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Study of Substrate-recognition Mechanism of Zn-metalloprotease

锌金属蛋白酶底物识别机制的研究

基本信息

批准号：
05680580
负责人：
HARADA Shigeharu
金额：
$ 1.15万
依托单位：
University of Tokyo (1994)Osaka University (1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

The three-demensional structure and amino acid sequence of a zinc metalloprotease produced by Streptomyces caespitosus (SCNP) have been determined in order to clarify the relationship between structure and function of SCNP.The amino-acid sequence has been determined by Edman degradation. SCNP consists of a single polypeptide chain of 132 amino acid residues with one disulfide bond between residues 99 and 112. The deduced amino acid sequence indicated that it is much shorter than other ones from metalloproteases previously reported. Although a zinc-binding motif, HEXXH,found at the active sites of most metalloproteases, was found in the sequence, SCNP did not share overall significant similarity to the sequences of other zinc metalloproteases. The three-demensional structure of SCNP has been determined by X-ray crystal structure analysis and refined to R-factor of 0.18 (2.0A resolution). A five stranded beta-sheet and three alpha-helices are found in the structure. The zinc-binding moti … More f (H83-E-T-G-H87) is located on the second alpha-helix from N-terminal. About 40 amino acid sequences of zinc-containing metalloproteases have been determined so far and classified into five distinct families according to the sequence homology : thermolysin, astacin, serratia, matrixin and snake venom. Crystal structures available for six enzymes have revealed that two histidine residues located in the the sequence of HEXXH are the first two zinc ligands. Gly observed as the 8th residue from the first His in the motif, which is conserved in all but thermolysin family, has been reported to be important for structural reason, becaluse this residue allows the 11th His to be the third zinc ligand by terminating the second alpha-helix and bending a main chain sharply. SCNP also has Gly at this position. However, the residue corresponding to the 11th His is Asp. The three-dimensional structure of SCNP revealed that this Asp is the third zinc lingand. It is deduced that SCNP may represent a new subfamily of zinc-containing metalloprotease, with respect to both the new type of ligand organization for Zn and a very small molecular size distinct from other known metalloproteases in the five families. Less

为了阐明簇状链霉菌（Streptomycescaespitosus，SCNP）锌金属蛋白酶的结构与功能的关系，测定了SCNP的三维结构和氨基酸序列。SCNP由132个氨基酸残基的单一多肽链组成，在残基99和112之间具有一个二硫键。推导的氨基酸序列表明，它比以前报道的其他金属蛋白酶短得多。虽然锌结合基序，HEXXH，发现在大多数金属蛋白酶的活性位点，被发现在序列中，SCNP没有共享整体显着的相似性，其他锌金属蛋白酶的序列。用X射线晶体结构分析确定了SCNP的三维结构，并将其精细化到R因子为0.18（2.0A分辨率）。在该结构中发现了五股β-折叠和三股α-螺旋。锌结合动机 ...更多信息 f（H83-E-T-G-H87）位于从N-末端起的第二个α-螺旋上。迄今为止，已测定了约40个含锌金属蛋白酶的氨基酸序列，并根据序列同源性将其分为5个不同的家族：嗜热菌蛋白酶、虾红素、沙雷氏菌属、基质蛋白酶和蛇毒。六种酶的晶体结构表明，位于HEXXH序列中的两个组氨酸残基是前两个锌配体。Gly作为基序中第一个His的第8个残基，在除嗜热菌蛋白酶家族以外的所有家族中是保守的，已经报道了Gly对于结构原因是重要的，因为该残基通过终止第二个α-螺旋和急剧弯曲主链而允许第11个His成为第三个锌配体。SCNP在该位置也具有Gly。然而，对应于第11个His的残基是Asp。SCNP的三维结构表明，该Asp是第三锌灵。据推断，SCNP可能代表一个新的含锌金属蛋白酶的亚家族，相对于新类型的配体组织的锌和一个非常小的分子大小不同于其他已知的金属蛋白酶在五个家庭。少