HOW DO ENZYMES GENERATE AND CONTROL FREE RADICALS

描述：本提案涉及腺苷钴胺(辅酶)的研究 B-12)催化谷氨酸异构化为3-甲基天冬氨酸 谷氨酸变位酶作为模型系统研究两个基本方面 酶介导的自由基催化：I)酶是如何产生自由基的，以及 二)底物活化的关键步骤--氢的去除情况如何， 催化的？ 腺苷钴胺是腺苷自由基的来源，它被 它的弱钴-碳键的均解。当被酶结合时，一个 组氨酸残基与钴碳横向配位 键；组氨酸反过来又参与与一个 天冬氨酸残留物。据推测，CoHis-Asp黑社会 在辅酶的结合和激活中起重要作用 同种溶血。组氨酸突变对酶活性的影响 将对钴-碳键进行研究。组氨酸的PK 将通过核磁共振和pH依赖的辅酶结合和 我们将考察其催化作用。天冬氨酸的突变体将被制造成 确定它们对组氨酸PK的影响，并调查它们的变化 在PK中与酶活性和/或辅酶结合的变化相关。 在谷氨酸变位酶反应中，底物被激活 重排：当氢原子被腺苷自由基移走时；之后 重排，氢被转移回来产生产物并再生 腺苷自由基。氚底物和辅酶将用于 用停流实验测量氚在不同气体之间的转移速率 底物、辅酶和产物都是双向的。同位素效应将是 为这些步骤确定的，从而确定氢转移的速率。通过 将这些结果与稳态动力学测量相结合，自由 将构建完整反应的能量分布。 将尝试捕获假定的反应中间体以进行测试 关于底物重排的机械假说 激进的。X射线结晶学和蛋白质核磁共振研究将启动 阐明该酶的三维结构。

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.