COBALT CHEMISTRY RELATED TO METHIONINE SYNTHASE FUNCTION

我的长期目标是发展有洞察力和信息丰富的光谱 用于评估涉及钴酰胺辅因子的动态过程的方法 （Cbas）结合酶或转运蛋白，并阐明 与功能相关的基础化学。 Cbas的构象变化 通过辅因子-蛋白质接触和蛋白质构象 酶功能的变化很重要。 为了实现这些目标，研究 与酶、天然B12化合物的衍生物一起提出， 与模型，包括一对新的拉里亚特型模型最近 在我的实验室里合成的 我们的重点是近红外FT-Raman， 核磁共振光谱法。 我们最近取得的进展 应用于依赖于Cbas之一的酶，5 '- 脱氧腺苷钴胺素（Methylcobalamin，MECbl）。 我结束发言 在要求的续约期间，我们在光谱学方面的专业知识， 模型化学最适用于MeCbl依赖性蛋氨酸 合成酶（MS）和阐明硫醇/硫醇酯的相互作用 与模型和B12物种。 133-kDa MS的MeCbl结合结构域是 包括在一个相对较小（<28 kDa）的结构域，这是最近 结晶 这样的结构域蛋白质应该给出高质量的光谱， 甚至更小的结构域蛋白质也有可能被鉴定出来， PCR-克隆-表达或通过蛋白水解消化的替代方法。 更多地了解MeCbl结合是必不可少的，以便最终 了解MS的功能。 提出了几种方法， 在更容易表征的结构域蛋白质中与MS中的结合 全酶 这些方法包括FT-拉曼，CD和31 P和19 F NMR， 结合的Cbl类似物的光解，自旋标记，丙酰胺修饰的 类似物和新的切割/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 与这些结构域结合的Cbl类似物应该允许开发 用于检测更复杂和更大的BBC 1依赖性酶的方法。 硫醇或硫醇盐在MS功能中的参与具有一个明确的和 另一个可疑的未知角色 明确的作用涉及基质， 同型半胱氨酸，其被MeCbl甲基化。 半胱氨酸（可能是C772， 结合结构域中唯一的半胱氨酸）被保护免受硫醇试剂的影响 CBL绑定。 这一观察结果，沿着半胱氨酸是 在许多依赖于半胱氨酸的酶中是必需的，暗示了MS中的半胱氨酸 功能 此外，通过一些分离程序， 最后，B12的形式是前体， 辅酶形成是谷胱甘肽Cbl。 很明显我们需要了解 硫醇/硫醇盐-Cbl相互作用。 然而，我们对硫醇/硫醇盐的了解 有机钴化学是基本的;少数发表的研究是 矛盾而复杂。 降低B12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，阻碍了这一领域的进展。 提出了大量的实验与Cbl衍生物和合成模型 来阐明这种基本的有机钴化学。

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.