Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Sam Hay, Michael J. Sutcliffe, Nigel S. Scrutton

    Research output: Contribution to journalArticlepeer-review


    Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s-1 at 25°C) and deuterium (12 to 31 s-1 at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of "barrier engineering" by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions. © 2007 by The National Academy of Sciences of the USA.
    Original languageEnglish
    Pages (from-to)507-512
    Number of pages5
    JournalProceedings of the National Academy of Sciences of the United States of America
    Issue number2
    Publication statusPublished - 9 Jan 2007


    • Flavoprotein
    • Hydrogen tunneling
    • Morphinone reductase
    • Pressure dependence
    • Stopped flow


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