HOW DO ENZYMES GENERATE AND CONTROL FREE RADICALS

描述：该提案涉及腺苷钴胺素（辅酶A）的研究 B-12）依赖性谷氨酸异构化为3-甲基天冬氨酸 谷氨酸盐作为一个模型系统，研究两个基本方面， 酶介导的自由基催化：i）酶如何产生自由基，以及 ii）如何去除氢气，这是基底活化的关键步骤， 催化？ 腺苷钴胺素作为腺苷自由基的来源， 弱钴碳键均裂。 当被酶结合时， 组氨酸残基与钴-碳反轴配位 键;组氨酸，反过来，参与氢键与一个 天冬氨酸残基。 假设Co-His-Asp三联体是 在辅酶的结合及其活化中重要， 均裂 突变组氨酸对酶的能力的影响， 将研究切割钴-碳键。 组氨酸的pK 将通过NMR和辅酶结合的pH依赖性来测量， 催化剂将被检查。 天冬氨酸的突变体将被制成 确定它们对组氨酸pK的影响，并研究是否改变 与酶活性和/或辅酶结合的变化相关。 在谷氨酸盐还原反应中，底物被激活， 当氢原子被腺苷基除去时的重排; 重排，氢被转移回来得到产物并再生 腺苷自由基 氚化底物和辅酶将用于 停流实验，以测量氚在 底物、辅酶和产物。 同位素效应将是 确定了这些步骤，因此确定了氢转移的速率。 通过 结合这些结果与稳态动力学测量，一个免费的 将为完全反应构建能量分布。 将尝试捕获假定的反应中间体以进行测试 关于底物重排的机理假说 激进 将启动X射线晶体学和蛋白质NMR研究， 阐明酶的三维结构。

DESCRIPTION: This proposal involves study of adenosylcobalamin (coenzyme B-12) dependent isomerization of glutamate to 3-methylaspartate catalyzed by glutamate mutase as a model system to investigate two fundamental aspects of enzyme-mediated radical catalysis: i) how do enzymes generate radicals, and ii) how is the removal of hydrogen, the key step in substrate activation, catalyzed? Adenosylcobalamin serves as a source of adenosyl radical that is unmasked by homolysis of its weak cobalt-carbon bond. When bound by the enzyme, a histidine residue coordinates cobalt trans-axially to the cobalt-carbon bond; the histidine, in turn, participates in a hydrogen bond with an aspartate residue. It is hypothesized that the Co-His-Asp triad is important in the binding of the coenzyme and its activation towards homolysis. The effect of mutating the histidine on the enzyme's ability to cleave the cobalt-carbon bond will be investigated. The pK of the histidine will be measured by NMR and the pH dependence of coenzyme binding and catalysis will be examined. Mutants of the aspartate will be made to determine their effect on histidine pK, and to investigate whether changes in pK correlate with changes in enzyme activity and/or coenzyme binding. In the glutamate mutase reaction, the substrate is activated towards rearrangement when a hydrogen atom is removed by the adenosyl radical; after rearrangement, hydrogen is transferred back to give product and regenerate adenosyl radical. Tritiated substrates and coenzyme will be used in stopped-flow experiments to measure the rates of tritium transfer between substrate, coenzyme and product in both directions. Isotope effects will be determined for these steps and hence the rates for hydrogen transfer. By combining these results with steady state kinetic measurements, a free energy profile will be constructed for the complete reaction. An attempt will be made to trap putative reaction intermediates to test mechanistic hypotheses concerning the rearrangement of the substrate radical. X-ray crystallography and protein NMR studies will be initiated to elucidate the three-dimensional structure of the enzyme.