MODULATION OF ORGANOCOBALT REACTIVITY BY HAPTOCORRIN

本研究项目的最终目标是制定一个详细的 了解维生素B12的辅酶形式是如何通过 需要辅酶B12的酶引起的碳-钴键均裂。最近 工作表明，热不稳定的烷基钴的均裂 （苄基钴胺素和新戊基钴胺素）由空间相互作用驱动 在有机配体和向上突出的a、c和g之间 乙酰胺侧链。如果这一假设被证明是正确的， 用于5 ′-脱氧腺苷钴胺素的酶促活化，其中 酶增加5 '-脱氧腺苷配体的空间相互作用 乙酰胺侧链会变得引人注目。拟议 研究被指定为严格测试碳的假设， 在这种烷基钴醇中的钴键均裂是空间位阻的结果 乙酰胺和有机配体之间的相互作用。 这一假设将通过两类研究进行检验。一是 烷基钴胺素类似物的热均裂，其中空间体积 和/或向上突出的侧链的数量已经改变 化学研究。二、乙酰胺方的重要性 碳-钴键均裂上的链移动性将通过 利用我们最近观察到的B12结合蛋白 （haptocorrin）从鸡血清稳定的碳钴键 热不稳定的烷基钴酸盐约三个数量级。我们 我曾假设，钴酸盐与触可林的结合涉及 侧链酰胺与蛋白质中受体的氢键 结合口袋，并随之而来的热固定的 乙酰胺是观察到的稳定化的原因。所以 烷基钴胺素类似物的治疗，其中氢键 蛋白质的向上定向侧链的能力已经被 还原的应显示结合后Co-C键的稳定性降低 结合可林。这些衍生物将包括a、c和g N- 甲基酰胺和单羧酸酯、C8和C13差向异构体（其中甲基酰胺和单羧酸酯是甲基酰胺和单羧酸酯的衍生物）、C8和C13差向异构体（其中甲基酰胺和单羧酸酯是甲基酰胺和单羧酸酯的衍生物）、C8和C13差向异构体（其中甲基酰胺和单羧酸酯是甲基酰胺和单羧酸酯的衍生物）、C8和C13差向异构体（其中甲基酰胺和单羧酸酯是甲基酰胺和单羧酸酯的衍生物）。 D或E侧链向上突出）以及其它类似物， 产生与有机配体的改变的空间相互作用。的 氢键在钴离子缔合中的重要性 结合咕啉将通过研究的热力学的 卡巴氨及其类似物的结合反应， 氢键供体已被化学除去。补充证据 将寻求从1H和15 N NMR研究的配合物的结合咕啉 用通过发酵获得的均匀富集15 N的氰钴胺 使用灰色链霉菌。这些测量将包括偏振 转移和多量子相干光谱以及15 N- 光谱编辑的1H NMR。这些实验将允许观察 酰胺氮和质子以及质子的化学位移 化学位移热梯度和H-D交换率。

The ultimate goal of this research project is to develop a detailed understanding of how the coenzyme form of vitamin B12 is activated via carbon-cobalt bond homolysis by coenzyme B12-requiring enzymes. Recent work suggests that homolysis of thermally labile alkylcobalamins (benzyl- and neopentylcobalamin) is driven by steric interactions between the organic ligand and the upward projecting a, c, and g acetamide side chains. Should this hypothesis prove correct, a mechanism for enzymatic activation of 5' -deoxy- adenosylcobalamin in which the enzyme increases the steric interactions of the 5'-deoxyadenosyl ligand with the acetamide side chains would become compelling. The proposed research is designated to rigorously test the hypothesis that carbon cobalt bond homolysis in such alkylcobalamins is the result of steric interactions between the acetamides and the organic ligand. This hypothesis will be tested by two types of studies. First, the thermal homolysis of alkylcobalamin analogs in which the steric bulk and/or the number of upward projecting side chains has been altered chemically will be studied. Second, the importance of acetamide side chain mobility on carbon-cobalt bond homolysis will be studied by capitalizing on our recent observation that a B12 binding protein (haptoccorrin) from chicken serum stabilizes the carbon-cobalt bond of thermaly labile alkylcobalamins by some three orders of magnitude. We have postulated that the binding of cobalamins to haptocorrin involves hydrogen bonding of the side chain amides to acceptors in the protein binding pocket and that the consequent thermal immobilization of the acetamides is responsible for the observed stabilization. Hence, the thermolysis of alkylcobalamin analogs in which the hydrogen bonding ability of the upward directed side chains to the protein has been reduced should show reduced stabilization of the Co-C bond upon binding to haptocorrin. Such derivatives will include the a, c, and g N- methylamides and monocarboxylates, the C8 and C13 epimers (in which the d or e side chain projects upward) as well as other analogs which may yield altered steric interactions with the organic ligand. The importance of hydrogen bonding in the association of cobalamins with haptocorrin will be determined by studies of the thermodynamics of the binding reactions of cabalamins and their analogs from which specific hydrogen bond donors have been removed chemically. Additional evidence will be sought from 1H and 15N NMR studies of the complex of haptocorrin with cyanocobalamin uniformly enriched in 15N obtained by fermentation using Streptomyces griseus. These measurements will include polarization transfer and multiple quantum coherence spectroscopy as well as 15N- spectral edited 1H NMR. These experiments will permit observation of the chemical shifts of the amide nitrogens and protons as well as the proton chemical shift thermal gradients and H-D exchange rates.