Sowadski J M, Handschumacher M D, Murthy H M, Kundrot C E, Wyckoff H W
J Mol Biol. 1983 Oct 25;170(2):575-81. doi: 10.1016/s0022-2836(83)80162-4.
Diffraction analysis reveals three metal ion binding sites, M1, M2 and M3, in each of two symmetric active centers 32 A apart in alkaline phosphatase from Escherichia coli with intermediate distances within the center of 4, 5 and 7 A for M1-M2, M2-M3 and M1-M3, respectively. A fourth site, M4, has been reported 25 A away. Arsenate, a product analog, binds adjacent to M1 and M2. The active serine residue, 102, which is phosphorylated during normal enzymatic turnover, is also adjacent to M1 and M2 and arginine 166 is adjacent to the arsenate. The implication with respect to the mechanism is that M1, M2 and Arg 166 neutralize and redistribute charges within the phosphate group, activate the serine hydroxyl, and stabilize transition states during bond formation and breakage. Three sites, A, B and C, have been deduced from solution studies and defined specifically on the basis of nuclear magnetic resonance data, binding studies and activity data. The evidence suggests correspondence of A to M1, B to M2, and C to M3. Strong antagonism between binding at M1 and M2 is evidenced crystallographically by a pseudo-saturation, which is relieved by phosphate binding. Local destabilization of the protein, particularly residues 323 through 333, is produced by removal of metals from the crystal.
衍射分析表明，在来自大肠杆菌的碱性磷酸酶中，两个对称的活性中心各自存在三个金属离子结合位点，即M1、M2和M3，两个活性中心相距32 Å，中心内部M1 - M2、M2 - M3和M1 - M3的中间距离分别为4 Å、5 Å和7 Å。据报道，第四个位点M4在25 Å之外。砷酸盐作为产物类似物，与M1和M2相邻结合。在正常酶促周转过程中发生磷酸化的活性丝氨酸残基102也与M1和M2相邻，精氨酸166与砷酸盐相邻。关于机制的含义是，M1、M2和精氨酸166中和并重新分布磷酸基团内的电荷，激活丝氨酸羟基，并在键形成和断裂过程中稳定过渡态。通过溶液研究推导得出三个位点A、B和C，并根据核磁共振数据、结合研究和活性数据进行了具体定义。证据表明A对应于M1，B对应于M2，C对应于M3。M1和M2处结合之间的强烈拮抗作用在晶体学上表现为假饱和现象，而磷酸盐结合可缓解这种现象。从晶体中去除金属会导致蛋白质局部不稳定，特别是323至333位的残基。
