Chemically-Rich Structure and Dynamics in the Active Site of Tryptophan Synthase

色氨酸合酶活性位点的化学丰富结构和动力学

基本信息

批准号：
8523915
负责人：
Leonard J Mueller
金额：
$ 27.47万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8523915
关键词：
Active Sites Adopted Animals Bacteria Biochemistry Boxing Catalysis Catalytic Domain Charge Chemical Structure Chemicals Chemistry Coenzymes Communicable Diseases Complex Data Development Diamines Drug Design Electrostatics Environment Enzymes Equilibrium Goals Herbicides Homologous Gene Housing Human Hydroxyl Radical Indoles Kinetics Label Ligands Location Measures Methodology Modeling Molds Nuclear Magnetic Resonance Organic Chemistry Pathway interactions Plants Positioning Attribute Process Proteins Protons Pyridoxal Phosphate Reaction Resolution Roentgen Rays Salmonella typhimurium Scanning Scheme Schiff Bases Side Site Solutions Specific qualifier value Spin Labels Staging Structure System Tryptophan Synthase Vitamin B6 Water Work X-Ray Crystallography Yeasts analog base computational chemistry electronic structure enzyme mechanism indoline insight molecular mechanics nanomachine novel protonation serine containing aminolipid solid state nuclear magnetic resonance success three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to provide the chemical level details necessary to understand the enzymatic mechanism in tryptophan synthase at atomic resolution. Enzymes have evolved to achieve remarkably efficient and specific chemical transformations that enable a diverse biochemistry. Yet atomic level details of enzyme mechanisms remain elusive; the intermediates are transient and the chemistry that drives the transformation, such as changes in hyrbridizaton and protonation states, is difficult to characterize in functioning enzyme systems. The pyridoxal-phosphate (vitamin-B6)-dependent tryptophan synthase 1222 bienzyme complex catalyses the last two steps in the synthesis of L-Trp, consecutive processes that require channeling of the common metabolite, indole, between the 1- and 2-subunits. Tryptophan synthase homologues are found in bacteria, yeasts, molds, plants, and some protozoans. The absence of a synthetic pathway for L-Trp in higher animals and in humans makes the tryptophan synthase nanomachine a potential target both for the development of herbicides, and for the design of drugs to treat infectious disease. Consequently, understanding the catalytic mechanism could provide useful insights for developing tryptophan synthase as an important target for drug design, or for the development of herbicides. Recent X-ray structure determinations of complexes with substrates, intermediates, and substrate analogues have resulted in a significant breakthrough concerning identification of the linkages between the bienzyme complex structure and catalysis. This effort combined organic synthetic work and solution kinetic/spectroscopic studies with X-ray crystal structure determinations of 10-15 different ligand complexes with tryptophan synthase at 1.7 to 2.4 A resolution. Despite these successes, significant chemical questions remain as the resolution of these structures does not allow for a detailed chemical mechanism to be established for the substrate transformation. Yet chemical level details such as protonation and hybridization states are critical for understanding enzymatic mechanism and function. Even under moderately high resolution, these are difficult to determine from X-ray crystallography alone. The chemical shift in nuclear magnetic resonance (NMR), however, is an extremely sensitive probe of chemical environment, making solid- state NMR and X-ray crystallography a powerful combination for defining chemically-detailed three dimensional structures. Here we adopt a combined X-ray crystallography/solid state NMR/ab initio calculation approach to determine the chemically-rich crystal structures of several key intermediates in the multistep transformation of substrate to product in the 2-subunit of tryptophan synthase. Models of the active site are developed using a synergistic approach in which the structure of this reactive substrate/analogue is freely optimized using computational chemistry in the presence of side chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues can be uniquely distinguished by their calculated effect on the chemical shifts, measured at specifically 13C and 15N-labeled positions on substrates/analogues, coenzyme and site catalytic residues. This treatment provides an accurate chemically-detailed starting point for dynamics and reaction coordinate scans that have already provided unique insight into the connection between chemical structure and the resulting local electrostatic fields that help drive and direct the next step in the catalysis.

描述（由申请人提供）：该提案的目的是提供在原子分辨率下了解色氨酸合酶中酶促机制所需的化学水平细节。酶已经发展为实现极有效和特定的化学转化，从而使能够多样化的生物化学。然而，酶机制的原子水平细节仍然难以捉摸。中间体是短暂的，驱动转化的化学反应，例如杂化剂和质子化状态的变化，在功能性酶系统中很难表征。吡啶毒素磷酸（维生素-B6）依赖性色氨酸合酶1222生物酶复合物催化L-TRP合成的最后两个步骤，即连续的过程，这些过程需要在1-和2-亚基之间传播常见的代谢物，吲哚，吲哚，吲哚。色氨酸合酶同源物在细菌，酵母，霉菌，植物和一些原生动物中发现。在上等动物和人类中，L-TRP的合成途径的缺乏使得色氨酸合酶纳米机械成为开发除草剂的潜在靶标，以及设计用于治疗传染病的药物。因此，理解催化机制可以为开发色氨酸合酶作为药物设计的重要靶点或除草剂的开发提供有用的见解。 X射线结构与底物，中间体和底物类似物的复合物的最新结构确定导致了有关鉴定生物酶复合物结构与催化之间联系的显着突破。这项工作将有机合成工作和溶液动力学/光谱研究与X射线晶体结构进行了10-15的X射线晶体结构，以1.7至2.4 A分辨率为1.7至2.4的色氨酸合酶。尽管取得了这些成功，但由于这些结构的分辨率不允许为底物转换建立详细的化学机制，因此仍然存在重大的化学问题。然而，质子化和杂交状态等化学水平的细节对于理解酶机制和功能至关重要。即使在中等高的分辨率下，仅凭X射线晶体学也很难确定。但是，核磁共振（NMR）的化学位移是对化学环境的极其敏感的探测器，使固态NMR和X射线晶体学成为定义化学详细详细测定的三维结构的强大组合。在这里，我们采用了组合的X射线晶体学/固态NMR/ab从头计算方法来确定在色氨酸合成酶的2-亚基中底物向产物的多步转化中的几个关键中间体的化学晶体结构。活跃位点的模型是使用协同方法开发的，在这种方法中，该反应性底物/模拟的结构在存在固定在结晶确定的坐标处的侧链残基的情况下，使用计算化学自由优化。底物和附近催化残基的各种电荷和质子化状态可以通过其对化学移位的计算影响来区分，这些对化学位移的影响在特定的13C和15N标记的位置上测量了底物/类似物，辅酶和位点催化残基。该处理为动力学和反应坐标扫描提供了精确的化学详细起点，该扫描已经为化学结构与所得局部静电场之间的联系提供了独特的见解，这些静电场有助于驱动和指导下一步催化。