Driving force analysis of proton tunnelling across a reactivity series for an enzyme-substrate complex.

Parvinder Hothi, Sam Hay, Anna Roujeinikova, Michael J. Sutcliffe, Michael Lee, David Leys, Paul M. Cullis, Nigel S. Scrutton

    Research output: Contribution to journalArticlepeer-review


    Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme-substrate complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.
    Original languageEnglish
    Pages (from-to)2839-2845
    Number of pages6
    JournalChemBioChem: a European journal of chemical biology
    Issue number17
    Publication statusPublished - 24 Nov 2008


    • amine oxidation
    • enzyme catalysis
    • isotope effects
    • proton tunnelling
    • quinoprotein
    • AZURIN


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