Heidarsson Pétur O, Sigurdsson Snorri Th, Asgeirsson Bjarni
Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, Iceland.
FEBS J. 2009 May;276(10):2725-35. doi: 10.1111/j.1742-4658.2009.06996.x. Epub 2009 Apr 1.
EPR spectroscopy, performed after site-directed spin-labeling, was used to study structural dynamics in a cold-adapted alkaline phosphatase (EC 3.1.1.1). Differences in the structural environment of six spin-labeled side chains allowed them to be classified (with reference to previously obtained mobility maps) as belonging to loop positions (either relatively surface exposed or in structural contact) or helix positions (surface exposed, in contact, or buried). The mobility map constructed in the present study provides structural information that is in broad agreement with the location in the crystal structure. All but one of the chosen serine-to-cysteine mutations reduced activity considerably and this coincided with improved thermal stability. The effect of spin-labeling on enzyme function ranged from nonperturbing to an almost complete loss of activity. In the latter case, treatment with a thiol reagent reactivated the enzyme, indicating relief of steric hindrance to the catalytic process. Two mutations of an active-site residue W274 (K328 in Escherichia coli alkaline phosphatase), known to reduce activity and increase stability of Vibrio alkaline phosphatase, gave a coincidental reduction in mobility of a nearby spin-label located at C67, as determined by EPR spectroscopy. This suggests that movement of the helix carrying C67 and the closely positioned nucleophilic S65 is interconnected with catalytic events.
在定点自旋标记后进行的电子顺磁共振（EPR）光谱学，被用于研究一种冷适应碱性磷酸酶（EC 3.1.1.1）的结构动力学。六个自旋标记侧链的结构环境差异使得它们（参考先前获得的迁移率图谱）可被分类为属于环位置（要么相对暴露于表面，要么处于结构接触中）或螺旋位置（暴露于表面、处于接触中或埋藏）。本研究构建的迁移率图谱提供的结构信息与晶体结构中的位置大致相符。除了一个选定的丝氨酸到半胱氨酸突变外，其他所有突变都显著降低了活性，这与热稳定性的提高相吻合。自旋标记对酶功能的影响范围从无干扰到几乎完全丧失活性。在后一种情况下，用硫醇试剂处理可使酶重新激活，这表明对催化过程的空间位阻得到了缓解。活性位点残基W274（在大肠杆菌碱性磷酸酶中为K328）的两个突变，已知会降低弧菌碱性磷酸酶的活性并提高其稳定性，EPR光谱学测定结果显示，位于C67处的附近自旋标记的迁移率同时降低。这表明携带C67的螺旋以及位置相近的亲核性S65的移动与催化事件相互关联。
