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.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis

“…The magnitude of can therefore be used as an excellent proxy for changes to the enzyme FEL, and more specifically for the changes in vibrational modes during enzyme turnover (conformational sampling). Pressure effects are system specific but should perturb a large as pressure affects the pre‐existing equilibrium of conformational states, favouring smaller volumes .…”

“…Numerically fitting these data then gives Δ H ‡ , Δ S ‡ , Δ G ‡ , , Δ V ‡ , Δβ ‡ and Δα ‡ reflecting the changes in enthalpy, entropy, Gibbs free energy, heat capacity, activation volume, compressibility and expansivity between the enzyme–substrate complex and the enzyme–transition state complex respectively. The enzyme kinetics for only a relatively few enzymes has been treated by combined p / T studies . In these cases, the p /T plane has not been fitted with a numerical model meaning at least and ∆α ‡ are not determined.…”

Quantum Biology: An Update and Perspective

“…Earlier laser photoexcitation studies, which investigated the temperature dependence of the KIEs, showed that both H-transfer reactions proceed by tunnelling and are coupled to promoting vibrations in the enzyme-substrate complex [100]. In addition, the KIEs associated with both the hydride and proton transfer reactions decrease significantly at higher pressures, which suggests that the generic pressure response of hydride and proton transfers are likely to be similar [90]. It has been proposed that the role of light in the overall reaction is to overcome the barrier for the highly unfavourable hydride transfer chemistry [100].…”

“…Since the zero-point energy, which arises from the high-energy stretching frequency of the breaking bond, is largely invariant with pressure (over the experimental range) [86,87], while donor-acceptor fluctuations can be affected, pressure-dependent KIEs are indicative of quantum tunnelling [88,89]. Unfortunately, no simple trends have emerged between the pressure-and temperature-dependence of KIEs [90]. For example, the KIE of morphinone reductase (MR) was found to increase with pressure while ∆∆H ‡ remained constant, while both the KIE and ∆∆H ‡ decreased with pressure in aromatic amine dehydrogenase (AADH).…”

Special Issue: Flavins and Flavoproteins

“…Structural and functional studies of oxidases , oxygenases and dehydrogenases are also reported. Other papers in the issue include studies of redox reactions in electron transfer flavoprotein and photoreceptors , enzyme inhibition for drug development , flavoenzyme for production of useful chemicals , regulation of riboflavin biosynthesis , and the use of physical tools and principles for interpreting protein dynamics and hydride transfer reactions .…”