title: Probing active site geometry using high pressure and secondary isotope effects in an enzyme-catalysed 'deep' H-tunnelling reaction.
abstract: We report the first study of the effects of hydrostatic pressure on α-2° KIEs for an enzyme-catalysed H-transfer reaction that occurs by 'deep' tunnelling. High pressure causes a significant decrease in the observed α-2° KIE on the pre-steady-state hydride transfer from NADH to FMN in the flavoprotein morphinone reductase. We have recently shown that high pressure causes a reduction in macroscopic reaction barrier width for this reaction. Using DFT vibrational analysis of a simple active site model, we posit that the decrease in α-2° KIE with pressure may arise due to a decrease in the vibrational coupling between the NADH primary (transferred) and secondary hydrogens in the 'tunnelling ready configuration', which more closely resembles the reactant state than the transition state.
authors.affiliation: Manchester Interdisciplinary Biocentre and Faculty of Life Science, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Pudney
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton