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见地和信息丰富的光谱学 评估涉及共胺辅助因子的动态过程的方法 （CBA）与酶或转运蛋白结合并阐明 与功能相关的基本化学。 CBA的构象变化 通过辅助蛋白接触和蛋白质构象诱导 变化在酶功能中很重要。 为了实现这些目标，研究 用酶，具有天然B12化合物的衍生物，并提出 使用模型，包括新型的报告型型模型 在我的实验室中合成。 我们的重点一直放在近乎ft-raman和 在NMR光谱法上。 我们最近取得的进展 依赖于CBA的酶的酶应用5'-- 脱氧腺苷胆质（ADOCBL）或甲基钴胺素（MECBL）。 我得出结论 在要求的续签期间，我们在光谱和 模型化学将最好应用于MECBL依赖性蛋氨酸 合成酶（MS）和阐明硫醇/硫醇酯的相互作用 与型号和B12物种一起。 133 kDa MS的MECBL结合域是 包括在一个相对较小的（<28 kDa）的域中，最近是 结晶。 这样的域蛋白质应给出高质量的光谱，并且 甚至可能会通过 PCR粘结表达或通过蛋白水解消化的替代方法。 对MECBL结合的更多了解对于最终 了解MS功能。 提出了几种方法来联系 在更容易表征的域 - 蛋白质中绑定到MS中的蛋白质 全酶。 这些方法包括FT-Raman，CD和31p和19f NMR， 绑定的CBL类似物，自旋标签，丙酰胺修饰的光解 类似物和新颖的分裂/探针类似物。 在两个领域和 将比较全酶。 CBL-的光谱和化学研究 以及这些领域的cbl-Analogue结合应允许开发 检查更复杂和较大的依赖性酶的方法。 硫醇或硫醇在MS功能中的参与具有一个清晰的，并且 另一个怀疑，未知的角色。 明确的作用涉及基材， 同型半胱氨酸，由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）受到保护免受硫醇试剂的保护 通过CBL结合。 这种观察结果以及证据表明半胱氨酸是 在许多依赖ADOCBL的酶中必不可少的酶，这意味着MS中的半胱氨酸 功能。 此外，通过某些隔离程序，硫酸硫磺是 与MS分离。 最后，B12的形式是前体 辅酶形成是谷胱甘肽。 显然需要了解 硫醇/硫代硫酸盐-CBL相互作用。 但是，我们对硫醇/硫醇酸盐的了解 Organocobalt化学是基本的。少数发表的研究是 矛盾和复杂。 降低的B12和 模型物种，结合硫醇/硫代酸盐的正常困难 氧化还原过渡金属化学在该领域的进展迟缓。 通过CBL衍生物和合成模型提出了许多实验 阐明这种基本的有机体化学。