Distal Residues in Enzyme Catalysis and Protein Design

标题：酶催化和蛋白质设计酶工程中的远端残基-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。这个项目寻求开发酶工程的设计原则，使用可以计算的蛋白质结构的性质。许多相同的反应，当在实验室或工业环境中进行时，需要高温和苛性条件。这项工作的一个重要的最终目标是能够设计出能够执行工业化学反应的蛋白质催化剂，因为对于大多数工业化学过程来说，没有天然的酶可以作为催化剂。这种用于工业用途的蛋白质催化剂的开发将转化为更少的能源消耗、更低的成本、更少的浪费和更少的有害副产品。因此，设计蛋白质催化剂的能力对环境、经济和人类福祉都有许多潜在的好处。这个项目将探索末端残基如何对酶催化做出贡献，建立关于它们在催化中的作用的额外原则，并迈出将这些原则用于酶设计的第一步。该项目采取了多边方法，结合了理论、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关于氨基酸残基(包括远程残基)对催化的静电、结构和动态影响的信息。这个项目中要研究的具体例子，Y家族DNA聚合酶DinB和醛缩酶，之所以被选中，是因为它们引入并提供了对蛋白质设计问题的洞察。远端残基对活性中心质子转移平衡的影响，以及相关的催化要求，将被调查。研究Y-家族DNA聚合酶中单个残基的作用有助于加深对DNA延伸在DNA复制和DNA损伤修复中的作用机制的理解。这些结果将被用来解决是否可以将改进的延伸能力工程化为聚合酶DinB。研究天然醛缩酶中残基之间的相互作用将增加对其催化机理的了解；新出现的原理将用于提高人工设计的反醛缩酶的活性。介绍了解决酶设计问题的新特征，即远端残基参与锻造正确的催化性质和使用耦合质子化状态的可预测性和重要性。设计能够催化任何所需化学反应的酶的能力在科学上是一个巨大的挑战。该项目将制定设计原则，以建立创造这种酶所必需的知识库。因此，将在该项目中开发和测试的酶设计原理对生物技术、环境修复、农业和“绿色”经济的发展以及化学工业具有潜在的影响。

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.