Power S D, Adams R M, Wells J A
Proc Natl Acad Sci U S A. 1986 May;83(10):3096-100. doi: 10.1073/pnas.83.10.3096.
The sequence of the cloned Bacillus amyloliquefaciens subtilisin gene suggested that this secreted serine protease is produced as a larger precursor, designated preprosubtilisin [Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A. & Chen, E. Y. (1983) Nucleic Acids Res. 11, 7911-7925]. Biochemical evidence presented here shows that a subtilisin precursor is produced in Bacillus subtilis hosts. The precursor is first localized in the cell membrane, reaching a steady-state level of approximately equal to 1000 sites per cell. Mutations in the subtilisin gene that alter a catalytically critical residue (i.e., aspartate +32----asparagine), or delete the carboxyl-terminal portion of the enzyme that contains catalytically critical residues, block the maturation of this precursor. This block occurs when these mutant genes are expressed in B. subtilis hosts where the chromosomal subtilisin gene has been deleted. When the mutant B. amyloliquefaciens subtilisins are expressed in B. subtilis hosts that contain an intact chromosomal subtilisin gene, the mutant precursors are processed to a mature form and released to the medium. Such processing, in trans, of the precursor is also demonstrated in vitro by addition of active subtilisin. Thus, the release of subtilisin from the cell membrane is dependent on an autoproteolytic process that appears to be novel among secreted proteins.
克隆的解淀粉芽孢杆菌枯草杆菌蛋白酶基因序列表明，这种分泌型丝氨酸蛋白酶是以一种更大的前体形式产生的，命名为前原枯草杆菌蛋白酶[韦尔斯，J.A.，法拉利，E.，亨纳，D.J.，埃斯特尔，D.A.和陈，E.Y.（1983年）《核酸研究》11卷，7911 - 7925页]。本文提供的生化证据表明，枯草芽孢杆菌宿主中会产生枯草杆菌蛋白酶前体。该前体首先定位于细胞膜，每个细胞达到约1000个位点的稳态水平。枯草杆菌蛋白酶基因中改变催化关键残基（即天冬氨酸+32变为天冬酰胺）或缺失包含催化关键残基的酶的羧基末端部分的突变，会阻断该前体的成熟。当这些突变基因在已缺失染色体枯草杆菌蛋白酶基因的枯草芽孢杆菌宿主中表达时，就会出现这种阻断。当突变的解淀粉芽孢杆菌枯草杆菌蛋白酶在含有完整染色体枯草杆菌蛋白酶基因的枯草芽孢杆菌宿主中表达时，突变前体被加工成成熟形式并释放到培养基中。通过添加活性枯草杆菌蛋白酶，还在体外证明了这种前体的反式加工。因此，枯草杆菌蛋白酶从细胞膜的释放依赖于一种自蛋白水解过程，这在分泌蛋白中似乎是新颖的。
