TY - JOUR
T1 - Active site cysteine is protonated in the PAD4 michaelis complex
T2 - Evidence from born-oppenheimer Ab initio QM/MM molecular dynamics simulations
AU - Ke, Zhihong
AU - Zhou, Yanzi
AU - Hu, Po
AU - Wang, Shenglong
AU - Xie, Daiqian
AU - Zhang, Yingkai
PY - 2009/9/24
Y1 - 2009/9/24
N2 - 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 C § of the substrate, and leads to a covalent tetrahedral intermediate; then, the C§-Nη1 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.
AB - 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 C § of the substrate, and leads to a covalent tetrahedral intermediate; then, the C§-Nη1 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.
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U2 - 10.1021/jp903173c
DO - 10.1021/jp903173c
M3 - Article
C2 - 19507815
AN - SCOPUS:70349266325
SN - 1520-6106
VL - 113
SP - 12750
EP - 12758
JO - Journal of Physical Chemistry B
JF - Journal of Physical Chemistry B
IS - 38
ER -