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

描述（由申请人提供）：本提案的目标是提供必要的化学水平细节，以在原子分辨率上理解色氨酸合酶的酶促机制。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，如杂化和质子化状态的变化，很难在功能酶系统中表征。吡哆醛-磷酸（维生素b6）依赖色氨酸合成酶1222双酶复合体催化l-色氨酸合成的最后两个步骤，这两个连续的过程需要在1-亚基和2-亚基之间传递常见代谢物吲哚。色氨酸合成酶的同源物存在于细菌、酵母、霉菌、植物和一些原生动物中。在高等动物和人类中缺乏l -色氨酸的合成途径，这使得色氨酸合成酶纳米机器成为开发除草剂和设计治疗传染病药物的潜在目标。因此，了解其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点提供有用的见解。最近对底物、中间体和底物类似物配合物的x射线结构测定在鉴定双酶配合物结构和催化之间的联系方面取得了重大突破。这项工作结合了有机合成工作和溶液动力学/光谱研究，并在1.7至2.4 A分辨率下测定了10-15种不同的色氨酸合酶配体配合物的x射线晶体结构。尽管取得了这些成功，但重要的化学问题仍然存在，因为这些结构的解决不允许为底物转化建立详细的化学机制。然而，化学水平的细节，如质子化和杂交状态是理解酶的机制和功能的关键。即使在中等高分辨率下，这些也很难单独通过x射线晶体学来确定。然而，核磁共振（NMR）中的化学位移是一种对化学环境极其敏感的探针，使固态核磁共振和x射线晶体学成为定义化学细节三维结构的强大组合。本文采用x射线晶体学/固体核磁共振/从头计算相结合的方法来确定色氨酸合成酶2亚基从底物到产物的多步转化过程中几个关键中间体的富化学晶体结构。活性位点的模型是使用协同方法开发的，其中这种活性底物/类似物的结构是利用计算化学在固定在其晶体学确定的坐标上的侧链残基的存在下自由优化的。底物和附近催化残基的各种电荷和质子化状态模型可以通过计算它们对化学位移的影响来唯一地区分，这些化学位移是在底物/类似物、辅酶和位点催化残基上的13C和15n标记位置测量的。这种处理为动力学和反应坐标扫描提供了精确的化学细节起点，已经为化学结构与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。

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.