SECRETION AND AUTOPROTEOLYTIC MATURATION OF SUBTILISIN
SECRETION AND AUTOPROTEOLYTIC MATURATION OF SUBTILISIN
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DOI：
10.1073/pnas.83.10.3096
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发表时间：
1986-05-01
影响因子：
11.1
通讯作者：
WELLS, JA
中科院分区：
文献类型：
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作者：
POWER, SD;ADAMS, RM;WELLS, JA
克隆的解淀粉芽孢杆菌枯草杆菌蛋白酶基因的序列表明，这种分泌的丝氨酸蛋白酶是作为较大的前体产生的，称为前枯草杆菌蛋白酶原[威尔斯，J.A.，Ferrari，E.，Henner，D. J.，Estell，D. A.和Chen，E. Y.（1983）Nucleic Acids Res. 11，7911-7925]。这里提出的生物化学证据表明，枯草杆菌蛋白酶前体在枯草芽孢杆菌宿主中产生。该前体首先定位于细胞膜中，达到稳态水平apeq。每个单元1000个站点。枯草杆菌蛋白酶基因中改变催化关键残基的突变（即，天冬氨酸+32天冬酰胺），或缺失酶的羧基末端部分，其含有催化关键残基，阻断该前体的成熟。当这些突变基因在B中表达时，就会发生这种阻断。枯草杆菌宿主，其中染色体枯草杆菌蛋白酶基因已被删除。当突变体B.解淀粉菌枯草杆菌蛋白酶在B中表达。 在含有完整染色体枯草杆菌蛋白酶基因的枯草杆菌宿主中，突变体前体被加工成成熟形式并释放到培养基中。前体的这种反式加工也通过加入活性枯草杆菌蛋白酶在体外证明。因此，枯草杆菌蛋白酶从细胞膜的释放依赖于一个自动蛋白水解过程，这似乎是新的分泌蛋白质。
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. and 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 .apprxeq. 1000 sites per cell. Mutations in the subtilisin gene that alter a catalytically critical residue (i.e., aspartate +32 .fwdarw. 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.