Distal Residues in Enzyme Catalysis and Protein Design

酶工程——设计蛋白质作为特定的、期望的化学反应的催化剂的能力——目前还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，为酶工程开发设计原则。该项目建立在最近发现的基础上，揭示了大自然如何构建蛋白质分子的催化中心的新信息，其中蛋白质结构中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，当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目标是能够设计出用于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使用，更低的成本，更少的浪费和更少的不必要的副产品。因此，设计蛋白质催化剂的能力对环境、经济和人类福祉有许多潜在的好处。该项目将培养博士生和本科生研究实习生，包括代表性不足的群体成员，成为生物化学和计算生物学领域的高素质科学家；培养这些专业知识对地区高科技经济和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，建立关于它们在催化中的作用的附加原则，并采取将这些原则用于酶设计的第一步。本项目采用理论、计算、生化实验、x射线晶体结构测定、x射线溶液散射、高场电子自旋共振光谱等多边化方法。这些模拟和实验将提供有关氨基酸残基（包括远端残基）对催化的静电、结构和动态影响的信息。本项目要研究的具体例子是y家族DNA聚合酶DinB和醛缩酶，之所以选择它们，是因为它们会导致蛋白质设计问题，并为蛋白质设计问题提供见解。远端残基对活性位点质子转移平衡的影响，以及催化的相关要求，将被研究。对y家族DNA聚合酶中单个残基的作用的研究将增加对DNA复制和损伤DNA修复中的延伸机制的理解。该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。研究天然醛缩酶中残基之间的相互作用将增加对其催化机理的理解；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，即在锻造正确的催化性能和耦合质子化状态的使用中，远端残基参与的可预测性和重要性。设计能够催化任何期望的化学反应的酶的能力在科学界是一个巨大的挑战。该项目将开发设计原则，建立在创造这种酶所必需的知识基础上。因此，在这个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”经济的增长以及化学工业都有潜在的影响。

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.