Study of Enzymatic Phosphoryl Transfer

描述（由申请人提供）：本提案的广泛目标是了解酶在催化磷酰基转移反应（生物学中最常见的反应）中使用的策略。我们专注于这类酶的原型解开酶催化的基本特征。此外，磷酰基转移是生物学中的关键转化，在癌症和其他疾病状态中经常受到干扰，并且所涉及的酶是许多疾病的实际或潜在治疗靶标。这些结果不会直接导致抑制剂和潜在的治疗方法，但增加对催化原理的理解，超越其内在的基本重要性，可能最终有助于改变我们设计和操纵抑制剂作为药物的能力。本文提出的研究也与新的酶活性的演变，一个基本的生物学兴趣的问题，是不可分割的，这项研究有可能帮助开发策略，以设计或更好地选择具有医学或经济上理想的活性的酶。该提案侧重于碱性磷酸酶（AP）超家族，旨在了解该超家族的不同成员优先催化不同反应的潜在结构和功能原因。尽管结构同源性和不可区分的双金属锌位点，但碱性磷酸酶和AP-超家族磷酸二酯酶（核苷酸焦磷酸酶/磷酸二酯酶或NPP）在磷酸单酯和二酯水解的特异性上相差>1015倍。此外，这两种酶催化硫酸酯水解的速率大大降低，但具有显著的速率提高。因此，提出了一个问题：这些酶的哪些特征导致每个同源反应的优化？比较这些反应的催化作用（其底物的电荷和过渡态性质不同）的能力提供了一种强有力的手段来区分特定催化机制中涉及的酶特征。将使用高度多学科的方法：同源和非同源底物与野生型和突变酶反应的稳态和预稳态动力学比较，以确定速率增强和分解催化作用;同源酶之间的结构比较，以指导和解释定点诱变; X射线晶体学和扩展X射线吸收精细光谱（EXAFS），以比较同源酶的结构，并确定突变的结构后果;底物、抑制剂和过渡态类似物与野生型和突变酶的结合，以进一步比较同源酶并确定突变的能量后果;以及线性自由能关系和重原子同位素效应，以获得关于反应的过渡态及其活性位点相互作用的信息。公共卫生相关性：揭示允许酶区分它们催化的反应的基本原理可以提供指导新酶的设计的知识，这些新酶催化的反应在医学上是有益的或在工业上是有用的。此外，这种理解可能最终有助于开发更好的药物，靶向疾病过程中的关键磷酸化事件。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to understand the strategies used by enzymes in the catalysis of phosphoryl transfer reactions, the most common reaction in biology. We focus on this class of enzymes as a prototype for unraveling fundamental features of enzymatic catalysis. Furthermore, phosphoryl transfer is a key transformation in biology, is often perturbed in cancer and other disease states, and enzymes involved are actual or potential therapeutic targets for numerous diseases. The results will not directly lead to inhibitors and potential therapeutics, but increased understanding of the principles underlying catalysis, beyond their intrinsic fundamental importance, may ultimately help transform our ability to design and manipulate inhibitors as drugs. The research proposed herein is also integrally tied to the evolution of new enzymatic activities, a question of fundamental biological interest, and this research has the potential to aid in the development of strategies to design or better select for enzymes with medicinally or economically desirable activities. This proposal focuses on the alkaline phosphatase (AP) superfamily, aiming to understand the underlying structural and functional reasons why different members of this superfamily preferentially catalyze different reactions. Despite structural homology and indistinguishable bimetallo zinc sites, alkaline phosphatase and the AP-superfamily phosphodiesterase (nucleotide pyrophosphatase/phosphodiesterase or NPP) differ in specificity for the hydrolysis of phosphate monoesters and diesters by >1015 fold. Further, both enzymes catalyze hydrolysis of sulfate esters at a greatly compromised rate but with a substantial rate enhancement. Thus, the question is raised: What features of these enzymes lead to optimization for each cognate reaction? The ability to compare catalysis of these reactions, whose substrates differ in charge and transition state nature, provides a powerful means to distinguish enzymatic features involved in specific catalytic mechanisms. A highly multidisciplinary approach will be used: steady state and pre-steady state kinetic comparisons of cognate and non-cognate substrate reactions with wild type and mutant enzymes to determine rate enhancements and to dissect catalysis; structural comparisons between homologous enzymes to guide and interpret site-directed mutagenesis; X-ray crystallography and extended X-ray absorbance fine spectroscopy (EXAFS) to compare structures of homologous enzymes and to determine the structural consequences of mutations; binding of substrates, inhibitors, and transition state analogs to wild type and mutant enzymes to further compare homologous enzymes and determine the energetic consequences of mutations; and linear free energy relationships and heavy atom isotope effects to obtain information about the reactions' transition states and their active site interactions. PUBLIC HEALTH RELEVANCE: Unraveling the fundamental principles that allow enzymes to distinguish between the reactions they catalyze may provide the knowledge to guide the design of new enzymes that catalyze reactions that are medically beneficial or industrially useful. Further, this understanding may ultimately aid in the development of better drugs that target critical phosphorylation events in disease processes.