SPECIFICITIES OF IDH AND IMDH

这个项目的目标是确定如何互动之间的积极 位点氨基酸残基和各种配体部分有助于酶 特异性和亲和力。 对这种相互作用的理解 关键的治疗药物的设计针对蛋白质， 已知的结构，通过工程酶设计新型催化剂， 对催化作用的理解，对蛋白质的理解 进化 该提案描述了两种用于生产酶的一般方法， 改变了特性。 一种方法使用定点诱变， 将特定序列和超二级结构替换为 相关酶 第二种方法模拟了长期的 通过将构建的E.大肠杆菌 由恒化器培养施加的强烈选择压力。 这些 这些方法，无论是单独使用还是联合使用， 特异性改变了 进化/突变的定点诱变 使用底物和底物类似物的酶和动力学研究， 然后将用于确定特异性变化的原因。 利用自然选择产生特异性改变的酶 是特别有吸引力的，因为没有先验的决定， 需要针对残留物进行研究。 因此， 发现特异性的不可预见的决定因素被最大化。 还有， 突变赋予生长率差异小至1.0%， 使用培养皿上的选择不能检测到的代， 在恒化器培养中可检测到。 因此，恒化器竞争实验 提供了一种通过小的增量来进化特异性的方法。 大肠杆菌异柠檬酸脱氢酶（IDH）及其相关酶 异丙基苹果酸脱氢酶（IMDH）将用作模型系统。 这些酶催化2 R-2 R的氧化脱羧反应。 苹果酸核心共同的他们的基板。 活性位点之间的相互作用 每种酶的残基和底物的各种γ-部分， 其附接在公共芯的3S位置处， 这是一种产生特异性的明显方法。 高分辨率结构 天然的和磷酸化的IDH，以及与 异柠檬酸盐和NADP。 还提供高 嗜热栖热菌IMDH的解析结构。 这些，一起 随着我们对动力学和催化机制的理解， 磷酸化调节IDH活性的方法，提供了基本的 详细解释结构-功能所需的信息 关系 制定新的一般性方法，以获得对具体问题的答案， 配体-蛋白质结合问题，并且不考虑预先设想的 实验主义者的观念，对于发现 不可预见的决定因素影响的亲和力和特异性， proteins. 这种方法的发展也将提供更大的 合理设计药物和新型生物制剂所需的见解 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.