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Studies on the Structure and the Mechanism of Radical Enzymes

自由基酶的结构与作用机制研究

基本信息

批准号：
10680611
负责人：
TORAYA Tetsuo
金额：
$ 2.37万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Electron paramagnetic resonance (EPR) measurements were conducted to elucidate the mode of binding of cobalamin to diol dehydratase. It was demonstrated that this enzyme binds cobalamin in the ゛base-on″mode, and that the bulkiness of the lower axial base is important for protection of reactive radical intermediates. Two forms of crystals of the diol dehydratase-cyanocobalamin complex were obtained, which diffracted up to 2.2 and 3.0 A resolution. The crystal structure of the complex revealed that cobalamin is bound to the enzyme in the base-on mode. The active site exists inside the TIM barrel which may protect reactive radical intermediates from side reactions. The two hydroxyl groups of the substrate coordinate directly to KィイD1+ィエD1 in the active site. Density functional theory computations indicated that the hydroxyl group migration from C-2 to C-1 proceeds by a concerted pathway through a cyclic transition state. KィイD1+ィエD1 seems to be important not only in stabilizing the transition state but also in labilizing the Co-C bond indirectly by increasing the substrate binding energy.The products of putative reactivating factor genes were purified and confirmed in vitro to function as a reactivating factor for glycerol-inactivated and oィイD22ィエD2-inactivated holoenzymes. The mechanism of action of the diol dehydratase-reactivating factor was investigated. It was demonstrated that ATP- and ADP -forms of this factor are low and high affinity forms for diol dehydratase, and that the modified coenzyme in the inactivation is released from the enzyme and, as a result, substituted by intact coenzyme, reconstituting catalytically active holoenzyme. This factor is thus considered as a sort of molecular chaperone. The two genes in proximity to the glycerol dehydratase genes were identified as the genes for a glycerol dehydratase-reactivating factor. Their amino acid sequences showed high homology with the diol dehydratase-reactivating factor.

电子顺磁共振（EPR）测量进行，以阐明钴胺素的二醇脱氢酶的结合模式。结果表明，这种酶与钴胺素的结合是以“轴向碱基-对”的方式进行的，并且下轴向碱基的庞大性对于保护活性自由基中间体是重要的。获得了二醇脱氢酶-氰钴胺复合物的两种形式的晶体，其衍射分辨率高达2.2和3.0 A。复合物的晶体结构表明，钴胺素是结合在碱基模式的酶。活性位点存在于TIM桶内，其可以保护活性自由基中间体免于副反应。底物的两个羟基基团直接与活性位点中的K D1+ K D1配位。密度泛函理论计算表明，羟基从C-2到C-1的迁移是通过一个环状过渡态的协同途径进行的。K重激活因子D_1 + K重激活因子D_1不仅对稳定过渡态有重要作用，而且通过增加底物结合能间接地使Co-C键不稳定，纯化了推测的重激活因子基因产物，并在体外证实其对甘油失活和O重激活因子D_2 ~（22）和O重激活因子D_2 ~（22）失活的全酶具有重激活因子的功能。研究了二醇脱氢酶再活化因子的作用机理。结果表明，ATP-和ADP -形式的这个因素是低和高亲和力的形式，二醇脱氢酶，并在失活的修饰的辅酶从酶中释放，因此，被完整的辅酶取代，重建催化活性全酶。因此，该因子被认为是一种分子伴侣。接近甘油脱氢酶基因的两个基因被鉴定为甘油脱氢酶再活化因子的基因。它们的氨基酸序列与二醇脱氢酶再激活因子具有很高的同源性。