Ramos-Guzmán Carlos A, Ruiz-Pernía J Javier, Zinovjev Kirill, Tuñón Iñaki
Departamento de Química Física, Universitat de Valencia, 46100 Burjassot, Spain.
Instituto de Materiales Avanzados, Universitat Jaume I, 12071 Castelló, Spain.
ACS Catal. 2023 Mar 15;13(7):4348-4361. doi: 10.1021/acscatal.3c00037. eCollection 2023 Apr 7.
Caspases are cysteine proteases in charge of breaking a peptide bond next to an aspartate residue. Caspases constitute an important family of enzymes involved in cell death and inflammatory processes. A plethora of diseases, including neurological and metabolic diseases and cancer, are associated with the poor regulation of caspase-mediated cell death and inflammation. Human caspase-1 in particular carries out the transformation of the pro-inflammatory cytokine pro-interleukin-1β into its active form, a key process in the inflammatory response and then in many diseases, such as Alzheimer's disease. Despite its importance, the reaction mechanism of caspases has remained elusive. The standard mechanistic proposal valid for other cysteine proteases and that involves the formation of an ion pair in the catalytic dyad is not supported by experimental evidence. Using a combination of classical and hybrid DFT/MM simulations, we propose a reaction mechanism for the human caspase-1 that explains experimental observations, including mutagenesis, kinetic, and structural data. In our mechanistic proposal, the catalytic cysteine, Cys285, is activated after a proton transfer to the amide group of the scissile peptide bond, a process facilitated by hydrogen-bond interactions with Ser339 and His237. The catalytic histidine does not directly participate in any proton transfer during the reaction. After formation of the acylenzyme intermediate, the deacylation step takes place through the activation of a water molecule by the terminal amino group of the peptide fragment formed during the acylation step. The overall activation free energy obtained from our DFT/MM simulations is in excellent agreement with the value derived from the experimental rate constant, 18.7 vs 17.9 kcal·mol, respectively. Simulations of the H237A mutant support our conclusions and agree with the reported reduced activity observed for this caspase-1 variant. We propose that this mechanism can explain the reactivity of all cysteine proteases belonging to the CD clan and that differences with respect to other clans could be related to the larger preference showed by enzymes of the CD clan for charged residues at position P1. This mechanism would avoid the free energy penalty associated with the formation of an ion pair. Finally, our structural description of the reaction process can be useful to assist in the design of inhibitors of caspase-1, a target in the treatment of several human diseases.
半胱天冬酶是一类半胱氨酸蛋白酶，负责切断天冬氨酸残基旁边的肽键。半胱天冬酶构成了参与细胞死亡和炎症过程的重要酶家族。包括神经和代谢疾病以及癌症在内的众多疾病都与半胱天冬酶介导的细胞死亡和炎症调节不良有关。特别是人类半胱天冬酶 -1 能将促炎细胞因子前白细胞介素 -1β 转化为其活性形式，这是炎症反应以及许多疾病（如阿尔茨海默病）中的关键过程。尽管其很重要，但半胱天冬酶的反应机制仍不清楚。适用于其他半胱氨酸蛋白酶且涉及催化二元组中离子对形成的标准机制提议未得到实验证据的支持。通过结合经典和混合 DFT/MM 模拟，我们提出了一种人类半胱天冬酶 -1 的反应机制，该机制解释了实验观察结果，包括诱变、动力学和结构数据。在我们的机制提议中，催化性半胱氨酸 Cys285 在质子转移到可裂解肽键的酰胺基团后被激活，这一过程由与 Ser339 和 His237 的氢键相互作用促进。催化性组氨酸在反应过程中不直接参与任何质子转移。在酰基酶中间体形成后，去酰化步骤通过酰化步骤中形成的肽片段的末端氨基激活水分子来进行。我们从 DFT/MM 模拟中获得的总活化自由能与从实验速率常数得出的值非常吻合，分别为 18.7 千卡·摩尔和 17.9 千卡·摩尔。对 H237A 突变体的模拟支持了我们的结论，并与报道的该半胱天冬酶 -1 变体活性降低一致。我们提出这种机制可以解释属于 CD 家族的所有半胱氨酸蛋白酶的反应性，并且与其他家族的差异可能与 CD 家族的酶对 P1 位置带电荷残基的更大偏好有关。这种机制将避免与离子对形成相关的自由能损失。最后，我们对反应过程的结构描述有助于辅助设计半胱天冬酶 -1 的抑制剂，半胱天冬酶 -1 是治疗几种人类疾病的靶点。
