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.

该项目的目的是对自然如何构建酶的活性位点进行更深入的了解。该项目旨在建立多层酶活性位点的原理，并了解第一层之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具有重要意义。实验和计算旨在了解第一个层以外的层如何促进催化。在近几十年中，结构和生化研究已经确定了直接参与化学转化步骤中底物结合和/或直接参与底物结合的酶的活性位点的残基。这些残基通常与反应底物分子直接接触，并且可能被认为位于反应物种周围的“第一壳”中。该项目基于初步证据，表明第一个外壳以外的残基对催化功能也很重要。这里的重点主要是第二层，即与第一壳残基接触的残基。 系统的实验研究确定了第二壳残基在酶催化中的重要性，以及计算研究以了解它们在酶促功能中所起的各种作用。在该项目中，将对选定的酶进行定向的诱变实验和动力学测定。这些蛋白质将被选为代表不同种类的酶，这些酶具有不同程度的预测参与的酶，这是第一个壳以外的残基。突变体的晶体结构将确定突变后测试结构变化。将进行静电计算和分子动力学模拟，以了解第二壳残基参与功能的机制。更好地理解酶如何影响催化可以帮助产生更清洁的，较低的成本，“绿色”工业过程以及生物燃料的商业可行酶合成。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方面的培训。该项目的一部分将集成到本科化学生物学实验室课程中。持续向少数族裔学生，特别是美洲原住民的宣传工作包括LSAMP学生参与该研究项目以及向中学生进行动手实验室实验室演示。将建立跟踪系统以跟随研究小组校友的职业，并试图量化本科研究对学士学位学生未来职业的影响。