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：
8728271
负责人：
Leonard J Mueller
金额：
$ 28.43万
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/8728271
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）：该提案的目标是提供在原子分辨率下理解色氨酸合酶的酶促机制所需的化学水平细节。酶已经进化到能够实现非常有效和特定的化学转化，从而实现多样化的生物化学。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，例如杂交和质子化状态的变化，很难在功能酶系统中表征。磷酸吡哆醛（维生素 B6）依赖性色氨酸合酶 1222 双酶复合物催化 L-Trp 合成的最后两个步骤，这是需要在 1- 和 2- 亚基之间引导常见代谢物吲哚的连续过程。色氨酸合酶同系物存在于细菌、酵母、霉菌、植物和一些原生动物中。高等动物和人类中缺乏 L-Trp 合成途径，这使得色氨酸合酶纳米机器成为除草剂开发和治疗传染病药物设计的潜在目标。因此，了解催化机制可以为开发色氨酸合酶作为药物设计或除草剂开发的重要靶标提供有用的见解。最近对底物、中间体和底物类似物的复合物的 X 射线结构测定在鉴定双酶复合物结构和催化之间的联系方面取得了重大突破。这项工作将有机合成工作和溶液动力学/光谱研究与 10-15 种不同配体复合物与色氨酸合酶的 X 射线晶体结构测定相结合，分辨率为 1.7 至 2.4 A。尽管取得了这些成功，但仍然存在重大的化学问题，因为这些结构的解析不允许为底物转化建立详细的化学机制。然而，质子化和杂交状态等化学水平细节对于理解酶机制和功能至关重要。即使在中等高分辨率下，仅通过 X 射线晶体学也很难确定这些。然而，核磁共振 (NMR) 中的化学位移是化学环境的极其敏感的探针，使得固态 NMR 和 X 射线晶体学成为定义化学详细三维结构的强大组合。在这里，我们采用X射线晶体学/固态NMR/从头计算相结合的方法来确定色氨酸合酶2-亚基中底物到产物的多步转化中几个关键中间体的化学丰富的晶体结构。活性位点模型是使用协同方法开发的，其中在固定在晶体学确定的坐标上的侧链残基存在的情况下，使用计算化学自由优化该反应底物/类似物的结构。底物和附近催化残基的电荷和质子化状态的各种模型可以通过计算它们对化学位移的影响来独特区分，在底物/类似物、辅酶和位点催化残基上的特定 13C 和 15N 标记位置进行测量。这种处理为动力学和反应坐标扫描提供了准确的化学详细起点，这些扫描已经为化学结构和由此产生的局部静电场之间的联系提供了独特的见解，有助于驱动和指导催化的下一步。