CRYSTALLOGRAPHIC STUDIES OF PROTEIN STRUCTURE/FUNCTION

描述：本提案的总体目标是了解 镁离子在碳水化合物加工催化机理中的作用 金属酶 研究人员还希望了解蛋白质动力学如何， 与金属蛋白的功能和稳定性相结合。 为实现这些目标 他们将使用他们在以前的预算中开发的新方法， 时间分辨结晶学（time resolved crystallography）。 在这一总体项目中有三个相互依存的分项目。 第一个目的是继续木糖异构酶的研究。 调查人员将 确定初始E-S复合物的结构，以推断 镁离子催化开环反应。 然后他们将 用组合量子化学模拟整个催化过程 力学/分子力学，了解独特的催化性能 桥接的中心。 第二个项目的重点是 3-异丙基苹果酸脱氢酶催化的反应中的镁。 研究人员将使用X射线晶体学和遗传学的结合， 选择映射热稳定性、蛋白质 这种必需氨基酸的弹性和金属依赖性活性 合成酶 最后，他们将确定结构， 双功能甘露糖-6-磷酸异构酶的催化机理 绿脓杆菌。 这种酶催化两种不相邻的反应 在藻酸盐生产中：两种反应都需要镁离子。 所有三 酶适合低温或劳厄晶体学，因此， 可以捕获瞬时E-S复合物和反应中间体， 以高分辨率研究，或者是单一底物/单一产物酶， 其中生产性米氏络合物可以直接在 在平衡条件下结晶。

DESCRIPTION: The overall objective of this proposal is to understand the role of magnesium ion in the catalytic mechanisms of carbohydrate-processing metalloenzymes. Investigators also wish to learn how protein dynamics if coupled to metalloprotein function, and stability. To achieve these aims they will use novel methods that they have developed over previous budget periods, including cryocrystallography and time-resolved crystallography. There are three interdependent sub-projects within this overall project. The first aims to continue studies of xylose isomerase. Investigators will determine the structure of the initial E-S complex to deduce the role of the magnesium ions in catalysis of the ring-opening reaction. They will then simulate the entire catalytic pathway by combined quantum mechanics/molecular mechanics, to understand the unique catalytic properties of bridged bimetallic centers. The second project focuses on the role of magnesium in the reaction catalyzed by 3-isopropylmalate dehydrogenase. Investigators will use a combination of X-ray crystallography and genetic selection to map the correlations between thermal stability, protein flexibility, and metal-dependent activity in this essential amino-acid synthesizing enzyme. Finally, they will determine the structure and catalytic mechanism of the bifunctional mannose-6-phosphate isomerase from Pseudomonas aeruginosa. This enzyme catalyzes two non-contiguous reactions in alginate production: both reactions require magnesium ion. All three enzymes are amenable to low temperature or Laue crystallography, so that transient E-S complexes and reaction intermediates can be trapped and studied at high resolution, or are single substrate/single product enzymes, where the productive Michaelis complex can be observed directly in the crystal under equilibrium conditions.