Are Enzyme Active Sites Built in Multiple Layers?

这个项目的目标是更深入地了解大自然是如何构建酶活性部位的。该项目旨在建立多层酶活性位点的原理，并了解第一层以外的残基如何影响催化活性和特异性。这一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层以外的层是如何对催化做出贡献的。近几十年来，结构和生化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活性部位的残基。这些残基通常与反应底物分子直接接触，并可被认为位于反应物种周围的“第一层壳”中。该项目建立在初步证据的基础上，即第一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，即与第一层壳残基接触的残基。将进行系统的实验研究，以确定第二壳残基在酶催化中的重要性，并进行计算研究，以了解它们在酶功能中扮演的不同角色。在这个项目中，将对选定的酶进行定点突变实验和动力学分析。这些蛋白质将被选为代表不同种类的酶，具有不同程度的预测参与第一壳外的残基。突变体的晶体结构将被确定，以测试突变后的结构变化。将进行静电学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。更好地了解酶是如何影响催化作用的，有助于产生更清洁、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的酶促合成。学生将接受计算方法、蛋白质表达、突变、纯化、动力学分析和晶体结构测定方面的培训。该项目的一部分将被整合到本科化学生物学实验室课程中。正在进行的针对少数族裔学生，特别是美洲原住民的外展努力，包括LSAMP学生参与这一研究项目，以及向中学生进行动手计算机实验室演示。将建立一个跟踪系统来跟踪研究组校友的职业生涯，并将试图量化本科生研究对学士学位学生未来职业生涯的影响。

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.