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

描述（由申请人提供）：本提案的目的是提供在原子分辨率下理解色氨酸合酶酶促机制所需的化学水平详细信息。 酶已经进化到能够实现非常有效和特异的化学转化，从而实现多样化的生物化学。然而，酶机制的原子水平的细节仍然难以捉摸;中间体是短暂的和化学驱动的转换，如在hyrbridizaton和质子化状态的变化，是很难在功能酶系统的特点。磷酸吡哆醛（维生素B6）依赖性色氨酸合酶1222双酶复合物催化合成L-Trp的最后两个步骤，这两个连续过程需要在1-和2-亚基之间引导共同代谢物吲哚。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。在高等动物和人类中缺乏L-Trp的合成途径，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的药物的潜在目标。因此，了解的催化机制可以提供有用的见解开发色氨酸合成酶作为一个重要的目标，药物设计，或开发除草剂。 最近的X-射线结构测定与底物，中间体和底物类似物的复合物已经导致了一个重大的突破，关于识别的双酶复合物的结构和催化之间的联系。这项工作结合了有机合成工作和溶液动力学/光谱研究，并以1.7至2.4 A的分辨率测定了10-15种不同配体与色氨酸合酶的复合物的X射线晶体结构。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，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，这些也很难单独从X射线晶体学中确定。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射线晶体学成为用于定义化学详细三维结构的强大组合。 在这里，我们采用相结合的X-射线晶体学/固态NMR/从头计算方法来确定化学丰富的晶体结构的几个关键中间体的多步转化的底物中的2-亚基的色氨酸合酶的产品。活性位点的模型是使用协同方法开发的，其中该反应性底物/类似物的结构在固定在其晶体学确定的坐标处的侧链残基的存在下使用计算化学自由地优化。底物和附近的催化残基的电荷和质子化状态的各种模型可以通过它们对化学位移的计算效果来唯一地区分，所述化学位移在底物/类似物、辅酶和位点催化残基上的特定13 C和15 N标记的位置处测量。这种处理为动力学和反应坐标扫描提供了一个精确的化学详细起点，这些动力学和反应坐标扫描已经为化学结构与所产生的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.