Simulation of Proton and Hydride Transfer in Enzymes

描述(申请人提供)：本研究的长期目标是阐明蛋白质和核糖核酸酶催化中氢转移的基本原理和机制。这些目标将通过广泛的理论和计算方法来实现，包括量子力学/分子力学计算、经典分子动力学模拟和量子/经典分子动力学混合模拟，这些模拟提供了关于催化化学反应中结构重排和构象运动的原子水平信息。这些计算将探索氢键、静电学、活性部位重组和构象采样在蛋白质和RNA酶催化中的作用。这些理论研究将与实验小组密切合作，协助解释实验数据并提供实验可验证的预测。前两个特异性目标集中在催化类固醇异构化的酮类类固醇异构酶(KSI)上。第一个具体目标是探讨氢键在KSI中的作用，重点是沿着氢键网络的诱导效应，氢键之间的耦合，以及水分子在活性中心的作用。第二个具体目的是研究酶运动在KSI中的意义，重点是活性位点重组、构象采样和远端突变的影响。这两个特定的目标将提供与几种不同类型的实验数据相关的预测，包括抑制剂的核磁共振化学位移和电子吸收光谱，结合光酸的随时间变化的斯托克斯位移，以及突变体的动力学。这种理论和实验之间的紧密联系为剖析与酶中氢转移反应有关的基本问题提供了机会。后两个特异性目标集中在丁型肝炎病毒(HDV)RNA酶(核酶)上，它催化内部磷酸二酯键的断裂。这两个特定目标的动机是最近解决了这种核酶的催化活性预裂晶体结构。第三个具体目的是阐明HDV核酶中的质子转移机制，重点是活性部位的胞嘧啶核苷酸和镁离子的催化作用。第四个具体目的是了解运动在HDV核酶中的作用，重点是相关的运动和在催化化学反应中发生的构象变化。这种核酶的生物医学意义在于，HDV增加了由乙肝病毒引起的肝脏疾病的严重程度，而HDV的复制依赖于HDV核酶的自我切割。所有这些研究都与公众健康有关，因为阐明酶催化的基本原理将有助于设计更有效的酶和抑制剂，从而可能有助于开发更有效的药物来治疗各种疾病。此外，对RNA催化的深入了解可能有助于核酶的开发，将其用作切割致病RNA的治疗剂。 与公众健康相关：这些研究与公众健康相关，因为阐明酶催化的基本原理将有助于设计更有效的酶和抑制剂，从而可能有助于开发更有效的药物来治疗广泛的疾病。此外，对RNA催化的洞察可能有助于RNA酶的开发，将其用作切割致病RNA的治疗剂。

DESCRIPTION (provided by applicant): The broad, long-term objectives of this research are to elucidate the fundamental principles and mechanisms of hydrogen transfer in both protein and RNA enzyme catalysis. These objectives will be accomplished with a broad range of theoretical and computational methods, including quantum mechanical/molecular mechanical calculations, classical molecular dynamics simulations, and hybrid quantum/classical molecular dynamics simulations that provide atomic-level information about structural rearrangements and conformational motions during the catalyzed chemical reaction. These calculations will probe the roles of hydrogen bonding, electrostatics, active site reorganization, and conformational sampling in both protein and RNA enzyme catalysis. These theoretical studies will be performed in close collaboration with experimental groups, assisting in the interpretation of experimental data and providing experimentally testable predictions. The first two specific aims center on the enzyme ketosteroid isomerase (KSI), which catalyzes the isomerization of steroids. The first specific aim is to probe the role of hydrogen bonding in KSI, focusing on inductive effects along the hydrogen-bonding network, coupling among the hydrogen bonds, and the role of water molecules in the active site. The second specific aim is to examine the significance of enzyme motion in KSI, focusing on active site reorganization, conformational sampling, and the impact of distal mutations. These two specific aims will provide predictions related to several different types of experimental data, including NMR chemical shifts and electronic absorption spectra of inhibitors, time-dependent Stokes shifts of bound photoacids, and kinetics of mutants. This strong connection between theory and experiment provides an opportunity to dissect fundamental issues pertaining to hydrogen transfer reactions in enzymes. The last two specific aims center on the hepatitis delta virus (HDV) RNA enzyme (ribozyme), which catalyzes the cleavage of an internal phosphodiester bond. These two specific aims are motivated by the recent solution of a catalytically competent pre-cleaved crystal structure of this ribozyme. The third specific aim is to elucidate the proton transfer mechanism in the HDV ribozyme, focusing on the catalytic roles of a cytosine nucleotide and a magnesium ion in the active site. The fourth specific aim is to understand the role of motion in the HDV ribozyme, focusing on the correlated motions and the conformational changes occurring during the catalyzed chemical reaction. The biomedical relevance of this ribozyme is that HDV increases the severity of liver diseases caused by hepatitis B virus, and replication of HDV depends on self-cleavage of the HDV ribozyme. All of these studies are relevant to public health because the elucidation of the underlying fundamental principles of enzyme catalysis will facilitate the design of more efficient enzymes and inhibitors, thereby potentially assisting in the development of more effective drugs for a wide range of diseases. Furthermore, insights into RNA catalysis may assist in the development of ribozymes for use as therapeutic agents to cleave pathogenic RNAs. PUBLIC HEALTH RELEVANCE: These studies are relevant to public health because the elucidation of the underlying fundamental principles of enzyme catalysis will facilitate the design of more efficient enzymes and inhibitors, thereby potentially assisting in the development of more effective drugs for a broad range of diseases. Furthermore, insights into RNA catalysis may assist in the development of RNA enzymes for use as therapeutic agents to cleave pathogenic RNAs.