BOMBYX ACID CYSTEINE PROTEINASE (BCP) : STRUCTURE AND MECHANISM OF ACTIVATION

家蚕（Bombyx mori）卵中的酸性半胱氨酸蛋白酶是一种无活性的前酶，并被糖基化。该47-kDa pro-BCP^1酶原分子可在体外加工成具有酶活性的39-kDa BCP分子(Takahashi et al., 1993， J.Biochem (Tokyo), 97,701-701；山本等，1994,j。(东京),116年,1330 - 1335)。在本研究中，在还原条件下，在pH约为4.0，温度为37℃的条件下，体外处理速度达到最大。前体加工被多种半胱氨酸蛋白酶抑制剂抑制，但EDTA和胃抑素不受抑制。原bcp浓度的增加不影响转化速率。我们制备了固定化BCP结合AH-Sepharose并检测了其活化情况。固定化的前bcp被自溶，尽管加工速度较慢，表明该反应可能是分子内反应。动力学实验表明，该机制可能涉及一个逐步反应，其中前bcp通过中间形式逐步释放小肽转化为活性酶。第一个主要中间体可能是通过去除pro-BCP^2的氨基末端27个残基（Val_<-105>-Glu_<-78>）产生的。然后两个肽（Asp_<-77>-Ser_<-44 >或Trp_<-43>和Trp_<-43>或Trp_<-42>-Ile_<-1>）可能依次从nh_2末端区域释放，酶最终被加工成活性形式（39-kDa）。结果表明，自催化裂解（分子内）是前bcp激活素早期的主要加工步骤。

Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme and is glycosylated. This 47-kDa pro-BCP^1 zymogen molecule can be processed in vitro into an enzymatically active 39-kDa BCP molecule (Takahashi et al., 1993, J.Biochem (Tokyo), 97,701-701 ; Yamamoto et al., 1994, J.Biochem. (Tokyo), 116,1330-1335). In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37ﾟC under reducing conditins. Precursor processing was inhibited by a variety of cysteine proteinase inhibitors, but not by EDTA and pepstatin. 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 reactin 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 first major intermediate might be generated by removal of the amino terminal 27 residues from pro-BCP^2 (Val_<-105>-Glu_<-78>). Then two peptides (Asp_<-77>-Ser_<-44'> or Trp_<-43> and Trp_<-43> or Trp_<-42>-Ile_<-1>) might be esequentially released from the NH_2-terminal region and the enzyme is finally processed to its active form (39-kDa).The results suggest that autocatalytic cleavage (intramolecular) is a major processing step in the early stage of pro-BCP activatin.