STRUCTURE AND FUNCTION OF RESTRICTION ENDONUCLEASES

结合序列特异性和催化化学的元素是 开发靶向特异性的化疗药物的中心问题 核酸序列和结构。限制性内切酶，其中EcoRI 核酸内切酶是最具代表性的，是最简单的生物制剂， 位点特异性核酸化学，然而这一点的许多机制方面 水解活性仍不确定。该项目采用了独特的 i）鉴定蛋白质和底物 所需金属离子（MG（II））的配体，并定义了 反应中的这些相互作用。这些目标将通过 定点诱变，金属离子取代，底物类似物， 惰性过渡金属络合物和磁共振光谱。ii）第二阶段 通过光谱法表征所需金属离子的水合作用 方法，并探讨质子转移在DNA水解中的重要性 利用动力学同位素效应。iii）探索构象的作用 介导EcoRI内切酶底物特异性和星星的变化 活动用3-氟-酪氨酸标记的EcoRI核酸内切酶将被 制备和表征。19/F NMR光谱将应用于此 酶衍生物，以确定底物特异性和星星活性 在构造上有物理基础。从这些来源获得的信息 实验将确定酶结构之间的关系， 所需的金属离子，以及基板以及澄清工作 EcoRI的活动模式。了解结构和功能 这种天然形式的具体方面，水解催化是一种可行的 最终设计序列特异性剪刀的策略， 针对与癌症和艾滋病相关的特定基因。

Combining elements of sequence-specificity and catalytic chemistry is the central problem in developing chemotherapeutics which target specific nucleic acid sequences and structures. Restriction enzymes, of which EcoRI endonuclease is most representative, are the simplest biological agents of site-specific nucleic acid chemistry, yet many mechanistic aspects of this hydrolytic activity remain undefined. This project employs a unique combination of techniques to: i) Identify the protein and substrate ligands of the required metal ion (MG(II)) and define the importance of these interactions in the reaction. These goals will be accomplished with site-directed mutagenesis, metal ion substitution, substrate analogs, inert transition metal complexes, and magnetic resonance spectroscopy. ii) Characterize the hydration of the required metal ion by spectroscopic methods and probe the importance of proton transfer in DNA hydrolysis using kinetic isotope effects. iii) Probe the role of conformational changes in mediating EcoRI endonuclease substrate specificity and star activity. EcoRI endonuclease labelled with 3-fluoro--tyrosine will be prepared and characterized. 19/F NMR spectroscopy will be applied to this enzyme derivative to determine if substrate specificity and star activity have a physical basis in conformation. Information obtained from these experiments will identify the relationships between the enzyme structure, the required metal ion, and the substrate as well as clarify working models of EcoRI activity. Understanding the structural and functional aspects of this natural form of specific, hydrolytic catalysis is a viable strategy to the eventual design of sequence-specific scissors which can target specific genes linked to cancer and AIDS.