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 degrees C) and deuterium (12 to 31 s(-1) at 25 degrees C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25 degrees 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.

Fig. 1.

The reductive half-reaction of MR at high pressure. (A) MR (20 μM) mixed with NADH (5 mM) at 2 kbar and 10°C. (Inset) Traces at 464 nm (marked with an arrow) extracted at 1 bar (i) and 2 kbar (ii). (B) Spectral deconvolution of the reaction of 20 μM MR mixed with 50 μM NADH at 2 kbar and 10°C by using an irreversible two-step reaction (a→b→c), where a is the fully oxidized enzyme, b is the enzyme–NADH CT complex, and c is the reduced enzyme. (Inset) The pressure dependence of the rate of CT formation (b) measured at 552 nm. This rate constant is second-order, and these values were determined at 50, 100, and 250 μM NADH concentrations.

Fig. 2.

The combined pressure and temperature dependencies of the reductive half-reaction of MR with NADH and NAD²H. (A) Plot of k_red versus p for the reaction with both cofactors at the limits of the temperature range, 5°C and 40°C. The solid lines are fits to Eq. 1; note the log scale for k. (B) Eyring plots of the reaction at atmospheric pressure and at 2 kbar. (C) The combined effect of pressure and temperature on the resulting KIE.

Fig. 3.

The effect of pressure on an environmentally coupled H-tunneling reaction. (A) The Kuznetov and Ulstrup model (20) (Eq. 5) showing the effects of the force constant of the gating mode (k_HO) and the donor–acceptor separation (r₀) on the KIE. If pressure reduces r₀ but not ΔΔH^‡, then the observed KIE will increase (red arrow, see text). (B) Orthogonal views of a model of the active site of MR based on the structure of Old Yellow Enzyme (Protein Data Bank ID code OYEA; see ref. 50) We propose that pressure will reduce the separation of the nicotinamide and flavin heavy atoms, [r₀(p)] along the gating coordinate X. Note that neither the compression of the nicotinamide/flavin separation nor the gating motion of the nicotinamide and flavin need to be exclusively along the gating coordinate X, but Δp (a vectoral component of any compression) and X (in the figure) need to be parallel.

Fig. 4.

Correlation of observed KIEs with calculated r₀ values as a function of pressure. (A) The calculated KIE dependence on r₀ from Eqs. 4 and 5 (shown in Fig. 3) when ΔΔH^‡ is fixed to 5.2 kJ·mol⁻¹. (B) The measured KIE versus p at 25°C. The solid line is a fit to KIE = k_0,H/k_0,D exp(−ΔΔV^‡p/R_pT). The increase in observed KIE is consistent with a decrease in the equilibrium separation, r₀, from ≈1.7 Å to ≈1.0 Å (depicted by the arrow).