Enzymatic single-molecule kinetic isotope effects

Christopher R. Pudney, Richard S K Lane, Alistair J. Fielding, Steven W. Magennis, Sam Hay, Nigel S. Scrutton

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    Ensemble-based measurements of kinetic isotope effects (KIEs) have advanced physical understanding of enzyme-catalyzed reactions, but controversies remain. KIEs are used as reporters of rate-limiting H-transfer steps, quantum mechanical tunnelling, dynamics and multiple reactive states. Single molecule (SM) enzymatic KIEs could provide new information on the physical basis of enzyme catalysis. Here, single pair fluorescence energy transfer (spFRET) was used to measure SM enzymatic KIEs on the H-transfer catalyzed by the enzyme pentaerythritol tetranitrate reductase. We evaluated a range of methods for extracting the SM KIE from single molecule spFRET time traces. The SM KIE enabled separation of contributions from nonenzymatic protein and fluorophore processes and H-transfer reactions. Our work demonstrates SM KIE analysis as a new method for deconvolving reaction chemistry from intrinsic dynamics. © 2013 American Chemical Society.
    Original languageEnglish
    Pages (from-to)3855-3864
    Number of pages9
    JournalJournal of the American Chemical Society
    Issue number10
    Publication statusPublished - 13 Mar 2013


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