Time-resolved X-ray Crystal Structure Analysis of Protein

为了研究催化必需的锌离子的几何性质，对一种产自丛生链霉菌（Streptomycescaespitosus）的锌内切蛋白酶（ScNP）进行了1 × 10 ~（-1）分辨率的晶体结构分析。锌离子由三个侧链（His 83、His 87和Asp 93）和一个水分子四面体配位。His 83 N ε、His 87 N ε和Asp 93 O δ的锌离子与配位原子的距离分别为2.01 nm、2.01 nm和1.95 nm。这些距离与存放在剑桥结构数据库中的小分子含锌化合物的晶体结构中通常发现的距离非常一致。另一方面，锌离子和配位水分子之间的距离（1.93 μ m）略短于数据库中发现的典型值（2.01 μ m）。此外，Glu 84 O ε与该水分子形成强氢键，距离为2.54 π。因此，水分子处于高度极化状态。Met 103的侧链与His 83和His 87的两个咪唑环之间存在两个氢键（His 83 N δ-Leu 102 O，His 87 N δ-Leu 91 O）和货车范德华相互作用。这些相互作用对于His 83和His 87构建锌离子的四面体配位排列可能是重要的。ScNP的这种晶体结构是迄今为止所确定的锌内切蛋白酶的晶体结构中分辨率和准确度最高的，并且不仅对于锌配位化学和在生物系统中的表现是重要的，而且对于包括锌的有机金属催化剂的设计也是有用的。对球孢链霉菌N-乙酰胞壁素酶的酶促反应机理进行了初步探讨。NAM和NAG之间的糖苷键被Asp 98切断，Asp 98作为酸催化剂将质子传递给连接NAM和NAG的氧原子。反应中间体氧碳正离子通过Asp 198的负电荷稳定。

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.