Probing active site geometry using high pressure and secondary isotope effects in an enzyme-catalysed 'deep' H-tunnelling reaction |
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Authors: | Hay Sam Pudney Christopher R Sutcliffe Michael J Scrutton Nigel S |
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Institution: | Manchester Interdisciplinary Biocentre and Faculty of Life Science, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK. |
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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. |
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