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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Redesigning allosteric activation in an enzyme
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Redesigning allosteric activation in an enzyme

机译：重新设计酶中的变构激活

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Enzyme activation by monovalent cations is widely documented in plants and the animal world. In type II enzymes, activation entails two steps: binding of the monovalent cation to its allosteric site and transduction of this event into enhanced catalytic activity. The effect has exquisite specificity for either Na~+ or K~+ , the most abundant cations present in physiological environments. Enzymes requiring K~+ such as kinases and molecular chaperones are not activated as well or at all by the larger cation Cs~+ or the smaller cations Na~+ and Li . Enzymes requiring Na~+ such as β-galactosidase and clotting proteases are not activated as well by Li~+, or the larger cations K~+ , Rb~+ , and Cs~+. Efforts to switch specificity between Na~+ and K~+ in this large class of enzymes and completely redesign the mechanism of allosteric transduction leading to enhanced catalytic activity have so far been unsuccessful. Here we show how muta-genesis of two loops defining the Na~+ binding site of thrombin, a Na~+ -activated clotting protease, generates a construct that is most active in the presence of K~+ toward synthetic and physiological substrates. The effect is the result of a higher binding affinity and more efficient allosteric transduction of binding into enhanced catalytic activity for K~+ compared to Na~+, which represents a complete reversal of the properties of wild type. In addition, the construct features altered specificity toward physiological substrates resulting in a significant anticoagulant profile. The findings are relevant to all Na~+ -activated proteases involved in blood coagulation and the complement system.
机译：单价阳离子对酶的激活已在植物和动物界广泛记载。在II型酶中，激活包括两个步骤：单价阳离子与其变构位点的结合以及将该事件转导为增强的催化活性。该作用对生理环境中存在的最丰富的阳离子Na〜+或K〜+具有极好的特异性。较大的阳离子Cs〜+或较小的阳离子Na〜+和Li都不会或完全不会激活需要K〜+的酶（例如激酶和分子伴侣）。 Li〜+或较大的阳离子K〜+，Rb〜+和Cs〜+不会激活需要Na〜+的酶（例如β-半乳糖苷酶和凝血酶）。迄今为止，尚未尝试在这种大类酶中在Na〜+和K〜+之间切换特异性并完全重新设计变构转导机制以增强催化活性的努力。在这里，我们显示了两个环的诱变如何定义凝血酶的Na +结合位点（Na +活化的凝结蛋白酶）的两个环的诱变如何生成在K +存在下对合成和生理底物活性最高的构建体。该效果是与Na〜+相比具有更高的结合亲和力和更有效的变构结合键转换成增强的K〜+催化活性的结果，这代表了野生型特性的完全逆转。另外，该构建体的特征是对生理底物的特异性改变，导致显着的抗凝特性。该发现与涉及血液凝固和补体系统的所有Na +活化的蛋白酶有关。

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