Elucidation of the mechanism and the free energy of a Morita-Baylis-Hillman type reaction catalyzed by enzymes using transition path sampling.

Abstract

Transition path sampling (TPS) is a Monte-Carlo importance sampling technique in the space of trajectories which is used to elucidate transition states, reaction coordinates and kinetic parameters of complex biomolecular reactions. Within TPS an ensemble of truly dynamic reactive trajectories are obtained with a Monte-Carlo type acceptance criterion in combination with the shooting and shifting algorithms. To sample the equilibrium properties such as free energies, the inclusion of non-reactive trajectories in the TPS ensemble is essential since these trajectories sample the long lived reactant and product states. With a modified acceptance criterion and in combination with a window based sampling algorithm, TPS can be used to obtain unbiased free energy profiles for chemical reactions that are catalyzed by enzymes. Here we use TPS to study the proton transfer mechanism and the corresponding reaction free energy of enzymes (MBHases) that catalyze a Morita-Baylis-Hillman (MBH) type reaction. These enzymes were developed through directed evolution and we aim to map the quantum mechanical tunneling (QMT) contribution in the proton transfer mechanism to laboratory evolution of the MBHases to determine the role played by QMT to rate improvements of evolved variants. The calculated free energies would also provide insights into the role of QMT in the reaction mechanism as the width and height of the free energy barrier determines the importance of the QMT contributions.