Institute for Drug Discovery, Leipzig University Medical School, Leipzig, Germany.
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia.
Protein Sci. 2024 Aug;33(8):e5100. doi: 10.1002/pro.5100.
Cholinesterases are well-known and widely studied enzymes crucial to human health and involved in neurology, Alzheimer's, and lipid metabolism. The protonation pattern of active sites of cholinesterases influences all the chemical processes within, including reaction, covalent inhibition by nerve agents, and reactivation. Despite its significance, our comprehension of the fine structure of cholinesterases remains limited. In this study, we employed enhanced-sampling quantum-mechanical/molecular-mechanical calculations to show that cholinesterases predominantly operate as dynamic mixtures of two protonation states. The proton transfer between two non-catalytic glutamate residues follows the Grotthuss mechanism facilitated by a mediator water molecule. We show that this uncovered complexity of active sites presents a challenge for classical molecular dynamics simulations and calls for special treatment. The calculated proton transfer barrier of 1.65 kcal/mol initiates a discussion on the potential existence of two coupled low-barrier hydrogen bonds in the inhibited form of butyrylcholinesterase. These findings expand our understanding of structural features expressed by highly evolved enzymes and guide future advances in cholinesterase-related protein and drug design studies.
胆碱酯酶是众所周知且广泛研究的酶，对人类健康至关重要，涉及神经学、阿尔茨海默病和脂质代谢。胆碱酯酶活性部位的质子化模式影响内部的所有化学反应，包括反应、神经毒剂的共价抑制和重激活。尽管其意义重大，但我们对胆碱酯酶的精细结构的理解仍然有限。在这项研究中，我们采用增强采样的量子力学/分子力学计算表明，胆碱酯酶主要作为两种质子化状态的动态混合物起作用。两个非催化谷氨酸残基之间的质子转移遵循质子转移机制，由一个介体水分子促进。我们表明，这种未被发现的活性部位的复杂性对经典分子动力学模拟提出了挑战，需要特殊处理。计算得到的质子转移势垒为 1.65kcal/mol，这引发了关于丁酰胆碱酯酶抑制形式中可能存在两个耦合低势垒氢键的讨论。这些发现扩展了我们对高度进化的酶表达的结构特征的理解，并指导了未来在胆碱酯酶相关蛋白质和药物设计研究方面的进展。
