Distal Residues in Enzyme Catalysis and Protein Design

标题：酶催化和蛋白质设计中的远端残基酶工程——设计蛋白质作为特定的、所需的化学反应的催化剂的能力——目前处于非常早期的阶段。该项目旨在利用可计算的蛋白质结构特性来开发酶工程的设计原理。该项目建立在最近的发现之上，揭示了大自然如何构建蛋白质分子催化中心的新信息，其中蛋白质结构内的多层氨基酸提供了必要的特性，使化学反应能够在生理温度和温和条件下在生物体内发生。许多相同的反应在实验室或工业环境中进行时，需要高温和腐蚀性条件。这项工作的一个重要的、最终的目标是能够设计蛋白质催化剂来执行工业化学反应，因为对于大多数工业化学过程来说，没有天然酶可以充当催化剂。开发这种工业用途的蛋白质催化剂将减少能源消耗、降低成本、减少浪费和减少不需要的副产品。因此，设计蛋白质催化剂的能力对环境、经济和人类福祉具有许多潜在的好处。该项目将培训博士生和本科生研究实习生，包括代表性不足群体的成员，成为生物化学和计算生物学领域的高素质科学家；培养此类专业知识对于地区高科技经济以及美国在全球经济中的竞争力至关重要。该项目将探索远端残基如何促进酶催化，建立有关其在催化中的作用的其他原则，并迈出使用这些原则进行酶设计的第一步。该项目采用多边方法，结合理论、计算、生化实验、X射线晶体结构测定、X射线溶液散射和高场电子自旋共振光谱。这些模拟和实验将提供有关氨基酸残基（包括远程残基）对催化的静电、结构和动态影响的信息。选择本项目中要研究的具体例子，Y 家族 DNA 聚合酶 DinB 和醛缩酶，是因为它们会导致并提供对蛋白质设计问题的深入了解。将研究远端残基对活性位点质子转移平衡的影响以及相关的催化要求。对 Y 家族 DNA 聚合酶中各个残基的作用的研究将增加对 DNA 复制和受损 DNA 修复延伸机制的理解。结果将用于解决是否可以将改进的延伸能力设计到聚合酶 DinB 中。研究天然醛缩酶残基之间的相互作用将增加对其催化机制的理解；新出现的原理将用于提高人工设计的逆醛醇酶的活性。介绍了解决酶设计问题的新特征，即远端残基参与形成正确催化特性的可预测性和重要性以及耦合质子化态的使用。设计能够催化任何所需化学反应的酶的能力是科学上的巨大挑战。该项目将开发设计原则，以创建此类酶所需的知识库为基础。因此，该项目中开发和测试的酶设计原理对生物技术、环境修复、农业、“绿色”经济以及化学工业的增长具有潜在影响。

Title: Distal Residues in Enzyme Catalysis and Protein DesignEnzyme engineering -the capability to design proteins to act as catalysts for particular, desired chemical reactions- is currently in its very early stages. This project seeks to develop design principles for enzyme engineering, using properties of the protein structure that can be computed. This project builds on very recent discoveries that reveal new information about how nature builds the catalytic center of a protein molecule, wherein multiple layers of amino acids within the protein structure provide the necessary properties that enable chemical reactions to happen within living organisms at physiological temperature and under mild conditions. Many of these same reactions, when performed in a laboratory or industrial setting, require high temperature and caustic conditions. An important, ultimate goal of this work is to be able to design protein catalysts to perform industrial chemical reactions, because for most industrial chemical processes, there is no natural enzyme that can serve as a catalyst. The development of such protein catalysts for industrial use will translate to less energy usage, lower costs, less waste, and fewer unwanted by-products. Thus the ability to design protein catalysts has many potential benefits to the environment, to the economy, and to human well-being. This project will train doctoral students and undergraduate research interns, including members of underrepresented groups, to become highly qualified scientists in the areas of biochemistry and computational biology; the cultivation of such expertise is vital to the regional high-tech economy and to U.S. competitiveness in the global economy. This project will explore how distal residues contribute to enzyme catalysis, establish additional principles about their role in catalysis, and take the first steps toward using these principles for enzyme design. The project takes a multilateral approach, combining theory, computation, biochemical experiments, x-ray crystal structure determination, x-ray solution scattering, and high-field electron spin resonance spectroscopy. These simulations and experiments will provide information about the electrostatic, structural, and dynamic effects of amino acid residues, including remote residues, on catalysis. The specific examples to be studied in this project, a Y-family DNA polymerase DinB and an aldolase, were chosen because they lead into - and provide insight into - protein design problems. The effects of distal residues on proton transfer equilibria in the active site, and the associated requirements for catalysis, will be investigated. Study of the roles of individual residues in Y-family DNA polymerases will increase understanding of the mechanism of extension in DNA replication and repair of damaged DNA. The results will be used to address whether improved extension capability can be engineered into the polymerase DinB. Investigation of the interactions between residues in a natural aldolase will increase understanding of its catalytic mechanism; the emerging principles will be used to improve activity of an artificially designed retroaldolase. New features to address the problem of enzyme design, namely the predictability and importance of distal residue participation in forging the right catalytic properties and the use of coupled protonation states, are introduced. The capability to design enzymes that can catalyze any desired chemical reaction is a grand challenge in science. This project will develop design principles to build on the knowledge base that is necessary to create such enzymes. Enzyme design principles to be developed and tested in this project thus have potential impact on biotechnology, environmental remediation, agriculture, and the growth of a "green" economy, as well as the chemical industry.