MECHANISM OF INOSINE MONOPHOSPHATE DEHYDROGENASE

肌苷-5 '-单磷酸脱氢酶氧化还原酶 催化鸟嘌呤生物合成中的限速反应 个核苷酸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 确定了活性中心的结构形式和催化机理 IMPDH存在于人类肿瘤中。该酶将从 大肠杆菌，表达大量的酶，从 克隆基因一个集成的动力学，光谱和亲和标记 在这些研究中将采用的方法。 IMPDH催化反应途径中的步骤和速率常数 对于这些步骤，将由稳态和 前稳态动力学研究和初级动力学同位素效应 决心。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 限制催化作用。 催化的化学机理将通过表征来确定 底物、产物和中间体的结构 活性部位适当富集的底物的13 C NMR和15 N NMR， 产品将与紫外分光光度计结合使用， 问题研究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 结合底物将由1H NMR转移的核 奥佛豪瑟效应。这些光谱研究将提供详细的 酶结合形式的结构表征 底物、产物和潜在的中间体。 由单价阳离子（例如K+）活化的机制将是： 测定动力学研究将阐明阳离子结合的影响 对酶的催化效率和对阳离子的选择性的影响 各种离子半径。平衡结合研究将揭示 化学计量的阳离子结合和核磁共振研究将揭示是否 离子在活性位点结合或者是变构激活剂。 通过以下方法鉴定对活性位点有贡献的氨基酸： 使用反应性IMP类似物的亲和标记和光亲和 IMP和NAD的衍生物。这些残基的定点突变 影响底物结合和催化效率。 这些研究的结果将构成理解 IMPDH与化疗重要化合物的相互作用，和 用于设计新的抑制剂。

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.