Mechanistic enzymology of phosphoryl transfer enzymes

描述（由申请人提供）：本项目的总体目标是了解酶催化磷酸化转移反应的具体化学策略，这是生物学中最常见和最重要的反应之一。我们目前的研究重点是酶，它是定义酶催化基本特征的原型。由于磷酰转移在生物学中是必不可少的，这类酶是人类疾病的潜在治疗靶点。当前项目期间的结果将增加对催化基本原理的理解，为可能导致新疗法的研究奠定基础。虽然对磷酰转移的研究已有几十年的历史，但关于这些酶的机制仍然存在长期存在的基本问题。许多这些酶的晶体结构已经被解决；然而，结构之间的显著差异和与功能实验的不一致仍然存在。这种缺乏理解是发现这类酶的生物催化的定义特征和实现基于机制的抑制剂设计潜力的关键障碍。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态相互作用的新方法。用该方法测定了用较重的稳定同位素取代原子和发生反应的原子对反应速率的影响。这些效应的大小提供了关于过渡态结构的详细信息。这些数据可以用来询问不同的生物催化剂之间的相互作用和过渡状态的磷酰转移反应。我们目前的工作重点是比较核酶和蛋白质磷酸化转移酶，旨在了解其潜在的独特和特殊的催化策略。在过渡态电荷分布水平上比较催化的能力，结合以确定的方式操纵活性位点相互作用的能力，为区分特定催化机制中涉及的酶的特征提供了有力的手段。虽然我们的主要重点是重原子同位素效应，但总体方法是多学科的。将整合来自高分辨率结构、野生型和突变型酶的稳态和预稳态动力学以及计算模拟的信息，以获得有关反应过渡态及其与催化剂相互作用的信息。

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.