MALIC ENZYME FROM ASCARIS SUUM

本研究计划的主要目的是阐明 NAD-苹果酸酶的完整三维结构 寄生线虫，猪蛔虫。 伴随着这个结构的解决方案 将是哺乳动物苹果酸酶的溶液。 的阐明 苹果酸酶的结构将通过X射线晶体学来确定， 圆二色性和荧光。 蛔虫酶晶体 已经获得了 2.5 埃单位的衍射，我们有 获得了几个2.5-3.0埃单位的原始数据集。 下一个 步骤是制造重金属衍生物，其中一种是用铀酰制成的 醋酸盐。 其余部分将需要收集至少两到三个 重金属衍生物，确定物相，制备 可解释的电子密度图并求解结构 苹果酸酶。 利用从 X 射线研究中获得的结构，我们 将利用基于比较结构的药物设计研究 计算机程序 DOCK 和各种苹果酸酶抑制剂。 这项研究还将涉及哺乳动物的结构 苹果酸酶。 我们还将对苹果的结构进行研究 酶的圆二色性和荧光技术。 由 研究效应器带来的结构变化，这些 变化可以与X射线结构解相关。 一个网站—— 还将利用该克隆进行定向诱变研究 苹果酸酶及其表达系统 这个实验室。 这项研究的目的有两个。 首先，要 识别参与催化机制的氨基酸，以及 其次，表征与已识别的抑制剂结合的氨基酸 在药物设计研究中。 改变位点体外诱变 系统将用于产生突变酶。 特定突变体 的氨基酸将在苹果酸结合域中产生 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的氨基酸组成 域以确定酶是否可以作为单体发挥作用。 最后，将在由基因组鉴定的那些氨基酸中产生突变体 结合抑制剂的药物设计研究，以便进一步了解 绑定。 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任何变化 可以通过该方法检测的结构。 此外， 晶体将由突变酶制成，其结构可以是 通过分子置换解决。

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.