STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

合成工程RNA酶的结构分析

• 批准号: 3298380
• 负责人: Brian Francis Edwards
• 金额: $ 9.96万
• 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 1991-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298380
• 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射线衍射分析将应用于a的单晶