STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

合成工程RNA酶的结构分析

• 批准号: 3298381
• 负责人: Brian Francis Edwards
• 金额: $ 11.43万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298381
• 关键词: X ray crystallography; active sites; chemical kinetics; conformation; crystallization; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; mutant; pancreatic ribonuclease; protein engineering; protein sequence; synthetic enzyme

X-ray diffraction analysis will continue to be applied to single crystals of a series of semisynthetic bovine pancreatic ribonucleases which exhibit altered catalytic efficiency or substrate specificity, with the intention of delineating further the roles played by aspartic acid-121 and phenylalanine-120 in establishing the catalytic power and substrate specificity of this enzyme. The parent, fully active semisynthetic enzyme consists of a non-covalent complex of residues 1-118, obtained by enzymatic digestion of the native enzyme, and a tetradecapeptide containing residues 111-124, obtained by chemical synthesis. A refined structure at 1.8 A (R = 20.4) of the parent complex has been obtained (Martin et al. (1987) J. Biol. Chem. 262, 15930-15938). If asp-121 is replaced by asn or ala, or phe-120 is replaced by leu, catalytic efficiency is reduced an order of magnitude. Refined 2.0-A structures of each of these three catalytically defective analogs have now been obtained, but interpretation of the structural basis for the loss of enzymatic activity is obscured by the multiplicity of structural changes that have occurred in all cases. In the expectation that more straightforward relationships will emerge in the presence of active site ligands, we plan to carry out structural analyses of the asn-121 and leu-120 analogs, in particular in the presence of the virtual substrate, 2;-deoxy-2'-fluorouridilyl-3'.5'-adenosine; the transition state analog, uridine vanadate; and the product, 3'-cytidylate. The semisynthetic analog in which his-119 is replaced by the very nearly isosteric 3-(3-pyrazolyl)-ala moiety is devoid of activity. Diffusion of true substrates into crystals of this analog should, therefore, permit direct examination of "enzyme-substrate" complexes. Of particular interest is a series of ribodinucleoside phosphate substrates that have been shown to exhibit kcat values ranging from 27 to 3000 sec-1 with the native enzyme. If the structures of the "enzyme-substrate" complexes obtained with the pyrazolyl analog reveal conformational features not observed with other active site ligands, double modified analogs containing 3-(3-pyrazolyl)-ala at position 119 and leucine at position 120 or asparagine at position 121 will be prepared, crystallized if possible, and analyzed in the presence of these true substrates.

X射线衍射分析将继续应用于单晶 一系列半合成牛胰腺核糖核酸酶，其表现出 改变催化效率或底物特异性，目的是 进一步描述天冬氨酸121所发挥的作用和 苯丙氨酸-120 在建立催化能力和底物中的作用 该酶的特异性。 母体，完全活性的半合成酶 由残基 1-118 的非共价复合物组成，通过酶促获得 天然酶的消化，以及含有残基的十四肽 111-124，通过化学合成获得。 1.8 A 的精细结构（R = 20.4) 的母体复合物已获得 (Martin et al. (1987) J. 生物。化学。 262、15930-15938）。 如果将 asp-121 替换为 asn 或 ala，或者 phe-120被leu取代，催化效率降低了一个数量级 震级。 催化这三种物质中每一种的精制 2.0-A 结构 现在已经获得了有缺陷的类似物，但对 酶活性丧失的结构基础被掩盖了 在所有情况下都发生了多种结构变化。 在 期望未来会出现更直接的关系 存在活性位点配体，我们计划进行结构分析 asn-121 和 leu-120 类似物，特别是在存在 虚拟底物，2；-脱氧-2'-氟尿嘧啶基-3'.5'-腺苷；这 过渡态类似物，尿苷钒酸盐；以及产物3'-胞苷酸。 半合成类似物，其中 his-119 被非常接近的取代 等排3-(3-吡唑基)-丙氨酸部分缺乏活性。 扩散 因此，将真正的底物转化为该类似物的晶体应该允许 直接检查“酶-底物”复合物。 特别感兴趣 是一系列核糖核苷磷酸底物，已被证明 与本机一起展示 kcat 值范围从 27 到 3000 sec-1 酶。 如果用获得的“酶-底物”复合物的结构 吡唑基类似物揭示了其他物质未观察到的构象特征 活性位点配体，含有 3-(3-吡唑基)-ala 的双修饰类似物 在位置 119 和亮氨酸在位置 120 或天冬酰胺在位置 121 如果可能的话，将进行制备、结晶，并在存在以下物质的情况下进行分析： 这些真实的基材。