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 replaced with aromatic amino acid residues aiming the perturbation of the electrostatics and the amplifying of hydrophobic interactions of the substrate binding site. The catalytic properties of the mutants K188F, K188Y, and K188W were measured at pH 7, 8, 9, and 10 using a pair of synthetic tetrapeptide p-nitroanilide substrates and beta-casein. The kinetic analysis reveals that all the mutants conserve the native trypsin capacity to split peptide bonds containing arginyl and lysyl residues. Surprisingly, however, depending on mutation, the optimum pH of activity changes. As demonstrated only by proteolysis of a natural substrate, all mutants cleave also peptide bonds involving asparagine and glutamine. These stuttered cleavage sites are close to the beta-casein fragments in beta-sheet according to Hydrophobic Cluster Analysis.
Chobert
J M
JM
Laboratoire d'Etude des Interactions des Molécules Alimentaires, Institut National de la Recherche Agronomique, Nantes, France. chobert@nantes.inra.fr
Briand
L
L
Tran
V
V
Haertlé
T
T
D000595:Amino Acid Sequence; D000598:Amino Acids, Cyclic / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002364:Caseins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D000598:Amino Acids, Cyclic; D002364:Caseins; D010446:Peptide Fragments; D010455:Peptides; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D014357:Trypsin; D008239:Lysine