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（由应用提供）：碳赤霉酶（CAS）催化了基本生化和物理重要性的反应，二氧化碳和碳酸氢盐离子CO2 + H2O的相互转换 - 所有CAS都是锌依赖性酶，以及具有良好的机械范围的酶含量 + Zn + catecantic zincion catecintic cateytic catalation catalatientic（ZN）（ZN）。基于观察到的锌的配位，在植物和细菌中常见的类碳酸酐酶（`-cas）确定的结构通常落入两个不同的子类中。一个子类的一个子类坐标为Zn2+四面体与四个蛋白质衍生的配体，并且在这种构造访问锌协调球的构型中显然被阻断。在另一个结构子类中观察到底物与锌协调的能力。最近的证据支持以下假设：如在ECCA中所见的阻塞构型（来自Escherichia coli的A€-CA）代表了酶的不活跃构象，并且所有这样的CA都可以经过过渡到活动会议的过渡。此外，在ECCA中发现了一种独特的非催化结合模式，该模式似乎在ECCA中发现了酶的阻塞，不活跃的酶形式，并且似乎代表了一种调节机制。该提案专门旨在表征许多长杆菌»-CAS可能共享的变构碳酸氢盐位点，包括许多病原体（例如结核分枝杆菌，肺炎菌，沙门氏菌）。靶向诱变对其破坏的结构和功能效应，应通过动力学测量支持的主要方法来研究X射线晶体学的主要方法。鉴于其作为治疗干预部位的潜力，将通过虚拟屏幕来促进变构位点的表征，以实现潜在的非基质配体。最后，将通过测试旨在移动会议等效的突变的效果来探测ECCA中变构碳酸氢盐结合与假设的结构转变之间的关系，这两个观察到的结构性亚类作为调节的显式两态模型。该项目将对对生物化学感兴趣的学生，尤其是蛋白质结构和酶学具有吸引力。它整合了一个非常快速的酶的教科书示例，并与酶活性的变构调节进行了调节。该项目还强调了计算方法，包括分子图形，建模和信息发现的信息方法。这项研究将收集有关细菌的重要基本信息 - 钙赤霉酶，即已知的许多病理微生物所必需的酶。变构的配体结合位点及其对酶活性的影响将以其发展为治疗药物的靶标发育。