Enzymatic Hydroxylation in p-Hydroxybenzoate Hydroxylase:  A Case Study for QM/MM Molecular Dynamics

We investigate the OH transfer step of the hydroxylation reaction of p-hydroxybenzoate in the enzyme p-hydroxybenzoate hydroxylase (PHBH) using QM/MM molecular dynamics methods. The QM region (49 atoms) is treated at the AM1 level, while the MM part (ca. 23 000 atoms) is described by the GROMOS force field. Performing pointwise thermodynamic integration from 10 starting structures, we have obtained an average value of the free-energy barrier for this reaction of 101 kJ mol(-)(1). The simulations provide insight into the dynamics of the hydrogen bonding network in the active site along the course of the reaction. In addition, we describe statistical techniques to analyze molecular dynamics data that assess the convergence of averages and yield an error measure. We discuss the effect of different error sources on the free energy.