MALIC ENZYME FROM ASCARIS SUUM

这项研究提案的主要目的是阐明 黄曲霉NAD-苹果酸酶的完整三维结构 寄生线虫猪蛔虫。随附于此结构解决方案 将是哺乳动物苹果酸酶的溶液。阐明了 苹果酸酶的结构将通过X射线结晶学进行测定， 圆二向色性和荧光。蛔虫酶的结晶 已经得到了衍射率为2.5埃的单位，我们有 获得了几个2.5-3.0埃单位的原始数据集。下一个 第一步是制造重金属衍生品，其中一种已经用铀酰制造 醋酸盐。剩下的将需要筹集至少两到三个 重金属衍生品，测定相，制备 可解释的电子密度图及其结构的求解 苹果酸酶。利用从X射线研究中获得的结构 将开展基于比较结构的药物设计研究 计算机程序与苹果酸酶的各种抑制剂对接。 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 用圆二色谱和荧光技术测定了酶的活性。通过 研究效应器带来的结构变化，这些 变化可以与X射线结构溶液相关联。一处地点- 还将利用克隆进行定向诱变研究 苹果酸酶及其表达系统已在 这个实验室。这项研究的目的有两个。第一，要 确定参与催化机制的氨基酸，以及 第二，鉴定与已鉴定的抑制物结合的氨基酸。 在药物设计研究中。体外诱变中的突变位点 系统将被用来产生突变酶。特定突变体 的氨基酸将在苹果酸结合区域中生成以 确定各种残基在催化序列中的作用。其他 突变体将由亚基相互作用中的氨基酸组成。 以确定该酶是否可以作为单体发挥作用。 最后，将在这些氨基酸中进行突变 结合抑制剂的药物设计研究以进一步了解 有约束力的。这些突变的酶也将通过循环研究 二色性和荧光来确定是否发生了任何变化 可以通过这些方法检测到的结构。此外, 晶体将由突变的酶组成，它们的结构可以 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.