title: Enzymatic single-molecule kinetic isotope effects.
abstract: 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.
authors: Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors: Christopher R
authors: CR
authors: Pudney
authors: Richard S K
authors: RS
authors: Lane
authors: Alistair J
authors: AJ
authors: Fielding
authors: Steven W
authors: SW
authors: Magennis
authors: Sam
authors: S
authors: Hay
authors: Nigel S
authors: NS
authors: Scrutton
mesh_terms: D055162:Biocatalysis; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010088:Oxidoreductases / Q000378:metabolism*; D011789:Quantum Theory
chemical_list: D010088:Oxidoreductases; C098939:pentaerythritol tetranitrate reductase