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速率限制反应 核苷酸。 IMPDH是抗肿瘤和抗病毒的既定目标 化学治疗剂。拟议的研究的目标是 确定形式的主动位点结构和催化机制 IMPDH存在于人类肿瘤中。酶将与 大肠杆菌表达大量的酶 克隆基因。集成的动力学，光谱和亲和力标记 这些研究将采用方法。 IMPDH催化反应途径和速率常数的步骤 对于这些步骤，将通过稳态和 presteady状态动力学研究和主要动力学同位素效应 确定。这些研究将揭示底物结合的顺序 和产品释放以及氢转移的程度 催化中的限制。 催化的化学机制将由表征确定 底物，产品和中间体结构的结构 活性站点。适当富集的底物的13C NMR和15N NMR 产品将与紫外线光谱法一起使用 研究。嘌呤环互变异物和IMP的电离状态和电离状态 将阐明与酶结合的产品XMP。构象 绑定的底物将从1H NMR转移的核定核确定 过度刺激效应。这些光谱研究将提供详细的 酶结合形式的结构的表征 底物，产品以及潜在的中间体。 单价阳离子（例如K+）激活的机制将是 决定。动力学研究将阐明阳离子结合的影响 关于催化效率和酶对阳离子的选择性 各种离子半径。平衡结合研究将揭示 阳离结合和NMR研究的化学计量法将揭示是否揭示 离子在活性位点结合或是变构激活剂。 促成活性位点的氨基酸将通过 使用反应性IMP类似物和光附属物的亲和力标记 Imp和Nad的衍生物。这些残基的位置定向突变如何 将确定影响底物结合和催化效率。 这些研究的结果将构成理解的基础 IMPDH与化学上重要化合物的相互作用，以及 用于设计新抑制剂。