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Rice Bowman-Birk inhibitors (RBBI), with one (8 kD) or two homologous domains (16 kD), were found to be effective trypsin inhibitors in vitro. In this study, we demonstrate that the 25-kD protein corresponding to the three-domain RBBI indeed ex- ists in rice in planta, and that the RBBIs are regulated by development and wounding. We also found by inhibitory activity assay that the 3:13 disulfide bond, but not the 4:5 disulfide bond, suppresses the tryp- sin-inhibitory activity, and the D3 domain of RBBI3-1 has no inhibitory activity against trypsin, chymotryp- sin, paparin or subtilisin. Mutation analyses showed that conversion from Lys to Leu or Tyr in the N-terminal P1 site in D1 domain did not create chy- motrypsin-inhibitory activity, suggesting that the structure of the reactive loop in D1 domain hinder the new inhibitory specificity at P1 site, and the chy- motrypsin-inhibitory activity might need the participa- tion of other structures, e.g. 3:13 disulfide bond.
In this study, we demonstrated that the 25-kD protein corresponding to the three (8 kD) or two homologous domains (16 kD), were found to be effective trypsin inhibitors in vitro. -domain RBBI indeed ex-ists in rice in planta, and that the RBBIs are regulated by development and wounding. We also found by inhibitory activity assay that the 3:13 disulfide bond, but not the 4: 5 disulfide bond, suppresses the tryp - sin-inhibitory activity, and the D3 domain of RBBI3-1 has no inhibitory activity against trypsin, chymotryp-sin, paparin or subtilisin. Mutation analyzes showed that conversion from Lys to Leu or Tyr in the N- terminal P1 site in D1 domain did not create chy-motrypsin-inhibitory activity, suggesting that the structure of the reactive loop in D1 domain hinder the new inhibitory specificity at P1 site, and the chy-motrypsin-inhibitory activity might need the participation- other of structures, eg 3 : 13 disulfide bond.