Probing active site geometry using high pressure and secondary isotope effects in an enzyme-catalysed ‘deep’ H-tunneling reaction

Sam Hay, Christopher Pudney, Michael J Sutcliffe, Nigel S Scrutton

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12 Citations (Scopus)

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
Original languageEnglish
Pages (from-to)696-701
JournalJournal of Physical Organic Chemistry
Volume23
Issue number7
DOIs
Publication statusPublished - Jul 2010

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