Mechanistic Enzymology of Phosphoryl Transfer Enzymes

描述(由申请人提供)：了解磷酸基转移酶的机制和过渡状态对于了解生物催化、促进新型催化剂的设计和支持酶抑制剂作为潜在药物的开发非常重要。在溶液中，这些反应可以通过几种不同的机制发生，具有特征的过渡态。过渡态质子化、亲核和离基成键以及总电荷分布对磷酸转移酶催化模型中提出的相同催化模式(静电稳定、Bronsted酸/碱、Lewis酸/碱)高度敏感。这些见解有力地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？具有不同活性部位几何构型(同工酶)的酶能稳定不同的过渡态吗？过渡态结构的差异能促进竞争性抑制剂作为潜在药物的发展吗？回答这些问题需要一个以溶液反应的理论和实验为基础的机理框架，以及将该框架应用于结构-功能研究以确定典型磷酸转移酶的过渡态和催化模式的能力。理解酶机制的一个有效途径是分析动力学同位素效应(Kies)，该效应衡量基态和过渡态成键的差异，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并集中实验努力。到目前为止，技术障碍禁止将这一强大的方法应用于涉及核酶和蛋白质磷酸转移酶的天然RNA寡核苷酸底物的反应，使得上面强调的问题对于一种重要的酶类没有得到回答。现在，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转移酶的机制。与约瑟夫·皮奇里利博士(芝加哥大学)和达林·约克博士(罗格斯大学)的合作放大了这些实验的影响，他们提供了互补的技术优势，并重要地贡献了独立的智力观点。我们的共同努力旨在为酶的活性部位环境如何稳定反应过渡态提供新的见解。所获得的信息将为活性中心化学和化学机理之间的相互作用提供新的线索，这将显著影响我们对生物催化的理解，并广泛支持在设计新的催化剂和发现具有潜在治疗应用的抑制剂方面的进展。

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.