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.

中文翻译：

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通过EPR光谱研究冷适应的碱性磷酸酶的结构特征和动力学。

在定点旋转标记后进行的EPR光谱用于研究冷适应的碱性磷酸酶的结构动力学（EC 3.1.1.1）。六个自旋标记侧链在结构环境上的差异使它们可以归类为环位置（相对表面暴露或结构接触）或螺旋位置（表面暴露，接触）（参考先前获得的迁移率图） ，或埋藏）。在本研究中构造的迁移率图提供了与晶体结构中的位置大体一致的结构信息。除了一种选择的丝氨酸至半胱氨酸突变外，所有突变都大大降低了活性，这与提高的热稳定性相吻合。旋转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活化，表明消除了对催化过程的空间位阻。活性位点残基W274的两个突变（大肠杆菌碱性磷酸酶中的K328）已知会降低活性并增加弧菌碱性磷酸酶的稳定性，通过EPR确定，该突变同时降低了位于C67的自旋标记的迁移率光谱学。这表明携带C67和紧密定位的亲核S65的螺旋的运动与催化事件相互联系。活性位点残基W274的两个突变（大肠杆菌碱性磷酸酶中的K328）已知会降低活性并增加弧菌碱性磷酸酶的稳定性，通过EPR确定，该突变同时降低了位于C67的自旋标记的迁移率光谱学。这表明携带C67和紧密定位的亲核S65的螺旋的运动与催化事件相互联系。活性位点残基W274的两个突变（大肠杆菌碱性磷酸酶中的K328）已知会降低活性并增加弧菌碱性磷酸酶的稳定性，通过EPR确定，该突变同时降低了位于C67的自旋标记的迁移率光谱学。这表明携带C67和紧密定位的亲核S65的螺旋的运动与催化事件相互联系。