Li Dongmei, Liu Cui, Lin Jianping
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China; Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China.
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China; Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China.
J Mol Graph Model. 2015 Feb;55:25-32. doi: 10.1016/j.jmgm.2014.10.014. Epub 2014 Nov 7.
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 Sγ to the Cζ 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, Sγ forms a three-membered ring with Cζ and Cη 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.
蛋白质精氨酸脱亚氨酶4（PAD4）在翻译后修饰过程中催化肽基精氨酸残基水解，形成瓜氨酸残基和氨。这一过程在类风湿性关节炎（RA）和基因调控中起着关键作用。F-脒属于一系列卤代脒化合物，是迄今为止描述的最有效的PAD4抑制剂。F-脒作为一种基于机制的PAD4抑制剂，通过对活性位点Cys645的共价修饰使PAD4失活。在本论文中，使用QM/MM方法研究了F-脒抑制PAD4的基本机制。我们的模拟表明，在PAD4-F-脒反应物复合物中，活性位点Cys645以硫醇盐形式存在，His471质子化。这与反向质子化机制一致，即PAD4中的活性位点亲核试剂Cys645在酶的活性形式中以硫醇盐形式存在。F-脒对PAD4的抑制作用始于Sγ对F-脒的Cζ进行亲核加成，导致形成四面体中间体。His471作为质子供体，帮助F离开F-脒的氟乙脒部分；同时，Sγ与F-脒的Cζ和Cη形成三元环。随后，三元硫鎓环崩塌并重排为最终的硫醚产物。His471在过渡态作为质子供体，促进PAD4的抑制反应。
