SPECIFICITIES OF IDH AND IMDH

这个项目的目标是确定活动之间的相互作用 位点氨基酸残基和各种配基部分对酶有贡献 专一性和亲和力。对这种相互作用的理解是 对靶向蛋白的治疗药物的设计至关重要 已知的结构，到通过工程酶设计新型催化剂， 为了理解催化作用，为了理解蛋白质 进化论。 本提案描述了两种生产酶的一般方法。 改变了具体情况。一种方法是使用定点突变来 将特定的序列和超二级结构替换为 相关的酶。第二种方法模拟了长期的 将构建的大肠杆菌菌株置于 恒化器培养施加的强烈的选择压力。这些 无论是单独的还是联合使用的方法，都会产生酶。 有不同的细节。进化/突变的定点突变 使用底物和底物类似物的酶和动力学研究， 然后将被用来确定变化的原因的特殊性。 利用自然选择产生特异性改变的酶 特别有吸引力，因为没有关于哪一个的先验决定 残留物到靶点进行研究是必要的。因此，有可能 发现不可预见的特异性决定因素被最大化。另外， 导致增长率差异小至1.0%的突变 使用培养皿上的选择不能检测到的产生 在恒化器培养中可检测到。因此，恒化器竞争实验 提供一种方法，通过小的增量来发展专一性。 大肠杆菌异柠檬酸脱氢酶(IDH)及其相关酶 以苹果酸异丙酯脱氢酶(IMDH)为模型体系。 这些酶在2R-上催化氧化脱羧基反应。 它们的底物共有的苹果酸盐核。活动站点之间的相互作用 每种酶的残基和底物的各种伽马部分， 其附接在公共核心的3S位置，提供了 产生特异性的明显手段。的高分辨率结构 天然的和磷酸化的IDH，以及与 有异柠檬酸和NADP可供选择。此外，还有一款高 嗜热嗜热菌IMDH的拆分结构。这些加在一起 随着我们对动力学和催化机理的了解，以及 磷酸化调节IDH活性的方式，提供了基本的 详细解释结构-功能所需的信息 关系。 开发新的通用方法来获得具体问题的答案 配体-蛋白质结合问题，而不考虑先入为主的问题 实验者的概念，对于发现其他方式是至关重要的 不可预见的决定因素影响着 蛋白质。这种方法的发展也将提供更大的 合理设计药物和新生物所需的洞察力 催化剂，以及对催化和蛋白质进化的理解。

The goal of this project is to determine how interactions between active site amino acid residues and various ligand moieties contribute to enzyme specificity and affinity. An understanding of such interactions is pivotal to the design of therapeutic drugs targeted towards proteins of known structure, to the design of novel catalysts by engineering enzymes, to an understanding of catalysis, and to an understanding protein evolution. This proposal describes two general approaches for producing enzymes with changed specificities. One approach uses site directed mutagenesis to replace specific sequences and super secondary structures with those of related enzymes. A second approach mimics the processes of long-term adaptive evolution by placing constructed strains of E. coli under the intense selective pressures imposed by chemostat culture. These approaches, either independently or in combination, will yield enzymes with altered specificities. Site directed mutagenesis of evolved/mutated enzymes and kinetic studies using substrates and substrate analogues, will then be employed to determine the causes of changes in specificity. The use of natural selection to produce enzymes of changed specificity is particularly attractive because no a priori decisions regarding which residues to target for study are necessary. Hence the chances of discovering unforeseen determinants of specificity are maximized. Also, mutations conferring differences in growth rates as small as 1.0% per generation, not detectable using selection on petri plates, are readily detectable in chemostat culture. Thus, chemostat competition experiments provide a means to evolve specificity by small increments. The isocitrate dehydrogenase (IDH) of Escherichia coli and the related isopropylmalate dehydrogenase (IMDH) will be used as a model system. These enzymes catalyze an oxidative decarboxylation reaction at the 2R- malate core common to their substrates. Interactions between active site residues of each enzyme and the various gamma-moieties of the substrates, which are attached at the 3S position of the common core, provide an obvious means to generate specificity. High resolution structures of native and phosphorylated IDH, and of the binary complexes with isocitrate and with NADP, are available. Also available is a high resolution structure of IMDH from Thermus thermophilus. These, together with our understanding of the kinetic and catalytic mechanisms and the means by which phosphorylation regulates IDH activity, provide the basic information necessary for detailed interpretations of structure-function relations. The development of new general approaches to obtain answers to specific ligand-protein binding problems, and without regard for the preconceived notions of the experimentalist, is crucial to discovering how otherwise unforeseen determinants influence the affinities and specificities of proteins. The development of such approaches will also provide greater insights necessary for the rational design of drugs and novel biological catalysts, and an understanding of catalysis and protein evolution.