CAREER: Establishing the Roles of Multi-enzyme Complexes in Metabolic Network Regulation

在代谢途径中催化顺序反应的酶经常形成多酶复合物。这种复合物允许中间代谢物在酶之间传递。理论和基于试管的实验研究表明，多酶复合物的形成在代谢网络的调节中起重要作用。然而，尽管经过40年的研究，多酶复合物在活细胞内的功能仍存在争议，而且由于难以控制这些复合物所在的高度可变的亚细胞微环境，因此难以证明其功能。本项目的目标是通过描述多酶复合物作为活细胞代谢过程调节剂的功能和作用来解决这一争议。所获得的知识将有助于科学家了解生物体如何在波动环境下维持其代谢稳态，并促进合成生物学应用的关键途径的代谢工程。作为该项目更广泛影响的一部分，研究者将开发一种创造性的纸工艺实践活动，向广泛的受众（包括本科生、高中生和成年人）教授基于酶的代谢调节原理。这种新颖的更广泛的影响活动将积极影响新陈代谢的素养，并增加公众对代谢研究的参与。此外，研究者将通过该项目的研究和教育活动培训研究生和本科生。快速、灵活和协调的代谢调节是所有生物对其不断变化的环境作出反应所必需的，这些环境有时在几秒钟内波动。目前已知的分子机制还不能完全解释这一调控过程。本项目将以酵母三羧酸（TCA）循环中苹果酸脱氢酶/柠檬酸合成酶/乌头酸酶多酶复合物的功能为模型系统进行研究。本项目的总体目标是通过实验确定TCA循环多酶复合物在酵母细胞代谢调节中的作用。研究者将探索的假设是，TCA循环多酶复合物相对于相关酶复合物的比例增强和/或重定向代谢通量。通过两个研究目标来评估TCA循环中酶关联率与代谢通量动力学之间的双向关系：1)评估多酶复合物亲和力变化对代谢通量的影响，2)评估代谢通量动力学变化对多酶复合物形成的影响。利用同位素示踪实验和定量建模方法分析TCA循环和邻近途径中的代谢通量。酶复合物关联将使用多种技术进行量化，包括一种新型的基于生物发光的实时分析，这将作为该项目的一部分开发。这项工作的结果将增强对生化系统中酶动力学与通量动力学耦合的基本理解，并进一步阐明亚细胞微环境对系统水平结果的影响。该奖项反映了美国国家科学基金会的法定使命，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。

Enzymes that catalyze sequential reactions in a metabolic pathway often form a multi-enzyme complex. The complex allows intermediate metabolites to be channeled between enzymes. Theoretical and test tube-based experimental studies have shown that multi-enzyme complex formation is important in the regulation of metabolic networks. However, despite 40 years of research, the function of multi-enzyme complexes within a living cell are controversial and difficult to demonstrate due to difficulties controlling highly variable subcellular micro-environments, where these complexes reside. The goal of this project is to address this controversy by characterizing the function and role of multi-enzyme complexes as regulators of metabolic processes in living cells. The knowledge gained will help scientists understand how organisms maintain their metabolic homeostasis under fluctuating environments, and facilitate metabolic engineering of key pathways for synthetic biology applications. As part of the broader impacts of the project, the investigator will develop a creative paper craft hands-on activity to teach principles of enzyme-based metabolic regulation to a wide audience, including undergraduate and high school students, and adult audiences. This novel broader impact activity will positively impact literacy in metabolism and increase public engagement with metabolic research. In addition, the investigator will train graduate and undergraduate students through the project's research and educational activities. Rapid, flexible and coordinated regulation of metabolism is essential for all living organisms to respond to their changing environments which fluctuate sometimes within an order of seconds. Currently known molecular mechanisms cannot fully explain this regulatory process. In this project, the functionality of the malate dehydrogenase/ citrate synthase/aconitase multi-enzyme complex of the yeast Krebs tricarboxylic acid (TCA) cycle will be investigated as a model system. The overarching goal of this project is to experimentally establish the roles of the TCA cycle multi-enzyme complex in metabolic regulation in yeast cells. The hypothesis the investigator will explore is that the TCA cycle multi-enzyme complex enhances and/or redirects metabolic flux relative to the ratio of the associated enzyme complex. The bi-directional relationship between the rate of enzyme association and metabolic flux dynamics within the TCA cycle will be assessed through two research objectives: 1) assessment of the effects of variations in multi-enzyme complex affinity on metabolic flux, and 2) assessment of the effects of changes in metabolic flux dynamics on multi-enzyme complex formation. Metabolic flux in the TCA cycle and adjacent pathways will be analyzed using isotope tracer experiments and quantitative modeling methods. Enzyme complex association will be quantified using multiple techniques, including a novel bioluminescence-based real-time assay, which will be developed as part of this project. Results of this work will enhance the fundamental understanding of how enzyme kinetics couples with flux dynamics in a biochemical system, and further clarify the impact of subcellular micro-environments on systems level outcomes.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.