METALLOBIOCHEMISTRY OF METHIONINE SYNTHASE

人类高水平的同型半胱氨酸被认为是导致心血管疾病的一个因素。蛋氨酸合成酶，一种依赖甲基钴胺的酶，将同型半胱氨酸转化为蛋氨酸；因此，这种酶是控制人类同型半胱氨酸水平的关键。本提案旨在提供详细的化学先例和酶的研究，集中在酶的一个方面，组氨酸和钴胺素在活性位点的协调。在蛋氨酸合酶中，甲基钴胺中钴的第六个配位体不是附加的5,6-二甲基苯并咪唑碱，而是由酶提供的组氨酸/咪唑残基。对于组氨酸轴向碱基的作用，一个有效的假设是质子化/去质子化循环，结合不同的Co-N键长度，对提高催化活性很重要。该提议通过结合化学先例和酶的研究来验证这一假设，这些研究旨在改变轴基的质子化状态和Co-N键的长度。甲基钴胺（甲基钴胺的无碱类似物）在外源轴基存在下的热力学和动力学研究将在溶液中完成。然后在溶液中完成有外源轴基和没有外源轴基的甲基甲酰胺动力学研究。然后在溶液中完成有外源轴碱和无外源轴碱的甲基甲酰胺动力学研究。然后在酶系统本身中完成有外源轴基和无外源轴基甲基甲酰胺的动力学研究，直接探讨轴基在蛋氨酸合酶中的作用。

High levels of homocysteine in humans have been implicated as a factor contributing to cardiovascular disease. Methionine synthase, a methylcobalamin-dependent enzyme, converts homocysteine to methionine; as such, this enzyme is key in controlling the level of homocysteine in humans. This proposal aims to provide detailed chemical precedent and enzymatic studies focusing on one aspect of the enzyme, the coordination of histidine to cobalamin in the active site. In methionine synthase, the sixth coordinating ligand to cobalt in methylcobalamin is not the appended 5,6-dimethylbenzimidazole base, but is a histidine/imidazole residue supplied by the enzyme. A working hypothesis for the purpose of the histidine axial base is that a protonation/deprotonation cycle, in combination with varying Co-N bond lengths, is important in enhancing catalytic activity. This proposal test the hypothesis by combining chemical precedent and enzymatic studies which are designed to vary the protonation state of the axial base and the length of the Co-N bond. Thermodynamic and kinetic studies of methylcobinamide (the base-free analog of methylcobalamin) in the presence of an exogenous axial base will be completed in solution. Then kinetic studies of methylcobinamide with and without an exogenous axial base will be complete with and without an exogenous axial base will be completed in solution. Then kinetic studies of methylcobinamide with and without an exogenous axial base will be completed in solution. Then kinetic studies of methylcobinamide with and without an exogenous axial base will be complete in the enzymatic system itself, directly probing the role of the axial base in methionine synthase.