STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

合成工程RNA酶的结构分析

• 批准号: 3298378
• 负责人: Brian Francis Edwards
• 金额: $ 10.87万
• 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/3298378
• 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