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

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. Copyright © 2010 John Wiley & Sons, Ltd.