Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Abstract

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar ‫؍‬ 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s ؊1 at 25°C) and deuterium (12 to 31 s ؊1 at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of ''barrier engineering'' by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.flavoprotein ͉ hydrogen tunneling ͉ morphinone reductase ͉ pressure dependence ͉ stopped flow E nzymes are efficient catalysts that can achieve rate enhancements of up to 10 21 over the uncatalyzed reaction rate (1). Our quest to understand the physical basis of this catalytic power, which is pivotal to our understanding of biological reactions and our exploitation of enzymes in chemical, biomedical, and biotechnological processes, is challenging and has involved sustained and intensive research efforts for Ͼ100 years (for reviews see, e.g., refs. 2-6). Recent experimental studies employing the temperature dependence of kinetic isotope effects (KIEs) have emphasized the role of quantum mechanical tunneling in enzymatic hydrogen-transfer (H-transfer) reactions (7-18). These data, which can be explained neither by conventional transition state theory (TST) nor by the Bell tunneling correction to TST (19), are in agreement with environmentally coupled models of hydrogen tunneling (H-tunneling) (12,20,21) and can be simulated computationally (22-30) within the framework of modern TST (31). In the Kuznetsov and Ulstrup environmentally coupled model of H-tunneling (20), as used quantitatively by Klinman and coworkers (32), temperatureindependent KIEs arise from Marcus-like (33) vibrations (collective thermally equilibrated motions) that lead to degenerate reactant and product states, whereas temperature-dependent KIEs arise from ''gating motions'' (motion along the reaction coordinate) that enhance the probability of tunneling at this configuration by ...

Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

“…[1] A number of recent experimental studies employing the temperature- [2][3][4][5][6][7][8][9][10][11][12][13] and pressure-dependence [14,15] of kinetic isotope effects (KIEs) have emphasized the role of quantum mechanical tunnelling in enzymatic hydrogen-transfer reactions. While some of these data have been explained by Bell-type corrections [16] to semi-classical transition state (TS) theory, or attributed to mechanical effects in the TS, [17,18] other data can only be described by environmentally-coupled tunnelling models, [19][20][21][22] where promoting motions in the enzyme are coupled to the reaction coordinate.…”

The enzyme aromatic amine dehydrogenase induces a substrate conformation crucial for promoting vibration that significantly reduces the effective potential energy barrier to proton transfer

“…Static protein structures show only the lowest energy or 'groundstate' conformation and the time averaged conformational ensemble, yet multiple protein conformations may exist in thermal equilibrium and function may depend on excursions to sparsely populated higher energy conformations of the enzyme and its substrates. Moreover, there is increasing recognition of the importance of non-equilibrated fast (sub-picosecond) dynamics in modifying the free energy barrier for a reaction (Caratzoulas et al 2002;Knapp et al 2002;Garcia-Viloca et al 2003;Masgrau et al 2006;Dybala-Defratyka et al 2007;Hay et al 2007;Johannissen et al 2007), which by definition influence the rate of an enzyme-catalysed reaction. There is poor understanding of structural change associated with this motion owing to the technical limitations of current structural biology methods (Wand 2001).…”

Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift

“…For example, the flash decay rate of firefly luciferase is reduced (5), the oxygen binding affinity of human hemoglobin is doubled (6), and oxidation rates by morphinone reductase are increased (7). Protein atomic structures at up to a few hundred MPa (8)(9)(10)(11)(12)(13)(14)(15)(16) indicate that atoms in protein molecules are typically displaced by Ϸ0.1-1.0 Å from their ambient pressure positions.…”