ENZYMATIC REPLACEMENT OF ARGINYL BY A LYSYL RESIDUE IN REACTIVE SITE OF SOYBEAN TRYPSIN INHIBITOR

The arginyl residue in the reactive site of soybean trypsin inhibitor was enzymatically replaced by a lysyl residue. The original arginyl residue was removed by treatment of virgin inhibitor (Arg(64)-Ile(65) bond intact) with trypsin to produce modified inhibitor (Arg-Ile bond hydrolyzed) followed by treatment with carboxypeptidase B. Addition of free lysine to inactive des-64-Arg-modified inhibitor was then catalyzed by large concentrations of carboxypeptidase B in the presence of free trypsin. The driving force for this peptide-bond synthesis was supplied by reaction of the trypsin with newly synthesized [64-lysine]-modified inhibitor to form trypsin-inhibitor complex. Free virgin [64-lysine]-inhibitor was obtained by dissociation of this complex in 6 M guanidine HC1. This new protein is almost fully active. It is indistinguishable from virgin authentic inhibitor by disc gel electrophoresis. It is converted by catalytic amounts of trypsin into modified [64-lysine]- inhibitor, although considerably more slowly than the authentic inhibitor. Carboxypeptidase B releases about 1 mole/ mole of lysine from this new modified. © 1969, American Chemical Society. All rights reserved.