title: Methyl-coenzyme M reductase from methanogenic archaea: isotope effects on label exchange and ethane formation with the homologous substrate ethyl-coenzyme M.
abstract: Ethyl-coenzyme M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a homologous substrate for the enzyme methyl-coenzyme M reductase (MCR) resulting in the product ethane instead of methane. The catalytic reaction proceeds via an intermediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-limiting step, many cycles of intermediate formation and reconversion to substrate occur before a substantial amount of ethane is released. In deuterated buffer, the intermediate becomes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables intermediate formation to be detected. Here, we present a comprehensive analysis of this pre-equilibrium. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the time course of each isotopologue was followed by NMR spectroscopy. A kinetic simulation including kinetic isotope effects allowed determination of the primary and α- and β-secondary isotope effects for intermediate formation and for the C-H/C-D bond activation in the ethane-containing intermediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. Am. Chem. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus imply the same catalytic mechanism for both substrates. The experiment by Floss and co-workers, demonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is compatible with the observed rapid isotope exchange if the isotope effects measured here are taken into account.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich , Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
authors.forename: Silvan
authors.initials: S
authors.lastname: Scheller
authors.forename: Meike
authors.initials: M
authors.lastname: Goenrich
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Thauer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Jaun
mesh_terms: D004980:Ethane / Q000378:metabolism*; D007554:Isotopes; D007700:Kinetics; D015080:Mesna / Q000737:chemistry* / Q000378:metabolism*; D017021:Methanosarcina barkeri / Q000201:enzymology*; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017385:Sequence Homology
chemical_list: D007554:Isotopes; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D004980:Ethane; D015080:Mesna