Mechanisms of Radical Catalysis in Vitamin B_<12> Enzyme and Reactivation by Molecular Chaperone-like Factor

1.(1)对重组甘油脱水酶进行了纯化，并对其酶学性质进行了研究。它的x射线结构首次被解开。(2)用可见光照射二醇脱水酶-腺嘌呤戊钴胺复合物，在晶体学上证实了腺嘌呤锚定自由基的形成。(3)确定了无底物型二醇脱水酶的结构。我们强烈认为，底物触发辅酶Co-C键的均裂是通过对已经产生一定张力的Co-C键诱导进一步的空间应变来实现的。(4)辅酶B12的碱基部分被其他碱基取代的辅酶类似物的辅酶活性与碱基的体积大小相关。也有人认为，核苷酸部分是稳定自由基中间体所必需的。(5)分析了二醇脱水酶与B12和r -或S-等对映体配合物的x射线结构，并根据x射线结构完整地阐明了各对映体底物转化为产物的立体化学过程。(6)简化模型的理论计算表明，二醇脱水酶反应各步骤的活化能很小，足以由底物结合能提供。分子伴侣样因子对B12酶的再激活机制(1)甘油脱水酶基因附近的两个orf被确定为可能的再激活因子基因。纯化的基因产物实际上通过分子伴侣样的方式重新激活了失活的全酶。(2)鉴定了乙醇胺氨还原活性再激活因子的基因。纯化的基因产物被证明在B12辅酶和ATP存在下作为再激活因子。本研究结果在《化学评论》的“自由基酶学”专刊上进行了综述。我被邀请为这份最权威的化学杂志撰写这篇评论，这一事实表明，这项研究的科学价值在该领域的国际社会中被评为优秀。少

1.Mechanism of radical catalysis by vitamin B12 enzymes(1) Recombinant glycerol dehydratase was purified, and its enzymological properties were investigated. Its X-ray structure was solved for the first time. (2) The formation of the adenine-anchored radical was crystallographically demonstrated upon illumination of the diol dehydratase-adeninylpentylcobalamin complex with visible light. (3) The structure of substrate-free form of diol dehydratase was determined. It was strongly suggested that substrate triggers the homolysis of the coenzyme Co-C bond by inducing further steric strain to the Co-C bond that had been already strained to some extent. (4) Coenzymic activity of coenzyme analogs in which the base moiety of the coezyme B12 was replaced by other bases was correlated with the bulkiness of the base. It was also suggested that the nucleotide moiety is required for stabilizing radical intermediates. (5) The X-ray structures of the complexes of diol dehydratase with B12 and R-or S- … More enantiomer were analyzed, and The stereochemical _courses of the steps of the conversion of each enantiomeric substrate to product was completely elucidated based on the X-ray structures. (6) Theoretical calculations with a simplified model indicated that the activation energies of each steps of diol dehydratase reaction are small enough to be supplied by substrate binding energy.2. Mechanism of reactivation of a B12 enzyme by molecular chaperone-like Factor(1) The two ORFs near the glycerol dehydratase genes were identified as putative reactivating factor genes. The purified gene products actually reactivated the inactivated holoenzyime by a molecular chaperone-like manner. (2) The gene encoding a reactivating factor for ethanolamine ammonia-lyric was identified. The purified gene product was shown to serve as a reactivating factor in the presence of B12 coenzyme and ATP.The results obtained by this study was summarized and published as a review in the special issue of "Radical Enzymology" in Chemical Reviews. The fact that I was invited to write this review for this most authoritative journal in chemistry indicates that the scientific merit of this study rated excellent among the international community in this field. Less