Mechanistic enzymology of phosphoryl transfer enzymes

磷酰基转移酶的机械酶学

• 批准号: 8329007
• 负责人: MICHAEL E. HARRIS
• 金额: $ 25.91万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8329007
• 关键词: Acids; Active Sites; Address; Affect; Biochemical Reaction; Biological; Biology; Catalysis; Catalytic RNA; Cell physiology; Charge; Chemicals; Clinical; Collaborations; Computer Simulation; DNA; DNA biosynthesis; Data; Development; Diagnostic; Drug Design; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Equilibrium; Goals; Health; Human; Hydrolysis; Introns; Investigation; Ions; Isotopes; Kinetics; Lead; Left; Measurement; Measures; Metabolism; Metals; Methods; Modeling; Oligonucleotides; Pancreatic ribonuclease; Pathogenesis; Pharmaceutical Preparations; Proteins; Protons; RNA; RNA biosynthesis; Reaction; Research; Resolution; Ribonuclease H; Ribonucleases; Role; Staging; Structure; Testing; Tetrahymena; Time; Transferase; base; catalyst; computer studies; design; enzyme mechanism; enzyme structure; functional group; human disease; inhibitor/antagonist; multidisciplinary; mutant; novel; novel therapeutics; prototype; reaction rate; research study; stable isotope; theories; therapeutic target

DESCRIPTION (provided by applicant): The broad goal of this project is to understand the specific chemical strategies used by enzymes to catalyze phosphoryl transfer reactions, which is one of the most common and essential reaction in biology. The current focus of our research is on enzymes that are prototypes for defining fundamental features of enzymatic catalysis. Because phosphoryl transfer is essential in biology, enzymes of this class are potential therapeutic targets for human diseases. The results from the current project period will increase understanding of the fundamental principles underlying catalysis setting the stage for studies that may lead to new therapeutics. While the study of phosphoryl transfer is decades old, there remain long standing, fundamental questions about the mechanisms of these enzymes. Crystal structures for many of these enzymes have been solved; yet, significant discrepancies between structures and inconsistencies with functional experiments remain. This lack of understanding is a key barrier to discovering the defining features of biological catalysis by this enzyme class and realizing the potential for design of mechanism-based inhibitors. To overcome this barrier, we developed new methods for analyzing enzyme transition state interactions by measuring kinetic isotope effects. In this method the effect on reaction rate of substituting and atom undergoing reaction with a heavier stable isotope is measured. The magnitude of these effects provides detailed information about the transition state structure. These data can be used to interrogate the interactions between diverse biological catalysts and the transition state for phosphoryl transfer reactions. Our current efforts focus on comparing ribozyme and protein phosphoryl transfer enzymes, aiming to understand their underlying unique and idiosyncratic catalytic strategies. The ability to compare catalysis at the level of transition state charge distribution, combined with the ability to manipulate active site interactions in defined ways provides a powerful means to distinguish enzymatic features involved in specific catalytic mechanisms. Although our main focus is on heavy atom isotope effects, the overall approach is multidisciplinary. Information from high resolution structures, steady state and pre-steady state kinetics with wild type and mutant enzymes, and computational simulations will be integrated to obtain information about the reactions' transition states and their interactions with catalysts.

描述（由申请人提供）：该项目的广泛目标是了解酶用于催化磷酸化转移反应的特定化学策略，这是生物学中最常见和最重要的反应之一。我们研究的当前重点是用于定义酶促催化基本特征的原型酶。由于磷酸化转移在生物学中至关重要，因此该类别的酶是人类疾病的潜在治疗靶标。当前项目时期的结果将增加对催化基本原理的理解，从而为可能导致新治疗剂的研究奠定了阶段。虽然对磷酸化转移的研究已有数十年的历史，但仍然存在有关这些酶机制的基本问题。许多这些酶的晶体结构已经解决。然而，结构与功能实验的不一致之间存在显着差异。缺乏理解是通过该酶类别发现生物催化的定义特征的关键障碍，并意识到设计基于机制的抑制剂的潜力。 为了克服这一障碍，我们开发了通过测量动力学同位素效应来分析酶过渡状态相互作用的新方法。在这种方法中，测量了对替代反应速率和原子与较重稳定同位素反应的影响。这些效果的大小提供了有关过渡状态结构的详细信息。这些数据可用于询问各种生物催化剂与磷酸转移反应的过渡态之间的相互作用。我们目前的努力着重于比较核酶和蛋白质磷酸化酶，旨在了解其潜在的独特和特质催化策略。在过渡状态电荷分布水平上比较催化的能力，结合以定义的方式操纵活性位点相互作用的能力提供了一种有力的方法，以区分特定催化机制涉及的酶促特征。尽管我们的主要重点是沉重的原子同位素效应，但总体方法是多学科的。来自高分辨率结构，具有野生型和突变酶的稳态和稳态动力学的信息以及计算模拟将集成，以获取有关反应的过渡状态及其与催化剂的相互作用的信息。