Structural Investigation of Allosteric Regulation in Bacterial Carbonic Anhydrase

细菌碳酸酐酶变构调节的结构研究

• 批准号: 7254495
• 负责人: Jeff D Cronk
• 金额: $ 18.85万
• 依托单位: GONZAGA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7254495
• 关键词: Active Sites; Address; Adopted; Affinity; Algorithms; Allosteric Regulation; Allosteric Site; Anti-Bacterial Agents; Bacteria; Bicarbonate Ion; Bicarbonates; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Carbon Dioxide; Characteristics; Class; Complement; Computing Methodologies; Crystallization; Crystallography; Data; Databases; Development; Disruption; Docking; Engineering; Enzymatic Biochemistry; Enzymes; Equilibrium; Escherichia coli; Escherichia coli Proteins; Evolution; Eye; Familiarity; Foundations; Haemophilus influenzae; Informatics; Investigation; Ions; Kinetics; Ligand Binding; Ligands; Light; Measurement; Medical; Methods; Modeling; Molecular; Molecular Biology; Molecular Conformation; Mutagenesis; Mutation; Mycobacterium tuberculosis; Nature; Numbers; Paper; Pharmaceutical Preparations; Physiological; Plants; Positioning Attribute; Proteins; Protocols documentation; Purpose; Reaction; Regulation; Reporting; Research; Salmonella typhimurium; Screening procedure; Shapes; Site; Structure; Structure-Activity Relationship; Students; System; Terminology; Testing; Textbooks; Therapeutic; Therapeutic Intervention; Training; Validation; Variant; Work; X-Ray Crystallography; Zinc; base; carbonate dehydratase; design; drug discovery; enzyme activity; experience; falls; interest; metalloenzyme; microorganism; mutant; pathogen; protein expression; protein structure; protein structure function; size; small molecule; therapeutic target; virtual; zinc hydroxide

DESCRIPTION (provided by applicant): The carbonic anhydrases (CAs) catalyze a reaction of fundamental biochemical and physiological importance, the interconversion of carbon dioxide and bicarbonate ion CO2 + H2O - All CAs are zinc-dependent enzymes and a well-established mechanistic paradigm requires the coordination of substrate to the catalytic zinc ion (Zn2+). The structures determined for the ¿ class carbonic anhydrases (¿-CAs), common in plants and bacteria, generally fall into two distinct subclasses based on the observed coordination of zinc. One subclass of ¿-CAs coordinate Zn2+ tetrahedrally with four protein-derived ligands, and in this configuration access of substrate to the zinc coordination sphere is apparently blocked. The ability of substrate to coordinate to zinc is observed in the other structural subclass. Recent evidence supports the hypothesis that the blocked configuration, as seen for example in ECCA, a ¿-CA from Escherichia coli, represents an inactive conformation of the enzyme, and that all such ¿-CAs can undergo a transition to an active conformation. In addition, a unique, non-catalytic binding mode for the substrate bicarbonate was discovered in ECCA that appears to stabilize the blocked, inactive form of the enzyme and seems to represent a regulatory mechanism. This proposal specifically aims to characterize the allosteric bicarbonate site that is likely shared by many eubacterial ¿-CAs, including a number of pathogens (e.g., Mycobacterium tuberculosis, Salmonella typhimurium). The structural and functional effects of its disruption by targeted mutagenesis are to be investigated by the primary method of X-ray crystallography, supported by kinetic measurements. In view of its potential as a site for therapeutic intervention, the characterization of the allosteric site will be furthered by a virtual screen for potential non-substrate ligands. Finally, the relationship between allosteric bicarbonate binding and the hypothesized structural transition in ECCA will be probed by testing the effects of mutations designed to shift the conformational equilibrium, The two observed structural subclasses serve as an explicit two-state model for regulation. This project will be attractive to students with interest in biochemistry, particularly protein structure and enzymology. It integrates a textbook example of an extremely fast enzyme with allosteric regulation of enzyme activity. The project also emphasizes computational methods, including molecular graphics, modeling, and informatics methods of drug discovery. This research will gather important basic information about eubacterial ¿-carbonic anhydrases, enzymes known to be required for a number of pathological microorganisms. An allosteric ligand binding site and its effect on enzyme activity will be characterized with an eye to its development as a target of therapeutic drugs.

描述（申请人证明）：碳赤霉酶（CAS）催化基本生化和生理学的反应重要性E二氧化碳和碳酸氢盐CO2 + H2O-H2O-ALL CAS的互连是锌依赖性的酶，以及良好的机械师ES ES均衡基材的催化锌离子（Zn2+）。基于观察到的脐带的碳酸酐酶（�-CAS），在植物和巴切利菌中常见，将其落入两个不同的亚类。 -cas坐标Zn2+四面体带有蛋白质衍生的配体，在底物访问锌源球的构型中，底物在其他结构子类中观察到了底物对锌I的能力。例如，在ECCA中， -ca来自大肠杆菌，代表了酶的不活跃概括，所有这些„ -CAS可以在CA中发现碳酸氢盐的非催化结合模式，从而稳定了酶的封闭式，似乎是一种调节机制旨在表征变构碳酸氢盐位点thate thate takely sareal» - 倾斜的病原体（例如，结核病，沙门氏菌，靶向型）。对于潜在的物质配体，将通过虚拟屏幕进一步进一步进一步，将ECCA中的碳酸氢盐结合与假设的结构过渡之间的关系。子类用作具有明确的两态模型或ES的教科书示例。关于Eubacterial»的基本信息 - 碳酸酐，许多病理微生物所必需的酶，将其作为治疗药物的靶向。