Stereochemical Micromapping of Enzyme Active Sites

MCB 9406644 Speckhard该项目将提供有关激酶活性位点的详细信息，特别是有关酶表面与惰性二齿铬三磷酸腺苷（ATP）和磷酸烯醇丙酮酸（PEP）复合物（细胞镁复合物的类似物）中配位沃茨之间的接触。在缺乏详细晶体学信息的情况下，确定金属离子配位球是否参与酶对底物的识别的最佳方法是在所有可能的位置构建一系列具有单个氨的铬络合物。然后将这些络合物用作镁络合物的竞争性抑制剂。通过比较抑制作用，可以绘制出金属周围区域的活性位点图。这种微作图技术，最近已被证明为己糖激酶，将被应用于甘油激酶，腺苷酸激酶，丙酮酸激酶和其他。结合显微成像和X射线晶体结构研究，如腺苷酸激酶的X射线晶体结构研究，将有可能获得关于活性位点的更详细的信息。通过定点诱变修饰的酶的分析将补充微图。一种新的微作图工具，惰性的磷酸烯醇丙酮酸单氨铬络合物，将被开发和用于丙酮酸激酶的PEP位点的地图。惰性铬PEP本身将有助于科学家寻求使用EPR和NMR弛豫技术来研究使用PEP的酶。这项建议的一个重要特点是，它将主要涉及本科生在本科院校的工作。 %许多酶是非常特异的催化剂。了解酶选择性地与底物相互作用的能力是生物化学的首要任务之一。这种理解可以通过允许构建具有高选择性的有效抑制剂来促进农业和医学的进步。这是合理设计农药和除草剂以及合理药物设计的基础。该项目将有助于识别和仔细“映射”一个称为激酶的酶家族中的特定区域，这些酶在细胞内传递能量。 在激酶上的某些部位可以附着特殊的化合物。如果这些化学物质以稳定分子的形式牢固地附着在细胞上，它们将干扰细胞的正常功能。这些分子有效地扰乱了细胞的内部通讯，损害了它们的功能。这可以创造一种新的物质，也可以改变细胞的行为方式。例如，一些可能具有破坏性的细胞现在可以变得无害。本科生将学习用于识别这些网站的化学物质，并进行“映射”实验。 ***

MCB 9406644 Speckhard This project will provide detailed information about kinase active sites, specifically about the contacts between the enzyme surface and the coordinated waters in inert bidentate chromium adenosine triphosphate (ATP) and phosphoenolpyruvate (PEP) complexes which are analogs of the cellular magnesium complexes. In the absence of detailed crystallographic information, the best way to determine if the metal ion coordination sphere is involved in the recognition of the substrate by the enzyme is to construct a series of chromium complexes with a single ammonia in all possible locations. These complexes are then used as competitive inhibitors of the magnesium complexes. Comparison of the inhibitions allows a map of the active site in the region around the metal to be constructed. This micromapping technique, recently demonstrated for hexokinase, will be applied to glycerokinase, adenylate kinase, pyruvate kinase and others. The potential for even more detailed information about the active site will result from the combination of micromapping and the study of x-ray crystal structures like that of adenylate kinase. Analysis of enzymes modified by site directed mutagenesis will complement the micromaps. A new micromapping tool, inert monoamminechromium complexes of phosphoenol pyruvate, will be developed and used to map the PEP site in pyruvate kinase. Inert chromium PEP itself will be useful to scientists seeking to use EPR and NMR relaxation techniques to study enzymes that use PEP. An important feature of this proposal is that it will primarily involve work by undergraduate students at an undergraduate institution. %%% Many enzymes are remarkably specific catalysts. Understanding the ability of enzymes to selectively interact with substrates is one of the top priorities in biochemistry. This understanding can lead to advances in agriculture and medicine, by allowing construction of potent inhibitors with high selectivity. This is the basis of r ational pesticide and herbicide design and rational drug design. This project will help to identify and carefully 'map' specific areas in a family of enzymes called kinases, which transfer energy within the cell. There are certain sites on the kinases where special chemical compounds can be attached. If these chemicals, in the form of stable molecules, can be attached firmly, they will interfere with the way the cell normally functions. These molecules effectively scramble the cell's internal communications and compromise their ability to function. This can create a new substance or it can change the way a cell behaves. As an example, some cells that may have been destructive can now be rendered harmless. Undergraduate students will learn about the chemicals used to identify these sites and conduct 'mapping' experiments. ***