title: Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.
abstract: Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, Alan Turing Building, Oxford Road, Manchester M13 9PL (UK). alex.jones-2@manchester.ac.uk.
authors.forename: Alex R
authors.initials: AR
authors.lastname: Jones
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN (UK).
authors.forename: Julius
authors.initials: J
authors.lastname: Rentergent
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN (UK).
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN (UK). sam.hay@manchester.ac.uk.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
mesh_terms: D002384:Catalysis; D003038:Cobamides / Q000737:chemistry*; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular
chemical_list: D003038:Cobamides; D004981:Ethanolamine Ammonia-Lyase
keywords: coenzyme B12; enzymes; ethanolamine ammonia lyase; kinetic isotope effects; reaction mechanisms