CRYSTALLOGRAPHIC STUDIES OF PROTEIN STRUCTURE/FUNCTION

描述：这项建议的总体目标是了解 镁离子在糖类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是如何 再加上金属蛋白的功能，而且稳定。为了实现这些目标 他们将使用他们在之前的预算中开发的新方法 周期，包括结晶学和时间分辨结晶学。 在整个项目中有三个相互依赖的子项目。 第一个目标是继续木糖异构酶的研究。调查人员将 初步确定E-S复合体的结构，以推断其作用 镁离子对开环反应的催化作用。到时候他们就会 用组合量子方法模拟整个催化过程 力学/分子力学，了解独特的催化性能 桥式双金属中心。第二个项目的重点是 镁在3-异丙基苹果酸脱氢酶催化下的反应。 研究人员将结合使用X射线结晶学和遗传学 选择绘制热稳定性、蛋白质之间的相关性图 这种必需氨基酸的灵活性和金属依赖性活性 合成酶。最后，他们将确定结构和 甘露糖-6-磷酸双功能异构酶的催化机理 铜绿假单胞菌。这种酶催化两个不连续的反应。 在藻酸盐生产中：这两个反应都需要镁离子。三个都是 酶易受低温或劳厄结晶学的影响，因此 瞬时的E-S络合物和反应中间体可以被捕获并 在高分辨率下研究，或者是单一底物/单一产物酶， 在这里可以直接观察到生产性的米氏复合体 平衡条件下的结晶。

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.