Time-resolved X-ray Crystal Structure Analysis of Protein

蛋白质的时间分辨 X 射线晶体结构分析

• 批准号: 09557187
• 负责人: HARADA Shigeharu
• 金额: $ 6.02万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557187/
• 关键词: dynamical structure; zinc protease; N-acetylmuramidase; structural biology; enzymatic reaction; nucleophic attack; hydrgogen bond; activation of water; ラウエ法; 白色X線; シンクロトロン放射光; 時間分割構造解析; カルシウム結合蛋白質; フローセル

The crystal structure of a zinc endoprotease from Streptomyces caespitosus (ScNP) determined at 1 Å resolution has been analyzed to investigate geometrical properties of a catalytically essential zinc ion. The zinc ion is tetrahedrally coordinated by three side-chains (His83, His87 and Asp93) and a water molecule. The distances between the zinc ion and the coordinating atoms are 2.01 Å, 2.01 Å and 1.95 Å for His83Nε, His87Nε and Asp93Oδ, respectively. These distances agree very well with those normally found in crystal structures of small zinc-containing compounds deposited in the Cambridge Structural Database. On the other hand, the distance between the zinc ion and the coordinating water molecule (1.93 Å) is slightly shorter than the typical value (2.01 Å) found in the Database. In addition, Glu84Oε makes a strong hydrogen bond to this water molecule with the distance of 2.54 Å. Thus, the water molecule is in a highly polarized state. Two hydrogen bonds (His83Nδ-Leu102O, His87Nδ-Leu91O) and van der Waals interactions between the side-chain of Met103 and the two imidazole rings of His83 and His87 are also observed. These interactions are probably important for His83 and His87 to construct the tetrahedral coordination arrangement to the zinc ion. This crystal structure of ScNP is the highest resolution and accuracy among crystal structures of zinc endoproteases ever determined, and is not only important for zinc coordination chemistry and manifestation in biological systems but useful for the design of organo-metallic catalyst including zinc as well. The enzymatic reaction mechanism of the N-acetylmuramidase produced by Streptomyces globisporus was also clarified. The glycosidic linkage between NAM and NAG is cut by Asp98, which as an acid catalyst hands proton to the oxygen atom linking NAM and NAG. The reaction intermediate, oxyocarbenium ion, is stabilized by the negative charge of Asp198.

在1 Å分辨率下，对caespitosus链霉菌（Streptomyces caespitosus, ScNP）中锌内源性蛋白酶的晶体结构进行了分析，以研究催化必需锌离子的几何性质。锌离子由三条侧链（His83、His87和Asp93）和一个水分子组成。对于His83Nε、His87Nε和Asp93Oδ，锌离子与配位原子的距离分别为2.01 Å、2.01 Å和1.95 Å。这些距离与通常在剑桥结构数据库中沉积的小含锌化合物的晶体结构中发现的距离非常吻合。另一方面，锌离子与配位水分子之间的距离（1.93 Å）略短于数据库中的典型值（2.01 Å）。此外，Glu84Oε与该水分子形成了一个距离为2.54 Å的强氢键。因此，水分子处于高度极化状态。还观察到Met103侧链与His83和His87的两个咪唑环之间的两个氢键（his83n - δ- leu102o和his87n - δ- leu91o）和范德华作用。这些相互作用可能对His83和His87与锌离子形成四面体配位排列很重要。该晶体结构是目前所确定的锌内源性蛋白酶晶体结构中分辨率和精度最高的，不仅对锌在生物系统中的配位化学和表现具有重要意义，而且对含锌的有机金属催化剂的设计也有重要意义。阐明了球孢链霉菌产n -乙酰化酶的酶促反应机理。Asp98作为酸性催化剂将质子传递到连接NAM和NAG的氧原子上，从而切断NAM和NAG之间的糖苷键。反应中间体氧羰基离子被Asp198的负电荷稳定。

项目成果

Genji Kurisu: "Structure of the Zinc Endoprotease from Streptomyces caespitosus" J.Biochem.121. 304-308 (1997)

Genji Kurisu：“来自链霉菌的锌内切蛋白酶的结构”J.Biochem.121。

松村浩由: "Ca^<2->結合蛋白質S100bの結晶構造と分子認識"日本結晶学会誌. 41. 347-352 (1999)

Hiroyoshi Matsumura：“Ca^2-结合蛋白S100b的晶体结构和分子识别”日本晶体学会杂志41. 347-352(1999)。

Genji Kurisu: "Structure of the zinc binding site in the crystal structure of a zinc endoprotease from Streptomyces caespitosus"J.Inorganic Biochemistry. (2000)

Genji Kurisu：“来自链霉菌的锌内切蛋白酶晶体结构中锌结合位点的结构”J.无机生物化学。

Genji Kurisu: "Structure of the zinc binding site in the crystal structure of a zinc endoprotease from Streptomyces caespitosus"J. iInorganic Biochemistry. (2000)

Genji Kurisu：“来自链霉菌的锌内切蛋白酶晶体结构中锌结合位点的结构”J。

Hiroyoshi Matsumura et al.: "A Novel Mode of Target Recognition Suggested by the 2.0 Å Structure of Holo S100B from Bovine Brain"Nihon Kessyougakkai-Si. Vol. 41. 347-352 (1999)

Hiroyoshi Matsumura 等人：“牛脑 Holo S100B 的 2.0 Å 结构提出的目标识别新模式”Nihon Kessyougakkai-Si 41. 347-352 (1999)。