Autocatalytic processing or recombinant human procathepsin L -: Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro
Autocatalytic processing or recombinant human procathepsin L -: Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro
复制标题
DOI：
10.1074/jbc.273.8.4478
复制
发表时间：
1998-02-20
影响因子：
4.8
通讯作者：
Mort, JS
中科院分区：
文献类型：
--
作者：
Ménard, R;Carmona, E;Mort, JS
利用在毕赤酵母中表达的重组蛋白酶原，研究了蛋白酶原L在体外的自催化过程。纯分子间加工研究了孵育的突变体蛋白酶原L（C25 S），它不能自动激活与少量的成熟活性组织蛋白酶L。结果清楚地表明，与最近的报道相反，前组织蛋白酶L的分子间加工是可能的。主要切割位点位于成熟酶的N末端或其附近，在前区的可接近部分，其含有对应于组织蛋白酶L的已知底物特异性的序列。与组织蛋白酶原B、K和S相反，组织蛋白酶原L的自催化加工可以产生酶的天然成熟形式。使用底物苄氧基羰基-Phe-Arg 4-甲基香豆素基-7-酰胺盐酸盐的连续测定也被用于获得关于野生型组织蛋白原L的自催化加工中涉及的步骤的性质的信息。通过将pH从8.0降低至5.3来开始处理。酶原浓度对加工速率的影响表明，在加工机制中存在单分子和生物分子步骤。触发加工的单分子事件的性质仍然难以捉摸。圆二色性和荧光测量表明，没有大规模的构象变化，在pH值的减少的蛋白酶原L的结构。然而，双分子反应可以归因于酶原的分子间加工。
The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and biomolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.