Toward a Comprehensive Model for Enzyme Catalysis

建立酶催化综合模型

基本信息

批准号：
8908014
负责人：
JUDITH P KLINMAN
金额：
$ 51.94万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1978
资助国家：
美国
起止时间：
1978-06-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8908014
关键词：
Acceleration Active Sites Address Alcohol dehydrogenase Anisotropy Basic Science Behavior Binding Biological Models Biology Catalysis Chemicals Chemistry Communication Copper Coupling Defect Deuterium Development Distal Dopamine Drug Design Electron Transport Electrons Enzymes Exhibits Family Fluorescence Future Goals Hydrogen Isotopes Kinetics Ligands Link Lipoxygenase Measurement Methods Mixed Function Oxygenases Modeling Mononuclear Motion Mutagenesis Nature Pathway interactions Play Process Property Protein Chemistry Proteins Raman Spectrum Analysis Reaction Roentgen Rays Role Sampling Series Side Site Site-Directed Mutagenesis Soybeans System Temperature Testing Time Tyramine Variant Water Work computer studies conformer design dimer driving force enzyme model flexibility insight link protein mutant oxidation peptidylglycine alpha-amidating monooxygenase pressure research study three dimensional structure uptake

项目摘要

Understanding how enzymes work has constituted the core of basic research in medically related fields for over 50 years. Such studies have led to the description of a vast array of different enzyme classes that includes their 3-dimensional structures and underlying chemical mechanisms. For most of this time, the fundamental assumption regarding the origin of enzyme catalysis has remained wedded to a mid-20th century proposal referred to as "enhanced transition state binding". For the last decade, this monolithic and static description of catalysis has given way to the challenging question regarding the role of protein size and motions in achieving huge rate accelerations. While the inherent flexibility of proteins had long been recognized as important to function, for example in models of allostery, the direct link of protein motions to the chemistry at enzyme active sites has been difficult to access experimentally. This proposal describes a series of experimental approaches aimed at tackling this challenging and central problem regarding enzyme function. The systems chosen for further characterization catalyze fundamental and pervasive processes in biology (hydride and hydrogen atom transfer reactions). Studies of this nature are central to the development of robust models for biological catalysis that can guide future efforts at drug design and de novo protein design. There are three main goals for this proposal. First, a family of temperature-adapted, tetrameric prokaryotic alcohol dehydrogenases (ADHs) has been described that includes highly homologous thermophilic (ht- ADH) and psychrophilic (ps-ADH) variants. Completed kinetic characterizations implicate hydride transfer via hydrogenic wave function overlap between donor and acceptor atoms that is linked to local hydrophobic side chains and conformational landscapes. The specific protein motions controlling H-tunneling will be studied using a combination of fluorescence lifetime and anisotropy measurements, resonance Raman spectroscopy and hydrogen deuterium exchange. A link of active site flexibility to a remote specific side chain at the protein dimer interface has been identified and will be tested using kinetic, spectroscopic and protein chemistry probes. Second, the role of protein motions in catalysis will be pursued for a paradigmatic hydrogen atom tunneling reaction, lipoxygenase, where experimental approaches will include paramagnetic NMR, high-pressure kinetic studies and room temperature X-ray characterization. Third, detailed studies of tyramine-b-monooxygenase, a model for the mammalian, mononuclear/two- copper enzymes (dopamine b-monooxygenase and peptidylglycine-a-monooxygenase) will be focused on the inter-domain communication that controls hydrogen abstraction at CuM and long-range electron transfer from CuH to CuM.

了解酶的工作如何构成医学相关的基础研究的核心田野超过50年。这样的研究导致描述了一系列不同的不同包括其三维结构和基础化学物质的酶类机制。在大多数情况下，关于酶起源的基本假设催化一直持续到20世纪中叶的提案，称为“增强过渡状态结合”。在过去的十年中，这种催化的整体和静态描述具有让位于有关蛋白质大小和运动在实现中的作用的挑战性问题巨大的加速度。虽然长期以来一直认为蛋白质的固有灵活性是对于功能很重要，例如在变构模型中，蛋白质运动与酶活性位点的化学性能很难实验。这个建议描述了一系列旨在应对这种挑战和中心的实验方法有关酶功能的问题。选择用于进一步表征的系统催化生物学的基本和普遍过程（氢化物和氢原子转移反应）。对这种性质的研究对于发展强大模型的生物催化模型至关重要。可以指导未来的药物设计和从头蛋白质设计。有三个主要目标这个建议。首先，一个适应温度的四聚体原核醇已经描述了脱氢酶（ADHS），其中包括高度同源的嗜热（HT- ADH）和精神病（PS-ADH）变体。完成的动力学特征暗示氢化物通过氢波函数在供体和受体原子之间重叠的转移，这些原子与局部疏水侧链和构型景观。特定蛋白质运动将使用荧光寿命和各向异性的组合来研究控制H隧道测量，共振拉曼光谱和氢氘交换。一个链接在蛋白质二聚体界面处的远程特定侧链的主动位点灵活性已经鉴定并将使用动力学，光谱和蛋白质化学探针进行测试。第二，蛋白质运动在催化中的作用将用于范式氢原子隧道反应，脂氧合酶，实验方法将包括顺磁性 NMR，高压动力学研究和室温X射线表征。第三，详细酪胺-B-单加氧酶的研究，哺乳动物，单核/两铜的模型酶（多巴胺B-单氧酶和肽基甘氨酸-A-偶加酶）将聚焦在控制暨和远距离的氢气中的域间通信上电子从CUH转移到暨。