Catalytic specificity of zinc phosphate esterases investigated by evolution

描述（由申请人提供）：酶在每一个生物功能和疾病过程中都起作用。可靠地设计新的酶催化剂的能力将对医学和生物医学研究有重大好处。然而，使酶成为优秀催化剂的结构特征尚未完全了解。拟议的研究旨在了解酶的结构如何定义功能。具体来说，定向实验室进化和机制分析的强大组合将用于确定定义两种相关酶的催化特异性的序列元素，并了解它们如何影响催化功能。碱性磷酸酶催化磷酸单酯的水解，其结构与核苷酸焦磷酸酶/磷酸二酯酶相似，后者是一种催化磷酸二酯水解的酶。这两种酶在结构保守区只有16%的序列相同，但它们具有几乎相同的锌离子反应中心。由于两种蛋白之间的序列一致性较低，因此无法确定是哪种序列差异导致了催化活性的差异。定向进化将用于提高现有的核苷酸焦磷酸酶/磷酸二酯酶对磷酸单酯的弱混杂催化活性。这一进化过程将使识别与单酯酶活性增加相关的序列变化成为可能。这些序列的变化将通过研究在断裂键处不同有效电荷的底物的影响来深入研究。这些分析将使研究序列变化是否影响反应的过渡态结构，双核锌反应中心如何根据底物的电荷区分底物，以及锌位点附近的静电环境如何影响它们的催化特异性成为可能。预计该项目将显示特定序列的变化如何导致催化活性的巨大变化。相关性：酶在各种生物功能和疾病过程中发挥作用，但其作用机制尚不完全清楚。研究酶结构变化的机制作用可以更好地理解这些基本的生物催化剂，从而可以开发具有治疗和诊断价值的新酶。

DESCRIPTION (provided by applicant): Enzymes have a role in every biological function and disease process. The ability to reliably design new enzyme catalysts would have major benefits for medicine and biomedical research. However, the structural features that make enzymes excellent catalysts are not yet fully understood. The proposed research seeks to understand how enzyme structure defines function. Specifically, a powerful combination of directed laboratory evolution and mechanistic analysis will be used to determine the sequence elements that define catalytic specificity in two related enzymes and to understand how they affect the catalytic function. Alkaline phosphatase catalyzes the hydrolysis of phosphate monoesters and has structural similarity to nucleotide pyrophosphatase/phosphodiesterase, an enzyme that catalyzes the hydrolysis of phosphate diesters. The two enzymes share only 16% sequence identity in a structurally conserved region, but they have almost identical zinc ion reaction centers. Because of the low sequence identity between the two proteins, it is impossible to determine which sequence differences lead to the differential catalytic activities. Directed evolution will be used to increase the existing weak promiscuous catalytic activity of nucleotide pyrophosphatase/phosphodiesterase toward phosphate monoesters. This evolutionary process will make it possible to identify sequence changes associated with increased monoesterase acivity. These sequence changes will be investigated in depth by studying the effect of substrates with varied effective charge at the breaking bond. These analyses will make it possible to investigate whether or not the sequence changes affect the transition state structure for the reaction, how the dinuclear zinc reaction center discriminates between substrates based on their charge, and how the electrostatic environment near the zinc sites affects their catalytic specificity. It is expected that the project will show how specific sequence changes can lead to dramatic changes in catalytic activity. Relevance: Enzymes have a role in every biological function and disease process, but their mechanisms of action are not fully understood. Studying the mechanistic effects of changes in enzyme structure permits greater understanding of these essential biological catalysts that may permit the development of new enzymes with therapeutic and diagnostic value.