Synchronizing enzyme-mediated radical chemistry with domain motion

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The research outlined in this proposal examines two relatively new and uncharted areas of enzymology: (1) radical-based reaction mechanisms and (2) the role of protein conformational motion in directing catalysis. A recent upsurge in genome sequencing and improved detection techniques has led to a rapid increase in the number of enzymes known or hypothesized to proceed by radical-based mechanisms. As many of these enzymes are key players in important biological processes, it is critical for us to understand, not only how they have evolved to control the trajectories of radicals, but also how this type of chemistry is exquisitely choreographed with varying levels and frequencies of protein motion. Using a coenzyme B12-dependent enzyme as a model system, the research program will strive to derive models for how these types of enzymes operate. The studies will compare the atomic structure of the enzyme "poised" at different states during catalysis, in order to pinpoint aspects of the protein that may play a role in radical stability and/or its dynamic properties. These structural features will be altered and the kinetic and functional behavior of the enzyme will be analyzed using spectroscopic (UV-visible and fluorescence) and fast reaction techniques. In collaboration with internationally recognized inorganic chemists (specializing in synthesis and design of radical-based catalysts) at UBC-Okanagan, novel coenzyme B12 analogs will be created that will allow us to investigate the reactive nature of the molecule. An understanding of how enzymes deploy radical-based chemistry is the first step towards engineering them to perform desired reactions that are applicable to industry and drug design, an objective that will ultimately improve the quality of life for Canadians in the future. Improved models for radical-based catalysis can also be applied to more medically relevant systems, that are often less accessible for investigation. Finally, the research will make a significant contribution to the field of enzymology, as it examines enzyme catalysis, not just in the context of "structure defines function" paradigm, but also from the emerging concept that conformational dynamics plays an integral role in its catalytic behavior.