MECHANISM OF INOSINE MONOPHOSPHATE DEHYDROGENASE

肌苷单磷酸脱氢酶的作用机制

• 批准号: 2190043
• 负责人: GEORGE Douglas MARKHAM
• 金额: $ 20.76万
• 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/2190043
• 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-5'-单磷酸脱氢酶（IMP:NAD）氧化还原酶； IMPDH） 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 化疗剂。拟议研究的目标是 确定活性位点结构和催化机制 IMPDH 存在于人类肿瘤中。该酶将从 表达大量酶的大肠杆菌 克隆基因。集成动力学、光谱和亲和标记 这些研究中将采用这种方法。 IMPDH催化反应途径的步骤和速率常数 对于这些步骤将由稳态和 前稳态动力学研究和初级动力学同位素效应 的决定。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 催化方面的限制。 催化的化学机制将通过表征来确定 结合在底物、产物和中间体的结构 活跃站点。适当富集底物的 13C NMR 和 15N NMR products will be used in conjunction with ultraviolet spectroscopic 研究。 IMP 的嘌呤环互变异构体和电离态 将阐明与酶结合的产物 XMP。的构象为 结合底物将通过 1H NMR 转移核确定 奥奥豪瑟效应。这些光谱研究将提供详细的 酶结合形式的结构表征 底物、产品，也可能是中间体。 单价阳离子（例如 K+）的活化机制为 决定。动力学研究将阐明阳离子结合的影响 酶对阳离子的催化效率和选择性 各种离子半径。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是否 离子结合在活性位点或者是变构激活剂。 有助于活性位点的氨基酸将通过以下方式鉴定 使用反应性 IMP 类似物和光亲和力进行亲和标记 IMP 和 NAD 的衍生物。这些残基如何进行定点突变 将确定影响底物结合和催化效率的因素。 这些研究的结果将构成理解的基础 IMPDH 与化疗重要化合物的相互作用，以及 用于设计新的抑制剂。