Pressure Effects on Enzyme-Catalyzed Quantum Tunneling Events Arise from Protein-Specific Structural and Dynamic Changes Sam Hay,* ,†,‡ Linus O. Johannissen, †,§ Parvinder Hothi, †,‡ Michael J. Sutcliffe, †,§ and Nigel S. Scrutton* ,†,‡ † Manchester Interdisciplinary Biocentre, ‡ Faculty of Life Sciences, and § School of Chemical Engineering and Analytical Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. * S Supporting Information ABSTRACT: The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor -acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature depend- ences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions. ■ INTRODUCTION Kinetic isotope effects (KIEs) are a useful probe of reaction mechanism, and inflated intrinsic KIEs remain the definitive hallmark of quantum mechanical hydrogen tunneling in enzymes. 1-3 The possibilities that tunneling during enzyme- catalyzed reactions may be catalytic and/or can be enhanced by the dynamic coupling of the H-transfer reaction coordinate to the environmenti.e., by promoting vibrationsremains contentious 4-7 and awaits a definitive experimental test. The observation of strongly temperature-dependent KIEs has been used to infer such environmental coupling, 8-10 but other experimental probes are needed. One potential probe is hydrostatic pressure. As semiclassical KIEs arise due to differences in vibrational zero-point energy, which have been shown to be insensitive to several kbar changes in pressure (the typical experimental range), 11,12 the pressure dependence of a KIE has been used as evidence for H-tunneling. 13-15 We have extended this approach to infer environmental coupling from the combined pressure and temperature (p-T) dependence of H-transfer reactions. 15-17 While p-T pressure-jump experi- ments are now established as a useful method of probing the free energy landscape of, e.g., protein folding, 18 the utility of p- T experiments as a probe of tunneling and/or environmental coupling during enzymatic H-transfer remains uncertain as, to date, this approach has only been used to study a small subset of enzymatic reactionshydride transfers catalyzed by a small number of reductase and dehydrogenase enzymes. 15,17,19,20 To investigate more generally the utility of variable pressure studies in probing such reactions, we have extended the p-T approach here to study an unrelated enzyme reaction, proton transfer during the reductive-half reaction (RHR) of bacterial aromatic amine dehydrogenase (AADH) with phenylethylamine (PEA). The RHR of AADH involves a rate-limiting proton transfer from a tryptophan tryptophylquinone (TTQ)-substrate iminoquinone adduct to an active-site aspartate (Scheme 1). 21-23 The RHR with the substrate tryptamine exhibits a H/D KIE of about 55one of the largest proton KIEs observed in an enzyme 21 and the proton transfer has a large tunneling component 21,24 assisted by a putative promoting vibration. 22,24,25 The RHR with para-substituted PEAs is about 100-fold slower (yet appears to be fully rate-limiting) than with tryptamine, possibly due to a reduction in the reaction driving force. 23 The KIE is also smaller (∼1020) with para- substituted PEAs and, unlike the reaction with tryptamine, is measurably temperature-dependent to varying degrees, depend- ing on the substrate and buffer conditions. 23 In this report, we characterize the p-T dependence of the RHR of AADH with PEA. We show that the origin of the pressure dependence of KIEs can be more complex than previously reported, attributed to anisotropic protein (de)compression mediated by hydro- static pressure. This has important implications for under- Received: March 12, 2012 Published: May 26, 2012 Article pubs.acs.org/JACS © 2012 American Chemical Society 9749 dx.doi.org/10.1021/ja3024115 | J. Am. Chem. Soc. 2012, 134, 9749-9754