Studies on the Structure and the Mechanism of Radical Enzymes

电子准磁性共振（EPR）测量方法是通过提取钴胺与二醇脱氢酶结合的模式来实现的。它被证明是因为这种酶绑定钴胺在“基-上”模式中，而低轴基的体积对于保护反应性激进介质是很重要的。二醇脱氢酶-青钴胺复合体的两种形式的晶体被发现，它们与2.2和3.0 A决议不同。复杂的晶体结构揭示了钴胺在基本模式下与酶绑定的晶体结构。活跃的网站存在于TIM桶中，它可能会保护反应性激进分子的副反应。The two hydroxyl groups of the substrate coordinate directly to KイD1+イエD1 in the active site。密度功能理论计算是指从C-2到C-1的羟基群迁移过程通过一个集中的途径通过一个循环过渡状态进行的。イイD1+イエD1 seems to be important not only in stManagement izing 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 D22-inactivated holoenzymes。二醇脱氢酶的作用机理-反应因子已被调查。这被证明是表明，该因子的ATP-和ADP-形式是二醇脱氢酶的低亲和力和高亲和力形式，并且在非激活中的修饰辅酶已从酶中释放，作为一个结果，由接触辅酶酶替代，重新构建催化活性的全氧酶。这一因素被认为是一组分子章节。两种基因的近似值被确定为glycerol去水解酶基因被确定为一种glycerol去水解酶-反应因子。他们的氨基酸序列展示了与二醇脱氢酶-反应因子的高同源性。

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.