Structural and proton dynamics of pyridoxal-5'-phosphate dependent enzymes (resubmission)

含有吡哆醛-5‘-磷酸(PLP)的酶参与了广泛的氨基反应 酸和胺，包括转氨基、外消旋化、脱羧基、β和γ消除、β和γ 取代，甚至，正如最近发现的，氧化和氧化。一些重要的洋流或 潜在的药物靶点是PLP依赖的酶，包括γ-氨基丁酸氨基转移酶 脱羧酶、丙氨酸外消旋酶、鸟氨酸脱羧酶和丝氨酸羟甲基转移酶。然而， 由于缺乏特异性，目前针对PLP依赖酶的许多药物都存在副作用 为了他们的目标。因此，了解这些酶与分子和原子的反应是重要的。 详细程度，以帮助设计新的、更有效和更有选择性的药物。利用X射线结晶学， 关于酶和辅因子在催化中的作用，人们已经了解了很多。尽管如此，仍有 我们对限制药物设计的PLP依赖的酶的理解存在严重差距。晶体结构本身就是 遗漏了两条重要的信息。首先，它们缺乏关于反应动力学的重要信息。 众所周知，蛋白质在配体结合和催化中的运动在酶中起着核心作用，但这是如何发生的 基本上是未知的。此外，在PLP催化中起关键作用的氢原子并不直接 通过X射线结晶学观察。这在我们对一般酸碱的理解上留下了很大的差距。 一般酶中的催化，特别是依赖PLP的酶中的催化，其中活性部位质子化状态 似乎在控制反应特异性方面起着关键作用。天冬氨酸的一种新的中子衍射结构 转氨酶在活性位置发现一个质子，形成一个低能垒氢。 底物羧酸盐和乙二胺氮之间的键。我们对质子化理解中的这一空白 电离状态阻碍了治疗药物的合理设计，例如，为特定药物量身定做的治疗药物 静电环境。拟议项目的目标是提供对PLP的非常详细的了解 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 结晶学、分子动力学(MD)和量子力学/分子力学(QM/MM) PLP依赖酶的计算、非弹性中子散射、稳态和快速动力学技术。 这一合作项目的结果将第一次提供一幅全球范围内由 以真正的原子分辨率获得稳定的中间体以及 它们之间的动态联系。从我们的结果和开发的技术中得到的见解将是 可转移到许多其他酶，并可能有助于改进合理的新型抗生素的药物设计， 抗糖尿病、抗疟疾和其他药物。

Enzymes containing pyridoxal-5'-phosphate (PLP) are involved in a broad range of reactions of amino acids and amines, including transamination, racemization, decarboxylation, β- and γ-elimination, β- and γ- substitution, and, as recently discovered, even oxidation and oxygenation. A number of important current or prospective drug targets are PLP-dependent enzymes, including γ-aminobutyrate aminotransferase, DOPA decarboxylase, alanine racemase, ornithine decarboxylase, and serine hydroxymethyltransferase. However, many of the current drugs that target PLP-dependent enzymes suffer from side effects due to lack of specificity for their targets. Thus, it is important to understand the reactions of these enzymes with molecular and atomic levels of detail to help in the design of new more potent and more selective drugs. Using X-ray crystallography, a great deal has been learned about the role of both enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding of PLP-dependent enzymes that limit drug design. Crystal structures alone are missing two essential pieces of information. First, they lack important information regarding reaction dynamics. Protein motion in ligand binding and catalysis is known to play a central role in enzymes, but how this occurs is essentially unknown. In addition, hydrogen atoms that play critical roles in PLP catalysis are not directly observed by X-ray crystallography. This leaves a significant gap in our understanding of general acid-base catalysis in enzymes in general and particularly in PLP-dependent enzymes, where active site protonation states appear to play critical roles in control of reaction specificity. A recent neutron diffraction structure of aspartate aminotransferase found a proton in an unpredicted position in the active site, forming a low barrier hydrogen bond between the substrate carboxylate and the aldimine nitrogen. This void in our understanding of protonation and ionization states impedes rational design of therapeutic agents that, for example, are tailored for specific electrostatic environments. The goal of the proposed project is to provide a very detailed understanding of PLP 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 crystallography, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations, inelastic neutron scattering, steady-state and rapid kinetics techniques of PLP dependent enzymes. The results of this collaborative venture will provide, for the very first time, a global picture of catalysis by a large and centrally important class of enzymes at true atomic-resolution for stable intermediates as well as the dynamic connections between them. The insights from our results and the techniques developed will be transferable to many other enzymes, and may contribute to improved rational drug design of novel antibiotic, antidiabetic, antimalarial, and other drugs.