SPECIFICITIES OF IDH AND IMDH

IDH 和 IMDH 的特殊性

• 批准号: 2536015
• 负责人: Antony M. DEAN
• 金额: $ 8.1万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2536015
• 关键词: Archaea; Escherichia coli; NAD(P)H dehydrogenase; active sites; alcohol dehydrogenase; bacterial proteins; biochemical evolution; chemical kinetics; cofactor; enzyme mechanism; enzyme model; enzyme substrate; enzyme substrate analog; enzyme substrate complex; isocitrate dehydrogenase; microorganism culture; protein engineering; protein structure function; site directed mutagenesis

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.

该项目的目的是确定活动之间的相互作用 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。 对这种互动的理解是 针对针对蛋白质的治疗药物设计的关键 已知结构，通过工程酶设计新型催化剂的设计， 了解催化和理解蛋白 进化。 该提案描述了两种与 改变了特异性。 一种方法使用定向诱变到 用特定序列和超辅助结构用 相关酶。 第二种方法模仿了长期的过程 通过将大肠杆菌的构造菌株放置在适应性演化下 化学仪培养施加的强烈选择性压力。 这些 独立或组合的方法将产生酶 特异性改变。 位点定向诱变的进化/突变 使用底物和底物类似物的酶和动力学研究， 然后，将被用来确定特异性变化的原因。 使用自然选择来产生变化的酶 特别有吸引力，因为没有关于哪个的先验决定 需要进行目标的残留物是必要的。 因此 发现不可预见的特异性决定因素是最大化的。 还， 突变赋予生长速率差异至每次1.0％ 很容易使用培养皿上的选择，无法检测到 在化学仪培养中可检测。 因此，化学稳定竞争实验 提供一种通过少量增量发展特异性的方法。 大肠杆菌和相关的异氯酸盐脱氢酶（IDH） 异丙丙酸酯脱氢酶（IMDH）将用作模型系统。 这些酶在2R-催化氧化脱羧反应 其底物共有的苹果酸核心。 活动站点之间的相互作用 每种酶的残留物和底物的各种伽马菌的残留物， 在公共核心的3s位置附加 明显的产生特异性的方法。 高分辨率结构 天然和磷酸化的IDH，以及带有二进制复合物 异氯酸盐和NADP可用。 也可用 IMDH的分辨率从热嗜热的结构。 这些，一起 我们了解动力学和催化机制以及 磷酸化调节IDH活性的方法，提供基本 详细解释结构功能所需的信息 关系。 开发新的一般方法以获取特定答案 配体 - 蛋白质结合问题，不考虑先入为主的 实验主义者的概念对于发现其他方式至关重要 不可预见的决定因素会影响 蛋白质。 这种方法的发展也将提供更大的 毒品和新型生物学的理性设计所需的见解 催化剂，以及对催化和蛋白质进化的理解。