Role of Coupled Amino Acids in the Mechanisms of Enzyme Catalysis

Northeastern University, Boston MA

Investigators

Abstract

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.