Unveiling the Mechanistic Singularities of Caspases: A Computational Analysis of the Reaction Mechanism in Human Caspase-1.

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