Role of the occluding loop in cathepsin B activity
Role of the occluding loop in cathepsin B activity
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DOI：
10.1074/jbc.272.2.1197
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发表时间：
1997-01-10
影响因子：
4.8
通讯作者：
Mort, JS
中科院分区：
文献类型：
--
作者：
Illy, C;Quraishi, O;Mort, JS
在溶酶体半胱氨酸蛋白酶家族中，组织蛋白酶B是独一无二的，因为它既可以作为内肽酶，也可以作为肽基二肽酶。后一种去除C-末端二肽的能力归因于20个残基的插入，称为闭塞环，它阻断了活性部位裂解的启动末端，人原蛋白B的变异体在毕赤酵母中表达，其中该元件的全部或部分被删除。一个突变体，其中闭塞环的12个中心残基被删除，自动处理，尽管比野生型酶原慢，以产生成熟形式的酶，其内肽酶活性与野生型组织蛋白酶B相当，但完全缺乏外肽酶活性，这个缺失突变体对抑制物cystatin C的亲和力高40倍，这表明闭塞环通常限制该抑制剂对活性部位的访问，此外，作为该酶的有效抑制剂的组织蛋白酶B前肽的结合亲和力增加了50倍，与环重定向于原酶的激活的发现一致这些结果表明，组织蛋白酶B的内肽酶活性是一个进化残留物，因为由于它是木瓜酶家族的成员，前肽必须能够通过活性部位裂隙的全长畅通无阻地结合。
Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft, Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris. A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wildtype cathepsin B, but totally lacking exopeptidase activity, This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site, In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme, These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.