Active site cysteine is protonated in the PAD4 Michaelis complex: evidence from Born-Oppenheimer ab initio QM/MM molecular dynamics simulations.

The protein arginine deiminase 4 (PAD4) catalyzes the citrullination of the peptidylarginine and plays a critical role in rheumatoid arthritis (RA) and gene regulation. Understanding its catalytic mechanism is not only of fundamental importance but also of significant medical interest for the rational design of new inhibitors. By employing on-the-fly Born-Oppenheimer ab initio QM/MM molecular dynamics simulations, we have demonstrated that it is unlikely for the active site cysteine and histidine to exist as a thiolate-imidazolium ion pair in the PAD4 Michaelis reactant complex. Instead, a substrate-assisted proton transfer mechanism for the deimination reaction step has been characterized: both Cys645 and His471 in the PAD4 active site are neutral prior to the reaction; the deprotonation of Cys645 by the substrate arginine occurs in concert with the nucleophilic addition of the Cys thiolate to Czeta of the substrate, and leads to a covalent tetrahedral intermediate; then, the Czeta-Neta1 bond cleaves and the resulted ammonia is displaced by a solvent water molecule. The initial deprotonation and nucleophilic attack step is found to be rate-determining. The computed free energy barrier with B3LYP(6-31G*) QM/MM MD simulations and umbrella sampling is 20.9 kcal.mol(-1), consistent with the experimental kinetic data. During the deimination, His471 plays an important role in stabilizing the transition state through the formation of the hydrogen bond with the guanidinium group. Our current studies further demonstrated the viability and strength of the ab initio QM/MM molecular dynamics approach in simulating enzyme reactions.