Lethal factor active-site mutations affect catalytic activity in vitro.
Lethal factor active-site mutations affect catalytic activity in vitro.
复制标题
致死因子活性位点突变影响体外催化活性。
DOI：
10.1128/iai.66.5.2374-2378.1998
复制
发表时间：
1998
影响因子：
3.1
通讯作者：
Hanna,PC
中科院分区：
文献类型：
--
作者：
Hammond,SE;Hanna,PC
炭疽杆菌致死毒素的致死因子（LF）蛋白含有嗜热菌蛋白酶样活性位点和锌结合共有基序HEXXH（K. R. Klimpel，N. Arora和S. H.莱普拉山Microbiol. 13：1093-1100，1994）。LF被假设为在巨噬细胞的细胞质中充当Zn 2+金属蛋白酶，但先前在任何靶底物上均未显示蛋白水解活性。在这里，合成肽在体外被LF水解。质谱和肽序列分离的裂解产物分离的反相高压液相色谱法表明，LF似乎更喜欢含脯氨酸的基板。一致的活性位点残基内的取代突变完全消除了所有的体外催化功能，如添加1，10-菲咯啉，EDTA和某些氨基酸异羟肟酸盐，包括新的锌金属蛋白酶抑制剂ZINCOV。相比之下，蛋白酶抑制剂bestatin和赖氨酸CMK，以前显示阻断LF活性的巨噬细胞，没有阻止LF活性在体外。这些数据提供了LF可能作为内肽酶的第一个直接证据。
The lethal factor (LF) protein ofBacillus anthracislethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.