COBALT CHEMISTRY RELATED TO METHIONINE SYNTHASE FUNCTION

与蛋氨酸合酶功能相关的钴化学

• 批准号: 2175441
• 负责人: LUIGI G. MARZILLI
• 金额: $ 14.87万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-09-01 至 1996-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175441
• 关键词: Raman spectrometry; allosteric site; catalyst; chemical kinetics; chemical models; chemical structure function; circular dichroism; cobalt; cobamide; conformation; cysteine; electron spin resonance spectroscopy; enzyme substrate complex; metalloenzyme; methionine; methyltransferase; molecular site; nuclear magnetic resonance spectroscopy; nutrition related tag; thiols; transport proteins; vitamin B12; vitamin B12 coenzyme; vitamin analog

My long-term goals are to develop insightful and informative spectroscopic methods for assessing the dynamic processes involving cobamide cofactors (Cbas) bound to enzymes or transport proteins and to elucidate the fundamental chemistry relevant to function. Conformational changes in Cbas induced through cofactor-protein contacts and protein conformational changes are important in enzyme function. To achieve these goals, studies are proposed with enzymes, with derivatives of natural B12 compounds, and with models, including a novel pair of lariat-type models recently synthesized in my laboratory. Our focus has been on near-IR FT-Raman and on NMR spectroscopic methods. The progress we have made recently has applications to enzymes dependent on one of the Cbas, 5'- deoxyadenosylcobalamin (AdoCbl) or methylcobalamin (MeCbl). I conclude that during the requested renewal period our expertise in spectroscopy and model chemistry would be best applied to MeCbl-dependent methionine synthase (MS) and to elucidation of the interaction of thiols/thiolates with both model and B12 species. The MeCbl-binding domain of 133-kDa MS is included in a relatively small (<28 kDa) domain, which was recently crystallized. Such domain-proteins should give high-quality spectra, and it is possible that even smaller domain-proteins will be identified by the alternative methods of PCR-cloning-expression or by proteolytic digestion. Greater knowledge of MeCbl binding is essential in order to eventually understand MS function. Several approaches are proposed to relate the binding in the more readily characterized domain-proteins to that in the MS holoenzyme. These approaches include FT-Raman, CD and 31P and 19F NMR, photolysis of the bound Cbl analogues, spin labels, propionamide-modified analogues, and novel cleaving/probe analogues. Results on both domains and holoenzyme will be compared. Spectroscopic and chemical studies of Cbl- and Cbl-analogue-binding to these domains should allow the development of methods for examining the more complex and larger AdoCbl-dependent enzymes. The involvement of thiols or thiolates in MS function has one clear and another suspected, unknown role. The clear role involves the substrate, homocysteine, which is methylated by MeCbl. A cysteine, (probably C772, the only cysteine in the binding domain), is protected from thiol reagents by Cbl binding. This observation, along with evidence that cysteines are essential in many AdoCbl-dependent enzymes, implicates a cysteine in MS function. Furthermore, by some isolation procedures, sulfitoCbl was isolated from MS. Finally, the form of B12 that is the precursor to coenzyme formation is glutathioneCbl. There is a clear need to understand thiol/thiolate-Cbl interactions. However, our knowledge of thiol/thiolate organocobalt chemistry is rudimentary; the few published studies are contradictory and complex. The high air sensitivity of reduced B12 and model species, combined with the normal difficulties of thiol/thiolate redox transition metal chemistry, has retarded progress in this field. Numerous experiments are proposed with Cbl-derivatives and synthetic models to elucidate this fundamental organocobalt chemistry.

我的长期目标是开发有洞察力和信息丰富的光谱 评估涉及钴酰胺辅因子的动态过程的方法 （Cbas）与酶或转运蛋白结合并阐明 与功能相关的基础化学。 Cbas构象变化 通过辅因子-蛋白质接触和蛋白质构象诱导 变化对于酶的功能很重要。 为了实现这些目标，研究 建议使用酶、天然 B12 化合物的衍生物，以及 模型，包括最近一对新颖的套索型模型 在我的实验室合成的。 我们的重点是近红外 FT-拉曼和 核磁共振波谱方法。 我们最近取得的进展 依赖于 Cbas 之一的酶的应用，5'- 脱氧腺苷钴胺素 (AdoCbl) 或甲钴胺 (MeCbl)。 我得出结论 在所要求的更新期内，我们在光谱学方面的专业知识和 模型化学最适用于 MeCbl 依赖性蛋氨酸 合成酶 (MS) 并阐明硫醇/硫醇盐的相互作用 具有模型和 B12 物种。 133-kDa MS 的 MeCbl 结合域是 包含在一个相对较小（<28 kDa）的结构域中，该结构域最近被 结晶。 这样的结构域蛋白质应该提供高质量的光谱，并且 甚至更小的结构域蛋白也可能被识别 PCR 克隆表达或蛋白水解消化的替代方法。 为了最终实现对 MeCbl 结合的更多了解至关重要 了解 MS 功能。 提出了几种方法来联系 更容易表征的结构域蛋白与 MS 中的蛋白结合 全酶。 这些方法包括 FT-拉曼、CD 以及 31P 和 19F NMR， 结合的 Cbl 类似物、自旋标记、丙酰胺修饰的光解 类似物，以及新颖的切割/探针类似物。 两个领域的结果 将比较全酶。 Cbl- 的光谱和化学研究 和 Cbl 类似物结合到这些域应该允许开发 检查更复杂和更大的 AdoCbl 依赖性酶的方法。 硫醇或硫醇盐参与 MS 功能有一个明确且明确的事实： 另一个可疑的未知角色。 明确的作用涉及底物， 同型半胱氨酸，被 MeCbl 甲基化。 半胱氨酸，（可能是 C772， 结合域中唯一的半胱氨酸），免受硫醇试剂的影响 通过 Cbl 结合。 这一观察结果以及半胱氨酸的证据 在许多 AdoCbl 依赖性酶中必不可少，暗示 MS 中存在半胱氨酸 功能。 此外，通过一些分离程序，sulfitoCbl 从 MS 中分离出来。 最后，B12 的形式是 形成的辅酶是谷胱甘肽Cbl。 有一个明确的需要了解 硫醇/硫醇盐-Cbl 相互作用。 然而，我们对硫醇/硫醇盐的了解 有机钴化学还很初级；少数发表的研究是 矛盾且复杂。 还原 B12 和的高空气敏感性 模型物种，结合硫醇/硫醇盐的正常困难 氧化还原过渡金属化学，阻碍了该领域的进展。 使用 Cbl 衍生物和合成模型进行了大量实验 阐明这一基本的有机钴化学。