Li Y, Inouye M
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
J Biol Chem. 1994 Feb 11;269(6):4169-74.
Subtilisin, an extracellular serine protease from Bacillus subtilis, requires the amino-terminal propeptide of 77 amino acid residues for the formation of the active enzyme. The propeptide is cleaved upon completion of folding. Serine 221 at the active center was substituted with cysteine, and the mutant enzyme (prothiolsubtilisin) was expressed in Escherichia coli under the control of a T7 promoter. Prothiosubtilisin, which was produced as inclusion bodies, was dissolved in 6 M guanidine HCl and purified to near homogeneity in the presence of 5 M urea. The purified protein was renatured by stepwise dialysis. In spite of the mutation at the active center, the propeptide was found to be autoprocessed with approximately 60-80% efficiency. However, protease activity could not be detected in the final product by the spectrophotometric assay. Moreover, the cleaved propeptide remained tightly bound to thiolsubtilisin without being digested, as evident by SDS-polyacrylamide gel electrophoresis. The amino-terminal sequence of the processed thiolsubtilisin was determined and proved that the propeptide was cleaved at a site identical to that of wild-type prosubtilisin. The processed thiolsubtilisin was also found to contain one free SH group/molecule. These results unambiguously demonstrate that the processing of prosubtilisin occurs by an intramolecular autoprocessing mechanism.
枯草杆菌蛋白酶是一种来自枯草芽孢杆菌的胞外丝氨酸蛋白酶，其活性酶的形成需要77个氨基酸残基的氨基末端前肽。前肽在折叠完成后被切割。活性中心的丝氨酸221被半胱氨酸取代，突变酶（前硫醇枯草杆菌蛋白酶）在T7启动子的控制下在大肠杆菌中表达。以前体形式产生的前硫醇枯草杆菌蛋白酶以包涵体形式存在，将其溶解在6M盐酸胍中，并在5M尿素存在下纯化至接近均一。纯化的蛋白质通过逐步透析复性。尽管活性中心发生了突变，但发现前肽的自我加工效率约为60-80%。然而，通过分光光度法测定，最终产物中未检测到蛋白酶活性。此外，如SDS-聚丙烯酰胺凝胶电泳所示，切割后的前肽紧密结合在前硫醇枯草杆菌蛋白酶上，未被消化。测定了加工后的硫醇枯草杆菌蛋白酶的氨基末端序列，证明前肽在与野生型前枯草杆菌蛋白酶相同的位点被切割。还发现加工后的硫醇枯草杆菌蛋白酶每个分子含有一个游离的SH基团。这些结果明确表明前枯草杆菌蛋白酶的加工是通过分子内自我加工机制进行的。
