Catalytic specificity of zinc phosphate esterases investigated by evolution

通过进化研究磷酸锌酯酶的催化特异性

• 批准号: 7432587
• 负责人: JONATHAN K LASSILA
• 金额: $ 4.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7432587
• 关键词: Address; Affect; Alkaline Phosphatase; Biological; Biological Process; Biomedical Research; Catalysis; Charge; Chemistry; Depth; Development; Diagnostic; Disease; Electrostatics; Elements; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Evolution; Family; Genomics; Hydrolysis; Ions; Laboratories; Lead; Link; Measures; Medicine; Nucleotide pyrophosphatase; Object Attachment; Process; Proteins; Reaction; Relative (related person); Research; Role; Site; Specificity; Structure; Techniques; Testing; Therapeutic; Training; Zinc; base; catalyst; design; directed evolution; enzyme structure; esterase; experience; improved; inorganic phosphate; phosphoric diester hydrolase; reaction rate; structural biology; zinc phosphate

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.

描述（由申请人提供）：酶在每个生物功能和疾病过程中发挥作用。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产生重大影响。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结构如何定义功能。具体而言，定向实验室进化和机理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件，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，与核苷酸焦磷酸酶/磷酸二酯酶（一种催化磷酸二酯水解的酶）具有结构相似性。这两种酶在结构保守区域中仅具有16%的序列同一性，但它们具有几乎相同的锌离子反应中心。由于两种蛋白质之间的低序列同一性，不可能确定哪些序列差异导致差异催化活性。定向进化将用于增加核苷酸焦磷酸酶/磷酸二酯酶对磷酸单酯的现有弱混杂催化活性。这一进化过程将使得有可能确定与单酯酶活性增加相关的序列变化。这些序列的变化将深入研究，通过研究不同的有效电荷在断裂键的基板的效果。这些分析将有可能调查序列的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其电荷区分底物，以及锌位点附近的静电环境如何影响其催化特异性。预计该项目将展示特定序列的变化如何导致催化活性的巨大变化。相关性：酶在每一种生物功能和疾病过程中都有作用，但其作用机制尚未完全了解。研究酶结构变化的机制效应可以更好地理解这些重要的生物催化剂，这些催化剂可以开发具有治疗和诊断价值的新酶。