Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution.
Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution.
复制标题
大肠杆菌碱性磷酸酶的精制结构，分辨率为 2.8 A。
DOI：
10.1016/0022-2836(85)90115-9
复制
发表时间：
1985
影响因子：
5.6
通讯作者：
Wyckoff,HW
中科院分区：
文献类型：
--
作者：
Sowadski,JM;Handschumacher,MD;Murthy,HM;Foster,BA;Wyckoff,HW
对大肠杆菌碱性磷酸酶的结构进行了测定，其分辨率为2.8？分子对称性平均化和溶剂平坦化显著改善了二聚体在3.4？处的多重同象取代电子密度图。根据这些图谱，使用已发表的氨基酸序列构建了该二聚体的多肽链。根据原始数据对该模型进行了立体化学限制的最小二乘修正，从3.4o数据开始，逐步扩展到2.8o分辨率，最终得到总体晶体R因数为0.256。碱性磷酸酶-磷酸单酯水解酶(EC 3.1.3.1)是一种金属酶，它形成具有两个相隔32？的反应中心的同源二聚体。该亚基的多肽折叠的拓扑结构属于αβ类蛋白质。尽管碱性磷酸酶在整个αβ折叠中与其他蛋白质有相似之处，但它并没有在平行板的羧基末端形成特征的结合裂隙，而是一个活性口袋，其中包含位于中央十链片面外的三个功能金属位点的簇。这个活性口袋位于四条链的羧基末端和反平行链的氨基末端附近，在片层的平面和同一侧的两个螺旋之间。碱性磷酸酶是一种非特异性的磷酸单酯酶，它能分解小分子磷酸单酯和DNA的磷酸末端。基于酶的精细坐标的可及性计算表明，活性口袋几乎容纳不了无机磷。因此，底物的醇类或酚类部分必须暴露在酶的表面。锌占据了两个金属位置，M1和M2，相距3.9？第三个点，M3，M2点的5？和M1点的7？，由镁占据，如果没有镁，则由锌占据。与其他含锌酶一样，组氨酸残基是锌位点M1(三个)和锌位点M2(一个)的配体。配位分配和金属择优表明，结晶学上发现的金属中心M1、M2和M3分别对应于光谱推断的金属中心A、B和C。砷是一种产物类似物和酶抑制剂，它结合在Ser102和锌的M1和M2位点之间。Arg166的胍基位置与砷酸根位置之间的氢键距离内。这些元素的结构配置表明，尽管缺乏蛋白质酸和蛋白质碱功能，但金属可以激活亲核试剂Ser102和水，这是磷上双线亲核置换所必需的。金属位M1和/或M2可以激活Ser102，并为亲核攻击准备磷酸基团。在成键和断裂过程中，它们能与Arg166一起稳定过渡态。在磷酸丝氨酸中间体的水解过程中，水进行第二次亲核攻击。将活性口袋的结构与最近的31P和113Cd共价和非共价磷酸酶中间体的核磁共振进行比较，表明水分子最有可能位于M1金属位。
The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 Å resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 Å was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 Å data and extending in steps to 2.8 Å resolution, proceeded to a final overall crystallographic R factor of 0.256. Alkaline phosphatase-phosphomonoester hydrolase (EC 3.1. 3.1) is a metalloenzyme that forms an isologous dimer with two reactive centers 32 Å apart. The topology of the polypeptide fold of the subunit is of the α β class of proteins. Despite the similarities in the overall α β fold with other proteins, alkaline phosphatase does not have a characteristic binding cleft formed at the carboxyl end of the parallel sheet, but rather an active pocket that contains a cluster of three functional metal sites located off the plane of the central ten-stranded sheet. This active pocket is located near the carboxyl ends of four strands and the amino end of the antiparallel strand, between the plane of the sheet and two helices on the same side. Alkaline phosphatase is a non-specific phosphomonoesterase that hydrolyzes small phosphomonoesters as well as the phosphate termini of DNA. The accessibility calculations based on the refined co-ordinates of the enzyme show that the active pocket barely accommodates inorganic phosphate. Thus, the alcoholic or phenolic portion of the substrate would have to be exposed on the surface of the enzyme. Two metal sites, M1 and M2, 3.9 Å apart, are occupied by zinc. The third site, M3, 5 Å from site M2 and 7 Å from site M1, is occupied by magnesium or, in the absence of magnesium, by zinc. As with other zinc-containing enzymes, histidine residues are ligands to zinc site M1 (three) and to zinc site M2 (one). Ligand assignment and metal preference indicate that the crystallographically found metal sites M1, M2 and M3 correspond to the spectroscopically deduced metal sites A, B and C, respectively. Arsenate, a product analog and enzyme inhibitor, binds between Ser102 and zinc sites M1 and M2. The position of the guanidinium group of Arg166 is within hydrogen-bonding distance from the arsenate site. The structural disposition of those elements suggests that, despite the lack of protein acid and protein base functions, metals can activate both nucleophiles, Ser102 and water, necessary for double in-line nucleophilic displacement on phosphorus. Metal sites M1 and/or M2 can activate Ser102 and prepare the phosphate group for nucleophilic attack. They can stabilize the transition state along with Arg166 during bond formation and breakage. A second nucleophilic attack is performed by water during hydrolysis of phosphoserine intermediate. Comparison of the architecture of the active pocket with recent 31 P and 113 Cd nuclear magnetic resonance on covalent and non-covalent phosphoenzyme intermediates indicates the water molecule is most probably located on the M1 metal site.