Abstract
The evolution and exploitation of dynamical processes — the coupling of specific protein motion to the reaction coordinate—remains a key question in the studies of enzyme mechanism.[1–4] It is now accepted that hydrogen (proton, hydrogen or hydride)-tunneling reactions often occur during enzyme-catalyzed reactions, [1, 5–10] but the role of non-equilibrium dynamical processes (or promoting motions) remains contentious.[3] This is despite compelling evidence from a number of kinetic and computational studies of such motions.[11–16] The experimental identification of promoting motions (or more generally environmental coupling) is challenging. Experimental evidence supporting a general role for protein motions in enzymatic H-tunnelling reactions has been gained primarily from observations of kinetic isotope effects (KIEs) with unusual temperature-dependences.[17–19] The most extreme cases of an unusual temperature-dependence of a primary (18) KIE are those with a ‘breakpoint’, where the KIE is weakly temperature-dependent at high temperatures and becomes strongly temperature-dependent below a critical breakpoint temperature. Such a trend has been demonstrated in two enzyme-catalyzed reactions: 1) the hydride transfer from benzyl alcohol to NAD+ catalyzed by the thermophilic alcohol dehydrogenase (ADH-ht) from Bacillus stearothermophilus, [18] and 2) the hydride transfer from NADPH to dihydrofolate catalyzed by the thermophilic dihydrofolate reductase (TmDHFR) from Thermotoga maritima. [19] Herein we explore the usefulness of a-secondary (28) KIEs (their magnitude and temperature dependence) as probes of environmentally-coupled H-transfer reactions in enzyme systems