MECHANISM OF INOSINE MONOPHOSPHATE DEHYDROGENASE

肌苷单磷酸脱氢酶的作用机制

• 批准号: 2190042
• 负责人: GEORGE Douglas MARKHAM
• 金额: $ 22.14万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190042
• 关键词: Escherichia coli; NAD(P)H dehydrogenase; active sites; catalyst; cations; chemical binding; chemical kinetics; chemical synthesis; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; inosine monophosphate; mutant; nuclear magnetic resonance spectroscopy; oncoproteins; potassium; protein purification; protein sequence; site directed mutagenesis; stoichiometry; tautomer; ultraviolet spectrometry

Inosine-5'-monophosphate dehydrogenase (IMP:NAD) oxidoreductase; IMPDH) catalyzes the rate limiting reaction in the biosynthesis of guanine nucleotides. IMPDH is an established target of anti-tumor and anti-viral chemotherapeutic agents. The goals of the proposed research are to determine the active site structure and catalytic mechanism of the form of IMPDH present in human tumors. The enzyme will be isolated from Escherichia coli which express large quantities of the enzyme from the cloned gene. An integrated kinetic, spectroscopic and affinity labelling approach will be employed in these studies. The steps in the IMPDH-catalyzed reaction pathway and the rate constants for those steps will be determined by a combination of steady state and presteady state kinetic studies and primary kinetic isotope effect determinations. These studies will reveal the order of substrate binding and product release, and the extent to which hydrogen transfer is rate limiting in catalysis. The chemical mechanism of catalysis will be determined by characterization of the structures of substrates, products and intermediate(s) bound at the active site. 13C NMR and 15N NMR of appropriately enriched substrates and products will be used in conjunction with ultraviolet spectroscopic studies. The purine ring tautomers and ionization states of IMP and the product XMP bound to the enzyme will be elucidated. The conformations of bound substrates will be determined from 1H NMR transferred nuclear Overhauser effects. These spectroscopic studies will provide a detailed characterization of the structures of the enzyme-bound forms of substrates, products and potentially also of intermediates. The mechanism of activation by monovalent cations, such as K+, will be determined. Kinetic studies will elucidate the effects of cation binding on catalytic efficiency and the selectivity of the enzyme for cations of various ionic radii. Equilibrium binding studies will reveal the stoichiometry of cation binding and NMR studies will reveal whether the ion binds at the active site or is an allosteric activator. Amino acids which contribute to the active site will be identified by affinity labelling using reactive IMP analogs and photoaffinity derivatives of IMP and NAD. How site-directed mutations of these residues affect substrate binding and catalytic efficiency will be determined. The results of these studies will form the basis for understanding the interaction of IMPDH with chemotherapeutically important compounds, and for the design of new inhibitors.

肌苷单磷酸脱氢酶(IMP：NAD)氧化还原酶 催化鸟嘌呤生物合成中的限速反应 核苷酸。IMPDH是抗肿瘤和抗病毒的既定靶点 化疗药物。拟议研究的目标是 确定了活性中心结构和催化机理。 IMPDH存在于人类肿瘤中。这种酶将从 在大肠杆菌中表达了大量来自 克隆的基因。一种集成动力学、光谱和亲和标记的方法 这些研究将采用研究方法。 IMPDH催化反应途径中的步骤和速率常数 对于这些步骤，将由稳定状态和 稳态期前动力学研究和初级动力学同位素效应 决定。这些研究将揭示底物结合的顺序 和产物释放，以及氢转移速率的程度 在催化方面有局限性。 催化的化学机理将通过表征来确定。 底物、产物和中间体(S)的结构 活动站点。适当富集底物的13C和15N核磁共振 产品将与紫外光谱分析结合使用 学习。嘌呤环互变异构体和IMP的电离态 与酶结合的产物XMP将被阐明。的构象 结合底物将从1H核磁共振转移核中确定 奥豪瑟效应。这些光谱研究将提供详细的 酶结合形式的结构表征 基材、产品以及潜在的中间体。 K+等单价阳离子的活化机理为 下定决心。动力学研究将阐明阳离子结合的影响。 关于该酶对阳离子的催化效率和选择性 不同的离子半径。平衡结合研究将揭示 阳离子结合的化学计量和核磁共振研究将揭示 离子结合在活性部位或是变构激活剂。 对活性部位有贡献的氨基酸将通过 反应性IMP类似物和光亲和标记的亲和标记 IMP和NAD的衍生物。这些残基的定点突变是如何 影响底物结合和催化效率将被确定。 这些研究的结果将成为理解 IMPDH与化学治疗重要化合物的相互作用，以及 用于设计新的抑制剂。