Autolytic activation mechanism of Bombyx acid cysteine protease (BCP)

Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme. This 47-kDa pro-BCP1 zymogen molecule can be processed in vitro into an enzymatically active 39-kDa BCP molecule. In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37 degrees C under reducing conditions. The rate of conversion was not affected by increasing concentrations of pro-BCP. We prepared immobilized BCP bound to AH-Sepharose and examined the activation. Immobilized pro-BCP was autolysed, although the rate of processing was slow, indicating that the reaction might be an intramolecular one. Kinetic experiments suggest that the mechanism is likely to involve a stepwise reaction, in which pro-BCP is converted to an active enzyme through intermediate forms releasing small peptides stepwise. The results suggest that autocatalytic cleavage (intramolecular) is a major processing step in the early stage of pro-BCP activation.