INTERACTIONS OF COBALAMINS

维生素B12研究中存在的主要问题之一是 5'端Co-C键均裂活化机理 脱氧腺苷钴胺素（B12-需要酶）。 我们 维生素C与钴配合物的初步研究 来自鸡血清的B12结合蛋白（haptocorrin）表明， 这些蛋白质将作为一个很好的模型， 用酶解Cb 1。 因此，我们建议设立一个 用于详细研究分子的实验系统 维生素的辅酶形式的活化机制 B12发生。 特别地，各种钴的络合物与 将研究鸡血清结合咕啉以确定 结合相互作用会影响 钴胺素的特性。 这些研究将直接针对 这类B12结合蛋白是否能“激活” 通过降低热传导的能量势垒， 碳-钴键均裂，并将提供直接的 酶促反应机理的实验研究 激活cb 1。 要进行的测量包括NMR光谱 测量蛋白质结合的钴，以阐明 结合相互作用对上下轴向的影响 配体键的钴。 这些将包括测量 的NMR参数的蛋白质结合的钴富集， 轴向核苷酸的咪唑碳（B2）中的13 C和 蛋白结合的氰基钴咕啉在上部富含15 N 氰化物配体 钴可啉与 脱触咕啉也将作为一种辅助研究， 碳-钴键热分解反应动力学 不稳定的，空间位阻的钴，当结合到这个B12上时， 结合蛋白 此类钴胺素将包括新戊钴胺素 和苄钴胺素以及β-环糊精。 本提案中描述的实验代表了第一个 详细的实验方法来解决分子 辅酶B12的酶促活化机制。 的 这项工作是我们以前研究的一个主要扩展， 模型维生素B12系统。 根据我们的经验和 直接的初步研究（如下所述），我们有信心 建议的实验，特别是同位素实验， 标记的NMR研究，将提供有价值的分子见解， 关键钴胺素激活机制的动力学。

One of the major remaining problems in vitamin B12 research is the mechanism of activation via Co-C bond homolysis of 5' deoxyadenosylcobalamin (AdoCb1) by B12-requiring enzymes. Our preliminary studies of complexes of cobalamins with a vitamin B12 binding protein (haptocorrin) from chicken serum suggest that such proteins will serve as excellent models of the interaction of AdoCb1 with enzymes. We thus propose to establish an experimental system for investigating in detail the molecular mechanism by which activation of the coenzyme form of vitamin B12 occurs. In particular, complexes of various cobalamins with the chicken serum haptocorrin will be studied to determine how the binding interaction affects the physical and chemical properties of the cobalamin. The studies will directly address the question of whether such B12 binding proteins can "activate" alkylcobalamins by lowering the energy barrier for thermal carbon-cobalt bond homolysis and will provide a direct experimental approach to a proposed mechanism of enzymic activation of AdoCb1. Measurements to be made include NMR spectroscopic measurements of protein-bound cobalamins to elucidate the effects of the binding interactions on the upper and lower axial ligand bonds of the cobalamins. These will include measurements of the NMR parameters of protein-bound cobalamins enriched in 13C in the imidazole carbon (B2) of the axial nucleotide and of protein-bound cyanocobalt corrins enriched in 15N in the upper cyanide ligand. Measurements of the kinetics of binding of cobalt corrins to the apohaptocorrin will also be made as an adjunct to studies of the kinetics of carbon-cobalt bond dissociation reactions of thermally labile, sterically hindered cobalamins when bound to this B12 binding protein. Such cobalamins will include neopentylcobalamin and benzylcobalamin as well as AdoCb1. The experiments described in this proposal represent the first detailed experimental approach to the question of the molecular mechanism of the enzymatic activation of coenzyme B12. The work represents a major extension of our previous studies on model vitamin B12 systems. Based on our experience and our direct preliminary studies (as described below), we are confident that the proposed experiments, particularly the isotopically labeled NMR studies, will provide valuable molecular insights into the dynamics of the critical cobalamin activation mechanism.