reveals that all the mutants conserve the native trypsi n capacity to split peptide bonds containing arginyl and lysyl residue s. Surprisingly, however, depending on mutation, the optimum pH of act ivity changes. As demonstrated only by proteolysis of a natural substr ate, all mutants cleave also peptide bonds involving asparagine and gl utamine. These stuttered cleavage sites are close to the beta-casein f ragments in beta-sheet according to Hydrophobic Cluster Analysis. (C) 1998 Academic Press.