Reconstitution of new inhibitors through mutual exchange of Ile(64)-Met(84) peptides of soybean trypsin inhibitors, Tia and Tib.
Singly modified soybean trypsin inhibitors (STIs), Tia* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDS-FAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDS-FAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs. The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its Ki value for bovine trypsin is around 10(-10) M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a Ki value of around 10(-9) M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10(-8) M similar to that of Tib for bovine trypsin.
Kim
S H
SH
Department of Chemistry, Osaka University College of Science.
Hara
S
S
Ikenaka
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D007532:Isoleucine / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000138:chemical synthesis*
D010455:Peptides; D007532:Isoleucine; D014360:Trypsin Inhibitor, Kunitz Soybean; D008715:Methionine