Role of Coupled Amino Acids in the Mechanisms of Enzyme Catalysis

该项目开发的概念将提高我们对酶如何工作的理解。酶是自然界的催化剂，使化学反应在温和的条件下发生。在实验室工作台或工业反应器上的相同反应可能需要高温和/或强酸或强碱。然而，每个生命系统中的酶都能在环境温度或体温以及中性条件下发生反应。该项目将为酶如何实现这一壮举提供有价值的新信息，并反过来为酶工程领域提供信息。目标是建立一个知识库，使酶的设计能够催化工业化学反应，比传统工艺能耗更低，副产物更少。数十名学生将在研究实验室和教室接受计算和实验技术培训，这对该地区的高科技经济和美国在全球经济中的竞争力至关重要。该项目将包括来自服务不足社区的学生。开发的所有方法和程序，包括生成的所有代码和数据将免费提供给社区。为了获得与天然酶相当的催化性能，工程酶必须具有与天然酶相同的静电、化学和动力学特性。该项目结合了理论，计算和生化实验，以实现三个目标。首先，将对进化的、设计的酶的途径进行回顾性研究，以确定哪些特性正在发展，因为催化能力从最初的设计增加到最新的、最有效的催化剂。磷酸葡萄糖异构酶（英语：Phosphoglucose isomerase，PGI）是一种酶，其实验结果已经表明，它的几种氨基酸，包括一些不直接与反应分子接触的氨基酸，参与了催化过程。PGI将被详细分析，以确定这些氨基酸中的每一种如何帮助催化。最后，从来自结核分枝杆菌（Mt）的未知功能的酶开始，该酶具有弱异构酶活性，但与来自恶臭假单胞菌的高活性酶甾酮异构酶具有高度结构相似性，计算工程将用于提高Mt酶中异构酶的活性，工程变体的活性将通过直接生物化学测定来测量。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project develops concepts that will improve our understanding of how enzymes work. Enzymes are nature’s catalysts that make chemical reactions happen under mild conditions. The same reactions on a laboratory bench or industrial reactor might require high temperature and/or strong acid or strong base. Yet enzymes in every living system enable reactions to happen at ambient temperature, or body temperature, and neutral conditions. This project will provide valuable new information into how enzymes achieve this feat and, in turn, inform the field of enzyme engineering. The goal is to build a knowledge base that will enable the design of enzymes that can catalyze industrial chemical reactions with less energy consumption and fewer by-products than conventional processes. Several dozen students, in research laboratories and classrooms, will be trained in computational and experimental techniques, a training vital to the high-tech economy of the region and U.S. competitiveness in the global economy. The project will include students from underserved communities. All methods and programs developed, including all code and data generated will be made freely available to the community. To achieve catalytic properties comparable to those of natural enzymes, engineered enzymes must incorporate the same kinds of electrostatic, chemical, and dynamic properties that occur in natural enzymes. This project combines theory, computation, and biochemical experiments to achieve three aims. First, a retrospective study will be performed of the pathways of evolved, designed enzymes to establish which properties are developing, as catalytic capabilities increase from the initial designs to the most recent, most effective catalysts. The human enzyme phosphoglucose isomerase (PGI) is an enzyme for which experimental results already exist to show that several of its amino acids, including some not in direct contact with the reacting molecules, participate in the catalytic process. PGI will be analyzed in detail to determine how each of these amino acids helps catalysis. Finally, starting with an enzyme of unknown function from Mycobacterium tuberculosis (Mt) with weak isomerase activity but high structural similarity to the highly active enzyme ketosteroid isomerase from Pseudomonas putida, computational engineering will be used to increase isomerase activity in the Mt enzyme and the activity of the engineered Variants will be measured through direct biochemical assay.This award was jointly funded by the Molecular Biophysics Cluster in the Molecular and Cellular Biosciences (MCB) Division and the Chemistry of Life Processes program in the Chemistry (CHE) Division.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.