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Theoretical study of the mechanism of protein arginine deiminase 4 (PAD4) inhibition by F-amidine


Dongmei Li, Cui Liua, Jianping Lin


Journal of Molecular Graphics and Modelling 55 (2015) 25–32


Protein arginine deiminase 4 (PAD4) catalyzes the hydrolysis of a peptidylarginine residue to form a citrulline residue and ammonia during posttranslational modification. This process plays a pivotal role in rheumatoid arthritis (RA) and gene regulation. F-amidine belongs to a series of haloacetamidine compounds that are the most potent PAD4 inhibitors described to date. F-amidine acts as a mechanism-based inhibitor of PAD4, inactivating PAD4 by the covalent modification of the active site Cys645. In this manuscript, the fundamental mechanism of PAD4 inhibition by F-amidine is investigated using a QM/MM approach. Our simulations show that in the PAD4-F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. This is consistent with the reverse protonation mechanism wherein the active site nucleophile, Cys645, in PAD4 exists as a thiolate in the active form of the enzyme. Inhibition of PAD4 by F-amidine is initiated by the nucleophilic addition of S-gamma to the C-zeta of F-amidine, leading to the formation of a tetrahedral intermediate. His471 serves as a proton donor, helping F to leave the fluoroacetamidine moiety of F-amidine; meanwhile, S-gamma forms a three-membered ring with C-zeta and C-eta of F-amidine. Subsequently, the three-membered sulfonium ring collapses and rearranges to the final thioether product. His471 acts as a proton donor in the transition state and facilitates the inhibition reaction of PAD4.