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Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex
Author(s) -
Hothi Parvinder,
Hay Sam,
Roujeinikova Anna,
Sutcliffe Michael J.,
Lee Michael,
Leys David,
Cullis Paul M.,
Scrutton Nigel S.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800408
Subject(s) - chemistry , kinetic isotope effect , quantum tunnelling , computational chemistry , proton , free energy perturbation , stereochemistry , molecular dynamics , atomic physics , deuterium , quantum mechanics , physics
Abstract Quantitative structure‐activity relationships are widely used to probe CH bond breakage by quinoprotein enzymes. [1–4] 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 CH bond breakage. [5] 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 (∼16) accompanying CH/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 CH 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.