MALIC ENZYME FROM ASCARIS SUUM

这项研究的主要目的是阐明 完整的三维结构的NAD-苹果酸酶从 寄生线虫，猪蛔虫。 该结构解决方案的服务员 将是哺乳动物苹果酸酶的溶液 阐明 苹果酸酶的结构将通过X射线晶体学来完成， 圆二色性和荧光性。 蛔虫酶的晶体 已经获得了它的单位为2.5埃，我们有 获得了2.5 - 3.0埃单位的几个天然数据集。 下一 第一步是制造重金属衍生物，一个是用铀酰制造的， 醋酸 剩下的就需要收集至少两三个 重金属衍生物，物相测定，制备 可解释的电子密度图和解决的结构， 苹果酸酶 利用从X射线研究中获得的结构， 将进行基于结构的药物设计比较研究， 计算机程序DOCK和苹果酸酶的各种抑制剂。 这项研究还将涉及哺乳动物的结构， 苹果酸酶 我们还将对苹果酸的结构进行研究 酶的圆二色谱和荧光技术。 通过 通过研究效应物带来的结构变化， 变化可以与X射线结构解决方案相关联。 一个地盘─ 还将使用克隆进行定向诱变研究， 苹果酸酶和表达系统， 这个实验室。 本研究的目的有两个方面。 一是 鉴定参与催化机制的氨基酸，和 第二，表征与所鉴定的抑制剂结合的氨基酸， 在药物设计研究中。 体外突变的位点改变 系统将用于产生突变酶。 特异性突变体 将在苹果酸结合结构域中产生氨基酸， 确定催化序列中各种残基的作用。 其他 突变体将由亚基相互作用中的氨基酸组成 结构域，以确定酶是否可以作为单体发挥作用。 最后，突变体将在那些由氨基酸序列鉴定的氨基酸中产生。 结合抑制剂的药物设计研究，以进一步了解 约束力 这些突变酶也将通过循环研究。 二色性和荧光，以确定是否发生了任何变化， 该结构可以通过所述方法检测。 此外，本发明还提供了一种方法， 晶体将由突变酶组成，它们的结构可以是 通过分子置换来解决。

The primary objective of this research proposal is to elucidate the complete three-dimensional structure of the NAD-malic enzyme from the parasitic nematode, Ascaris suum. Attendant to this structure solution will be the solution of the mammalian malic enzyme. The elucidation of the structure of malic enzyme will be done by X- ray crystallography, circular dichroism, and fluorescence. Crystals of the ascarid enzyme have been obtained which diffract to 2.5Angstrom units and we have obtained several native data sets at 2.5-3.0Angstrom units. The next step is to make heavy metal derivatives, one has been made with uranyl acetate. The remainder will entail collecting at least two or three heavy metal derivatives, determining the phases, preparing an interpretable electron density map and solving the structure of the malic enzyme. Utilizing the structure obtained from X-ray studies we will carry out a comparative structure-based drug design study utilizing the computer program DOCK and various inhibitors of the malic enzyme. This study will also involve working with the structure of the mammalian malic enzyme. We will also carry out a study on the structure of malic enzyme by the techniques of circular dichroism and fluorescence. By studying the change in structure brought about by effectors, these changes can be correlated with the X-ray structure solution. A site- directed mutagenesis study will also be carried out using the clone for the malic enzyme and the expression system which has been developed in this laboratory. The purpose of this study is two-fold. First, to identify amino acids that are involved in the catalytic mechanism, and second, to characterize amino acids that bind to inhibitors identified in the drug design studies. The Altered Sites in vitro mutagenesis system will be used to generate the mutant enzymes. Specific mutants of amino acids will be generated in the malate binding domain to determine the role of various residues in the catalytic sequence. Other mutants will be made of amino acids that are in the subunit interaction domain in order to ascertain if the enzyme can function as a monomer. Finally, mutants will be made in those amino acids identified by the drug design studies which bind inhibitors in order to further understand binding. These mutant enzymes will also be studied by circular dichroism and fluorescence to determine if any changes have occurred in the structure that can be detected by the methods. In addition, crystals will be made of the mutant enzymes and their structures can be solved by molecular replacement.