Preparation and characterization of the active derivative bovine trypsin-kallikrein inhibitor (Kunitz) with the reactive site lysine-15 -- alanine-16 hydrolyzed.
The derivative of the trypsin-kallikrein inhibitor (Kunitz), TKI+, was prepared with the reactive-site peptide bond Lys-15-Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis). The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1-15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10(8)-10(10). The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor. In the modified inhibitor the hydrolyzed peptide bone was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15--Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor--proteinase interactions. This presents new evidence that this model is generally applicable.
D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001894:Borohydrides; D002268:Carboxypeptidases / Q000378:metabolism; D002417:Cattle; D007700:Kinetics; D008239:Lysine / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D014357:Trypsin; D014360:Trypsin Inhibitor, Kunitz Soybean; D014361:Trypsin Inhibitors
D000596:Amino Acids; D001894:Borohydrides; D010446:Peptide Fragments; D014361:Trypsin Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean; D002268:Carboxypeptidases; D000626:Aminopeptidases; D014357:Trypsin; D008239:Lysine; D000409:Alanine