Role of Coupled Amino Acids in the Mechanisms of Enzyme Catalysis

这个项目开发了一些概念，将提高我们对酶如何工作的理解。酶是自然界的催化剂，能在温和的条件下发生化学反应。在实验室工作台或工业反应堆上进行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使反应在环境温度或体温和中性条件下发生。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。其目标是建立一个知识库，使酶的设计能够以比传统工艺更少的能源消耗和更少的副产品来催化工业化学反应。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，这一培训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。所有开发的方法和程序，包括生成的所有代码和数据，都将免费提供给社区。为了获得与天然酶相媲美的催化特性，工程酶必须包含与天然酶相同的静电、化学和动态特性。该项目将理论、计算和生化实验相结合，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，以确定随着催化能力从最初的设计增加到最新、最有效的催化剂，哪些特性正在发展。人的磷酸葡萄糖异构酶(PGI)是一种已有的实验结果表明，它的几个氨基酸，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。将对PGI进行详细分析，以确定这些氨基酸如何帮助催化。最后，从结核分枝杆菌(Mt)的一种功能未知的酶开始，它的异构酶活性较弱，但结构与恶臭假单胞菌的高活性酮类类固醇异构酶高度相似，将使用计算工程来提高Mt酶中的异构酶活性，并将通过直接生化分析来测量工程变体的活性。该奖项由分子和细胞生物科学(MCB)部门的分子生物物理学集群和化学(CHE)部门的生命过程化学项目共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的审查标准进行评估，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.