STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

合成工程RNA酶的结构分析

• 批准号: 3298377
• 负责人: Brian Francis Edwards
• 金额: $ 9.72万
• 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 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298377
• 关键词: X ray crystallography; aminoacid; aspartate; chemical group; chemical structure function; crystallization; electron density; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; nucleoside monophosphate; pancreatic ribonuclease; phenylalanine; protein engineering; synthetic enzyme

X-ray diffraction analysis will 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 various active site residues 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 III-124, obtained by chemical synthesis. Catalytically modified analogs to be studied include the phe-120 to leu derivative (k cat = 13%), the asp-121 to asn derivative (k cat = 1.8%) and the asp-121 to ala derivative (activity against cytidine cyclic 2',3'-phosphate = 10%). In the case of the leu-120 analog, ismorphous crystals have been obtained and a 2.0-A electron density map is presently undergoing further refinement. Diffraction-quality crystals of the asn-121 derivative which have the same space group and are very nearly isomorphous with crystals of the parent structure are in hand. Efforts are underway to obtain suitable crystals for the ala-121 analog, and also for the tyr-120 analog, which exhibits a two-fold enhancement in activity toward uridine cyclic 2',3'-phosphate, but is otherwise normal. The semisyntheitc 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 dinucleoside phosphate substrates that have been shown to exhibit k cat values ranging from 27 to 3000 sec-1 with the native enzyme. If fairly final and satisfactory hypotheses concerning the roles of phe-120 and asp-121 in the action of the enzyme have not emerged from the examination of singly modified derivatives, a series containing both the pyrazolyl-119 structure and one or the other of the 120 or 121 modifications will be prepared. If suitable crystals can be obtained for members of this series, it should be possible to determine structural interactions of true substrates in enzymes know to exhibit altered catalytic rates or substrate specificities.

X射线衍射分析将被应用于一种新型的单晶 系列半合成的牛胰腺核糖核酸酶 表现出不同的催化效率或底物专一性 目的是进一步界定不同组织所扮演的角色 活性中心残基在确定催化能力中的作用 该酶的底物专一性。父级，完全活动 半合成酶由一种非共价复合体组成 残基1-118，通过酶消化本地人获得 酶和含有残基III-124的十四肽， 通过化学合成获得的。催化修饰的类似物 被研究的包括Phe-120到Leu的衍生物(k CAT=13%)， Asp-121对Asn衍生物(k CAT=1.8%)和asp-121对丙氨酸 衍生物(对环状胞苷2‘，3’-磷酸的活性=10%)。 在LEU-120类似物的情况下，同构晶体已经 并且2.0-A电子密度图目前正在进行中 进一步完善。具有衍射性的ASN-121晶体 具有相同空间群的导数，并且非常接近 与母体结构同构的晶体都在手中。 目前正在努力获得适合ALA-121的晶体 模拟，也适用于TYR-120模拟，它表现出双重 对尿苷环2‘，3’-磷酸的活性增强，但 在其他方面是正常的。 一种半合成模拟器，其中HIS-119被非常 近等位3-(3-吡唑基)-丙氨酸部分失去活性。 真正的衬底扩散到这种模拟晶体中应该， 因此，允许直接检查“酶”--底物 复合体。特别令人感兴趣的是一系列二核苷 已被证明具有k CAT值的磷酸盐底物 用天然酶从27秒到3000秒-1不等。 如果相当最终和令人满意的假设是关于 Phe-120和asp-121在酶的作用下没有 从对单一修饰的衍生品的检查中出现了一个 同时含有吡唑基-119结构和一个或 将准备120或121个修改中的其他修改。如果合适的话 这个系列的成员可以获得晶体，它应该是 有可能确定真实底物的结构相互作用 已知的酶表现出不同的催化速率或底物 具体细节。