Reaction mechanism of caspases: insights from QM/MM Car-Parrinello simulations

Caspases are fundamental targets for pharmaceutical interventions in a variety of diseases involving disregulated apoptosis. Here, we present a quantum mechanics/molecular mechanics Car-Parrinello study of key steps of the enzymatic reaction for a representative member of this family, caspase-3. The hydrolysis of the acyl-enzyme complex is described at the density functional (BLYP) level of theory while the protein frame and solvent are treated using the GROMOS96 force field. These calculations show that the attack of the hydrolytic water molecule implies an activation free energy of ca. DeltaF(A) approximately equal 19 +/- 4 kcal/mol in good agreement with experimental data and leads to a previously unrecognized gem-diol intermediate that can readily (DeltaF(A) approximately equal 5 +/- 3 kcal/mol) evolve to the enzyme products. Our findings assist in elucidating the striking difference in catalytic activity between caspases and other structurally well-characterized cysteine proteases (papains and cathepsins) and may help design novel transition-state analog inhibitors.