The 1.9 Å crystal structure of heat-labile shrimp alkaline phosphatase
The 1.9 Å crystal structure of heat-labile shrimp alkaline phosphatase
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DOI：
10.1016/s0022-2836(02)00035-9
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发表时间：
2002-05-17
影响因子：
5.6
通讯作者：
Hough, E
中科院分区：
文献类型：
--
作者：
de Backer, M;McSweeney, S;Hough, E
碱性磷酸酶是非特异性磷酸单酯酶，广泛分布于从细菌到人类的物种中。这项研究主要集中在北极虾的组织非特异性碱性磷酸酶（虾碱性磷酸酶，SAP）上。 SAP 源自冷活性物种，不耐热，在体外广泛使用，例如由于碱性磷酸酶是含锌酶，因此在锌 K 边缘进行了多波长反常色散 (MAD) 实验，从而以 1.9 埃的分辨率确定了结构。异常数据清楚地表明活性位点存在锌三联体，而碱性磷酸酶的每个单体通常含有两个锌离子和一个镁离子。SAP 共享核心、侧翼为 α 螺旋的延伸 β 片层以及具有当前已知的碱性磷酸酶结构（大肠杆菌结构和人胎盘结构）的金属三联体。尽管 SAP 缺乏哺乳动物酶特有的一些特征，但它们的主链非常相似，因此可能是其他高等生物的典型特征。此外，SAP 具有其他结构所缺乏的显着特征：表面电势表示表明酶的净电荷为 -80，其分布使得表面除了带正电的活性位点外主要带负电。因此，带负电的底物必须强烈导向活性位点。人们普遍认为静电优化是与冷适应相关的特性之一。 SAP 非常清楚地展示了这一原则。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature.Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 Angstrom. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer.SAP shares the core, an extended beta-sheet flanked by alpha-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of -80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly. (C) 2002 Elsevier Science Ltd. All rights reserved.