Structural and proton dynamics of pyridoxal-5’-phosphate dependent enzymes Resubmission (Diversity Supplement)

项目摘要 (父项目范围不变) 依赖PLP的酶约占蛋白质组的2%，其中许多是现有的或潜在的 毒品目标。依赖吡哆醛-5‘-磷酸(PLP)的酶有四个主要家族，分别是 通过不同的三维折叠：天冬氨酸氨基转移酶或α家族(折叠I)，色氨酸 合成酶或β家族(折叠II)、丙氨酸外消旋酶家族(折叠III)和D-氨基酸转氨酶 家庭(文件夹四)。利用X射线结晶学，人们对这两者的作用已经有了很大的了解 催化中的酶和辅因子。尽管如此，在我们对限制药物的理解上仍然存在重大差距 设计。建议项目的目标是提供对PLP依赖的非常详细的理解 通过从全球到原子水平协调定义它们的结构和动力学来研究酶的作用机制。 为了实现这一点，我们将使用对以下生物物理技术敏感的协同组合 不同的大小和时间尺度。这些将包括联合X射线/中子结晶学、固体核磁共振 光谱学、分子动力学和QM/MM计算、非弹性中子散射、快速动力学 技术，以及重酶动力学同位素效应。我们将重点关注四个结构良好的特点 PLP依赖酶，天冬氨酸氨基转移酶，丝氨酸羟甲基转移酶，酪氨酸苯酚裂解酶 和色氨酸合成酶，但缺乏关于质子化和动力学的信息。这些酶是 药物靶点(天冬氨酸氨基转移酶、丝氨酸羟甲基转移酶)或作为药物模型 靶标(色氨酸合成酶、酪氨酸苯酚裂解酶)。这些酶催化不同的反应，但使用 在相似的活性部位有相同的辅因子。因此，我们假设反应的特异性必须由一个 蛋白质动力学和反应中间产物的选择性质子化的结合。联合X射线/中子 结晶学将是我们研究的基础，为蛋白质提供原子级的结构基础 中等分辨率蛋白质结构中氢原子的动力学和精确可视化。这个 X射线/中子结晶学的结果将与新的固体核磁共振结晶学和 用非弹性中子散射来表征这些酶在皮秒到皮秒的全局和局部运动 微秒时间刻度。压力跳跃松弛动力学和重酶动力学同位素效应 补充和提供皮秒到分钟时间尺度中域运动的动态信息。它 值得注意的是，非弹性中子散射、压力跳跃和重酶动力学是 互补的技术，因为它们都对酶的振动运动的变化敏感， 但在不同的时间尺度上审问。所有这些结果都将与分子计算相结合，以 提供了这些重要酶的动态特性的前所未有的完整图景。这 知识可能允许设计新的、有效的和选择性的酶抑制剂，这些酶抑制剂可能提供新药 靶向PLP依赖的酶。

Project Summary (NO CHANGE IN THE SCOPE OF THE PARENT PROJECT) PLP-dependent enzymes represent about 2% of the proteome, and a number of them are current or potential drug targets. There are four major families of pyridoxal-5’-phosphate (PLP)-dependent enzymes, distinguished by different three-dimensional folds: the aspartate aminotransferase or α-family (Fold I), the tryptophan synthase or β-family (Fold II), the alanine racemase family (Fold III), and the D-amino acid aminotransferase family (Fold IV). Using X-ray crystallography, a great deal has been learned about the role of both these enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding that limit drug design. The goal of the proposed project is to provide a very detailed understanding of PLP-dependent enzyme mechanisms by coordinately defining their structures and dynamics from the global to the atomic level. To accomplish this, we will employ a synergistic combination of biophysical techniques that are sensitive to different size- and time-scales. These will include joint X-ray/neutron crystallography, solid-state NMR spectroscopy, molecular dynamics and QM/MM calculations, inelastic neutron scattering, rapid kinetics techniques, and heavy enzyme kinetic isotope effects. We will focus on four structurally well-characterized PLP-dependent enzymes, aspartate aminotransferase, serine hydroxymethyltransferase, tyrosine phenol-lyase and tryptophan synthase, but for which information on protonation and dynamics is lacking. The enzymes are drug targets (aspartate aminotransferase, serine hydroxymethyltransferase) or serve as models for drug targets (tryptophan synthase, tyrosine phenol-lyase). These enzymes catalyze diverse reactions, but use the same cofactor in similar active sites. Thus, we postulate that the reaction specificity must be controlled by a combination of protein dynamics and selective protonation of reaction intermediates. Joint X-ray/neutron crystallography will be the foundation of our research, providing an atomic-level structural basis for protein dynamics and accurate visualization of hydrogen atoms in protein structures at moderate resolutions. The results of X-ray/neutron crystallography will be combined with novel solid-state NMR crystallography and with inelastic neutron scattering to characterize the global and local motions of these enzymes at picosecond-to- microsecond time scales. Pressure-jump relaxation kinetics, and heavy enzyme kinetic isotope effects will complement and provide dynamic information on domain motion in the picosecond to minute time-scales. It should be noted that the inelastic neutron scattering, pressure-jump and heavy enzyme kinetics are complementary techniques in that they all are sensitive to changes in the vibrational motions of the enzyme, but interrogate at different time scales. All these results will be integrated with molecular computations to provide an unprecedented complete picture of the dynamic properties of these important enzymes. This knowledge may allow the design of novel, potent and selective enzyme inhibitors that may provide new drugs targeted against PLP-dependent enzymes.