Structural Investigation of Allosteric Regulation in Bacterial Carbonic Anhydrase

描述(由申请人提供)：碳酸酐酶(CA)催化具有基本的生物化学和生理意义的反应，二氧化碳和碳酸氢根离子CO2+H2O的相互转化-所有的CA都是依赖于锌的酶，一个公认的机制范例需要底物与催化锌离子(锌离子)的配位。碳水解酶在植物和细菌中很常见，根据观察到的锌的配位情况，确定的结构通常分为两个不同的亚类。β-CaS的一个亚类与四个蛋白质衍生的配体以四面体配位方式配位，在这种构型下，底物对锌配位球的访问明显受阻。在另一个结构亚类中观察到底物与锌的配位能力。最近的证据支持这样的假设，即封闭的构型，例如在ECCA中看到的，来自大肠杆菌的？-CA代表酶的非活性构象，并且所有这些？-CA都可以经历到活性构象的转变。此外，在ECCA中发现了底物碳酸氢盐的一种独特的非催化结合模式，该模式似乎稳定了受阻的、不活跃的酶形式，似乎代表了一种调节机制。这项提议的具体目的是确定许多真细菌包括一些病原体(例如结核分枝杆菌、鼠伤寒沙门氏菌)可能共有的变构碳酸氢盐的特征。靶向突变破坏其结构和功能的影响将通过X射线结晶学的主要方法进行研究，并辅之以动力学测量。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确定变构部位的特征。最后，通过检验旨在改变构象平衡的突变的效果，探讨变构碳酸氢盐结合与ECCA中假设的结构转变之间的关系，这两个观察到的结构亚类作为一个显式的两态模型进行调节。这个项目将吸引对生物化学，特别是蛋白质结构和酶学感兴趣的学生。它结合了一个极快的酶与酶活性的变构调节的教科书例子。该项目还强调计算方法，包括药物发现的分子图形学、建模和信息学方法。这项研究将收集有关真细菌碳酸氢酶的重要基本信息，这些酶是许多病理微生物所需的已知酶。一个变构配体结合位置及其对酶活性的影响将被表征，并着眼于它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.