title: Alpha-secondary isotope effects as probes of "tunneling-ready" configurations in enzymatic H-tunneling: insight from environmentally coupled tunneling models.
abstract: Using alpha-secondary kinetic isotope effects (2 degrees KIEs) in conjunction with primary (1 degrees ) KIEs, we have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate reductase (PETNR). We find exalted 2 degrees KIEs (1.17-1.18) for both enzymes, consistent with hydrogen tunneling. These 2 degrees KIEs, unlike 1 degrees KIEs, are independent of promoting motions-a nonequilibrium pre-organization of cofactor and active site residues that is required to bring the reactants into a "tunneling-ready" configuration. That these 2 degrees KIEs are identical suggests the geometries of the "tunneling-ready" configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a clearly temperature-dependent 1 degrees KIE. The work emphasizes the benefit of combining studies of 1 degrees and 2 degrees KIEs to report on pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7ND, United Kingdom.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Pudney
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D004798:Enzymes / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry
chemical_list: D004798:Enzymes; D009243:NAD; D009249:NADP