Mechanistic Enzymology of Phosphoryl Transfer Enzymes

描述（由申请人提供）：了解磷酸化转移酶的机制和过渡状态对于理解生物催化、促进新型催化剂的设计和支持作为潜在药物的酶抑制剂的开发非常重要。在溶液中，这些反应可以通过几种具有特征过渡态的不同机制发生。过渡态质子化作用、亲核试剂和离去基团键和整体电荷分布是高度敏感的同一催化模式(静电稳定,布仑斯惕酸/碱,路易斯酸/碱)模型中,提出了磷酰转移酶的催化。这些见解强烈强调了解决生物催化领域长期悬而未决的问题的重要性：酶所采用的催化模式如何改变过渡态电荷分布？具有不同活性位点几何形状的酶（同工酶）稳定不同的过渡态吗？过渡态结构的差异能否促进竞争性抑制剂作为潜在药物的发展？回答这些问题将需要一个基于溶液反应理论和实验的机制框架，以及将该框架应用于结构-功能研究以确定代表性磷酰转移酶的过渡态和催化模式的能力。了解酶机制的一个有效方法是分析动力学同位素效应（KIEs），它测量基态和过渡态键的差异，并将这些信息与分子和量子力学模拟相结合，以评估特定的机制情景和重点实验工作。到目前为止，由于技术障碍，这种强大的方法无法应用于涉及核糖酶和蛋白质磷酸化转移酶的天然RNA寡核苷酸底物的反应，因此对一类重要的酶留下了上述突出的问题。现在，我们已经建立了KIE分析RNA和核苷酸反应的方法，我们使用理论和实验相结合的方法来全面了解磷酸化转移酶的机制。通过与Joseph Piccirilli博士（芝加哥大学）和Darrin York博士（罗格斯大学）的合作，这些实验的影响被放大了，他们提供了互补的技术优势，重要的是贡献了独立的知识视角。我们的共同努力旨在为酶的活性位点环境如何稳定反应过渡态提供新的见解。所获得的信息将揭示活性位点化学和化学机制之间的相互作用，这将显著影响我们对生物催化的理解，并广泛支持设计新的催化剂和发现具有潜在治疗应用的抑制剂的进展。

DESCRIPTION (provided by applicant): Understanding the mechanisms and transition states of phosphoryl transfer enzymes is important for understanding biological catalysis as well as facilitating the design of novel catalysts and supporting the development of enzyme inhibitors as potential drugs. In solution these reactions can occur by several different mechanisms with characteristic transition states. Transition state protonation, nucleophile and leaving group bonding and overall charge distribution are highly sensitive to the same catalytic modes (electrostatic stabilization, Bronsted acid/base, Lewis acid/base) that are proposed in models of catalysis by phosphoryl transferases. These insights strongly underscore the importance of addressing long standing unanswered questions in the field of biological catalysis: How do the catalytic modes employed by enzymes alter transition state charge distribution? Do enzymes with different active site geometries (isoenzymes) stabilize different transition states? Can differences in transition state structure facilitate the development of competitive inhibitors as potential drugs? Answering these questions will require a mechanistic framework grounded in theory and experiment for solution reactions, and the ability to apply this framework in structure-function studies to determine the transition states and catalytic modes for representative phosphoryl transfer enzymes. A powerful approach to understand enzyme mechanism is by analyzing kinetic isotope effects (KIEs), which measure the differences in ground state and transition state bonding, and integrating this information with molecular and quantum mechanical simulations to evaluate specific mechanistic scenarios and focus experimental efforts. Until now, technical barriers prohibited application of this powerful approach to reaction involving native RNA oligonucleotide substrates of ribozymes and protein phosphoryl transfer enzymes, leaving the questions highlighted above unanswered for an important enzyme class. Now, having established methods for KIE analyses RNA and nucleotide reactions, we using an integrated approach of theory and experiment to gain a comprehensive understanding of the mechanisms of phosphoryl transfer enzymes. The impact of these experiments is amplified by collaboration with Dr. Joseph Piccirilli (U Chicago) and Dr. Darrin York (Rutgers) who provide complementary technical strengths and importantly contribute independent intellectual perspectives. Our combined efforts are directed at providing new insights into how the active site environments of enzymes act to stabilize reaction transition states. The information gained will shed new light on the interplay between active site chemistry and chemical mechanism, which will significantly impact our understanding of biological catalysis and broadly support advances in design of new catalysts and discovery of inhibitors with potential therapeutic application.