Probing substrate/inhibitor binding to metalloenzymes using EPR

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With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Professors Michael Crowder and David Tierney of Miami University to study substrate and inhibitor binding to metalloenzymes using the tools of EPR (electron paramagnetic resonance) spectroscopy. Once thought to be rigid molecules, it is now understood that enzymes--arguably Nature's most important biological molecules--are dynamic and that enzyme motions are essential for catalysis. This proposal describes novel methods to probe enzyme motions as the enzymes catalyze chemical reactions; these motions occur on timescales less than one one-thousandth of a second. The techniques developed in this project can, in principle, be applied to all enzymes, and the information gleaned can possibly be used to alter existing enzymes to catalyze different reactions. This project will allow for the training of a large number of undergraduate and graduate students in molecular biology, enzymology, and spectroscopy and be integrated into an outreach program that provides hands-on STEM activities for elementary school students.This research project will assess new ways to generalize the use of EPR-based techniques to determine inter-spin distances in otherwise inaccessible systems/states. The techniques are designed to access a wide range of processes that span microseconds to milliseconds at room temperature, from inter-subunit motions to the motion of unstructured loops relative to the substrate and active site metal ions. Ultimately, the method is to be used to determine how concerted motions track with important catalytic events. Specifically, the project involves experiments that determine the reliability of rapid-freeze quench (RFQ)-EPR-derived distance changes that accompany substrate binding and turnover in a series of paramagnetic analogs of the metallo-beta-lactamase NDM-1, freeze-quenched at reaction times ranging from around 100 microseconds to greater than 500 milliseconds. Parallel studies at both X- and Q-band will be conducted to ensure internal consistency and to guarantee access to the interactions. Experiments will also be conducted to examine the scope of spin-bearing species combinations that yield detectable, and interpretable, dipolar couplings from cw-EPR and DEER, and several unique combinations that are best suited for ESEEM interrogation. This goal will be accomplished by examining available permutations within a set of singly- and doubly spin-labeled proteins, selectively loaded with diamagnetic Zn(II)/Cd(II) or paramagnetic Co(II) in either side of the dinuclear active site, and spin-labeled substrate/inhibitor analogs, including several triply-labeled species.