title: Does the pressure dependence of kinetic isotope effects report usefully on dynamics in enzyme H-transfer reactions?
abstract: The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental probe of enzymatic H-transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H-transfer chemistry to the protein environment, the so-called 'promoting motions/vibrations hypothesis'. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass-modulated enzymes and time-resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi-classical KIEs, which are defined by differences in zero-point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature (p-T) dependence studies of H-transfer reactions are, however, limited. Here, we extend and review the available p-T studies that have utilized well-defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light-activated enzymes and chemical model systems. We show that there is no clear general trend between the p-T dependencies of the KIEs in these systems. Given the complex nature of p-T studies, we conclude that computational simulations using determined (e.g. X-ray) structures are also needed alongside experimental measurements of reaction rates/KIEs to guide the interpretation of p-T effects. In providing new insight into H-transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case-by-case basis. Although individually informative, we conclude that p-T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs.
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, UK.
authors.forename: Robin
authors.initials: R
authors.lastname: Hoeven
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, UK.
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, UK.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, UK.
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D003198:Computer Simulation; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry*; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D011312:Pressure; D011789:Quantum Theory; D013696:Temperature
chemical_list: D004798:Enzymes; D005420:Flavoproteins; D007554:Isotopes; D006859:Hydrogen; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037193:aromatic amine dehydrogenase
keywords: dynamics; flavoprotein; hydrogen transfer; pressure; quantum tunnelling