Are Enzyme Active Sites Built in Multiple Layers?

酶活性位点是多层构建的吗？

• 批准号: 0843603
• 负责人: Mary Jo Ondrechen
• 金额: $ 41.02万
• 依托单位: Northeastern University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843603&HistoricalAwards=false
• 关键词: Enzyme; Active; Sites; Built; Multiple

The objective of this project is to obtain greater insight into how nature constructs enzyme active sites. The project aims to establish the principle of multilayer enzyme active sites and to gain understanding of how residues outside the first layer influence the catalytic activity and specificity. This understanding will have important implications for enzymology and for protein engineering. Experiments and calculations are designed to understand how the layers beyond the first one contribute to catalysis. In recent decades, structural and biochemical studies have identified residues in active sites of enzymes that directly participate in substrate binding and/or in the chemical transformation steps. These residues generally are in direct contact with the reacting substrate molecule and may be thought of as located in a "first shell" surrounding the reacting species. This project builds upon preliminary evidence that residues beyond the first shell also are important for catalytic function. The focus here is primarily on the second layer, the residues in contact with the first-shell residues. Systematic experimental studies to establish the importance of second-shell residues in enzyme catalysis, and computational studies to understand the varied roles that they play in enzymatic function will be pursued. In this project, site-directed mutagenesis experiments and kinetics assays will be performed on selected enzymes. These proteins will be chosen to represent different kinds of enzymes with different degrees of predicted participation by residues outside the first shell. Crystal structures of the mutants will be determined to test for structural changes upon mutation. Electrostatics calculations and molecular dynamics simulations will be performed to understand the mechanisms by which second-shell residues participate in function. A better understanding of how enzymes affect catalysis can help to produce cleaner, lower cost, "green" industrial processes and to the commercially viable enzymatic synthesis of biofuels. Students will be trained in computational methods, in protein expression, mutation, purification, kinetics assays, and crystal structure determination. Part of the project will be integrated into the undergraduate Chemical Biology laboratory course. Ongoing outreach efforts to minority students, particularly Native Americans, include LSAMP student participation in this research project and a hands-on computer lab demonstration to middle school students. A tracking system will be put into place to follow the careers of research group alumni and will attempt to quantify the impact of undergraduate research upon the future careers of bachelor's degree students.

这个项目的目的是获得更深入的了解自然界如何构建酶的活性位点。该项目旨在建立多层酶活性位点的原理，并了解第一层外的残基如何影响催化活性和特异性。这种理解将对酶学和蛋白质工程有重要的意义。实验和计算旨在了解第一层以外的层如何有助于催化。近几十年来，结构和生物化学研究已经确定了直接参与底物结合和/或化学转化步骤的酶的活性位点中的残基。这些残基通常与反应底物分子直接接触，并且可以被认为位于围绕反应物质的“第一壳”中。该项目建立在初步证据的基础上，即第一个外壳之外的残留物对催化功能也很重要。这里的重点主要是第二层，与第一壳残基接触的残基。 系统的实验研究，以建立第二壳残基在酶催化的重要性，和计算研究，以了解他们在酶功能中发挥的各种作用将被追求。在这个项目中，定点诱变实验和动力学分析将进行选定的酶。这些蛋白质将被选择来代表不同种类的酶，其具有不同程度的第一壳外残基的预测参与。将测定突变体的晶体结构，以检测突变后的结构变化。将进行静电计算和分子动力学模拟，以了解第二壳层残基参与功能的机制。更好地了解酶如何影响催化作用有助于生产更清洁、更低成本的“绿色”工业过程，并有助于商业上可行的生物燃料酶促合成。学生将接受计算方法，蛋白质表达，突变，纯化，动力学测定和晶体结构测定的培训。该项目的一部分将被整合到本科化学生物学实验室课程。正在进行的推广工作，以少数民族学生，特别是美洲原住民，包括LSAMP学生参与这一研究项目和动手计算机实验室演示给中学生。将建立一个跟踪系统，跟踪研究小组校友的职业生涯，并试图量化本科研究对学士学位学生未来职业生涯的影响。