Abstract: Cassia obtusifolia trypsin inhibitor (CoTI), a member of plant Kunitz protease inhibitor family, shows strong inhibitory activity against trypsin. In this paper, the three-dimensional model of CoTI was predicted by homologous modeling. The model adopted a β-trefoil fold with β-barrel at the bottom and the lid at the top. Then, compared with the amino acid sequences and three-dimensional structures of various Kunitz protease inhibitors, a conserved Trp114 that was located in the lid region and extended to the β-barrel was found in the CoTI model. Since Trp114 formed hydrogen bonds with Leu98 and Ser137, hydrophobic interaction with Val68, Leu98 and Phe100, it was suggested to play an important role in the formation and stability of β-trefoil fold. After mutation of Trp114 residue to Ala residue using site-directed mutagenesis technique, the inhibitory activities of Trp114Ala mutant against bovine trypsin and Pierisrapae midgut trypsin were decreased by 63.4% and 62.0%, respectively. As a result, the importance of Trp114 would lay a theoretical foundation for the study of structure-function of CoTI and its related applications.

Key words: Cassia obtusifolia, trypsin inhibitor, homology modeling, hydrophobic core, site-directed mutation