Structural Investigation of Allosteric Regulation in Bacterial Carbonic Anhydrase

描述（由申请人提供）：碳酸酐酶（CA）催化具有基本生物化学和生理学重要性的反应，即二氧化碳和碳酸氢根离子CO2 + H2O的相互转化。所有CA都是锌依赖性酶，并且公认的机理范式需要底物与催化锌离子（Zn 2+）的配位。碳酸酐酶（carbonic anhydrases，CA）是植物和细菌中常见的一类酶，根据观察到的锌的配位作用，其结构通常分为两个不同的亚类。的一个子类- CA与四个蛋白质衍生的配体四面体地配位Zn 2+，并且在这种配置中，底物对锌配位球的访问显然被阻断。在其他结构亚类中观察到底物与锌配位的能力。最近的证据支持这样的假设，即封闭构型，例如在ECCA中看到的，大肠杆菌的<$-CA，代表酶的非活性构象，并且所有这些<$-CA都可以经历向活性构象的转变。此外，在ECCA中发现了底物碳酸氢盐的独特的非催化结合模式，该模式似乎稳定了酶的封闭的无活性形式，并且似乎代表了一种调节机制。该提案特别旨在表征变构碳酸氢盐位点，该位点可能由许多真细菌的CA共享，包括许多病原体（例如，结核分枝杆菌、鼠伤寒沙门氏菌）。通过靶向诱变对其破坏的结构和功能影响将通过X射线晶体学的主要方法进行研究，并得到动力学测量的支持。鉴于其作为治疗干预部位的潜力，将通过虚拟筛选潜在的非底物配体来进一步表征变构部位。最后，将通过测试旨在改变构象平衡的突变的影响来探讨ECCA中变构碳酸氢盐结合和假设的结构转变之间的关系。观察到的两个结构子类作为明确的双态模型进行调节。本专题将吸引对生物化学，特别是蛋白质结构和酶学感兴趣的学生。它整合了一个教科书的例子，一个极快的酶与酶活性的变构调节。该项目还强调计算方法，包括分子图形，建模和药物发现的信息学方法。这项研究将收集有关真细菌碳酸酐酶的重要基础信息，已知这种酶是许多病理微生物所需的。变构配体结合位点及其对酶活性的影响将被表征为着眼于其作为治疗药物的靶点的发展。

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.