MODULATION OF ORGANOCOBALT REACTIVITY BY HAPTOCORRIN

本研究项目的最终目标是开发一种详细的 了解辅酶形式的维生素B12是如何通过 需要辅酶B12的碳钴键均裂。近期 研究表明，热不稳定的烷基钴胺类化合物的均解 (苄基和新戊基钴胺)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，一种机制 用于5‘-脱氧腺苷钴胺的酶激活，其中 酶增强5‘-脱氧腺苷配体的空间相互作用 带有乙酰胺侧链的化合物将变得很有吸引力。建议数 这项研究被指定用来严格检验碳 这种烷基钴胺中的钴键均裂是空间位阻的结果 乙酰胺与有机配体之间的相互作用。 这一假设将通过两种类型的研究来检验。首先， 空间体烷基钴胺类似物的热均解 和/或向上突出的侧链的数量已经改变 将对其进行化学研究。第二，乙酰胺方面的重要性 碳-钴键均解的链迁移率将通过 利用我们最近观察到的一种B12结合蛋白 鸡血清中的(触角蛋白)稳定碳-钴键 热不稳定的烷基钴胺大约三个数量级。我们 已经推测钴蛋白与触角蛋白的结合包括 蛋白质中侧链酰胺与受体的氢键作用 以及随之而来的热固定化 乙酰胺是导致观察到的稳定的原因。因此， 烷基钴胺类似物的热分解，其中氢键 向上定向的侧链对蛋白质的能力已经被 还原应显示结合时Co-C键的稳定性降低 为了高氯酸乙酯。这些衍生工具将包括a、c和g N- 甲胺和单羧酸盐，C8和C13同分异构体(其中 D或E侧链向上突出)以及可 产生改变的空间相互作用与有机配体。这个 氢键在钴胺与蛋白质结合中的重要性 通过对胡敏素的热力学研究，将确定其含量。 卡巴拉明及其类似物的结合反应 氢键供体已被化学移除。其他证据 将从~1H和~(15)N核磁共振研究中寻找到的目标化合物。 用发酵法获得均匀富含15N的氰钴胺 使用灰色链霉菌。这些测量将包括偏振 转移谱和多量子相干谱以及15N- 波谱编辑的~1H核磁共振。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、温度梯度和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.