Probing the high pressure effect on morphinone reductase using computational methods

Enzymes are highly efficient catalysts, achieving rate enhancements of up to 10 e21, however it is not fully understood how they work. Transition State Theory (TST) is the dominant model used to describe the catalytic power of enzymes. Although TST has proven to be successful for many biological reactions, a recent paradigm shift has attempted to incorporate the quantum behaviour of hydrogen into TST. The de Broglie wavelength of hydrogen is short enough that it needs to be considered in reactions that include hydrogen transfer - comprising over half of all known enzyme catalysed reactions. These transfers can occur by a partial or full quantum mechanical tunneling mechanism. Morphinone reductase (MR) is an enzyme which employs a deep tunneling mechanism in its reductive half-reaction ? the hydride transfer from the NADH-C4 atom to the FMN-N5 atom. This mechanism is thought to be assisted by a promoting vibration which decreases the distance between the donor and acceptor atoms. Experimental work using the pressure dependence of kinetic isotope effects (KIEs) as a probe for hydrogen tunneling has suggested an increase in pressure, from 1 bar to 2 kbar, decreases the donor-acceptor distance. Using pressure is a novel approach for probing hydrogen tunneling in enzymes, complementing the established use of temperature dependence. Pressure can be used as a tool to shift the equilibrium of systems ? in this instance it shifts the equilibrium of cofactor and substrate configurations towards the tunneling ready configuration. In this research, computational chemistry techniques are used to provide atomistic insight into the pressure effect on MR. Molecular Dynamics (MD) simulations were carried out to probe the rate of structural changes in MR as a function of pressure. The protein is shown to be stable across the range of pressures used. Trajectory analysis showed a decrease of 0.19 � in the average distance between the donor and acceptor atoms, as the pressure is increased from 1 bar to 2 kbar. This is due to the pressure restricting the conformational space in which the nicotinamide can move. These observations suggest that there is a compression of the reaction barrier as the pressure is increased. The research is extended to analyse the pressure effect on the active site residues in an attempt to identify which of these may be involved in the barrier compression. It is suggested that the pressure affects the configuration of several residues, and this combined effect is responsible for limiting the conformational space around the nicotinamide. The effect of the high pressure configuration on the reaction barrier is also studied, via a combined Quantum Mechanical/Molecular Mechanical (QM/MM) method. There is a decrease in the average barrier height for structures taken from the high pressure MD simulations, compared to the simulations at atmospheric pressure. This is accompanied by an increase in the KIE ? two observations that are consistent with experimental work. This work gives the first atomistic insight into the effect of pressure on the tunneling reaction, through the shortening of the donor-acceptor distance. It also provides an example of how pressure can be used as a probe to study a tunneling reaction, using a QM/MM method.