Computational Study of the Catalytic Mechanism of the Cruzain Cysteine Protease

Cysteine proteases of the papain family are involved in many diseases, making them attractive targets for developing drugs. In this paper, different catalytic mechanisms of cruzain cysteine protease have been studied on the basis of molecular dynamics simulations within hybrid quantum mechanics/molecular mechanics potentials. The obtained free energy surfaces have allowed characterizing every single step along the mechanisms. The results confirm that the full process can be divided into an acylation and a deacylation stage, but important differences with respect to previous studies can be deduced from our calculations. Thus, our calculations suggest that the acylation stage takes place in a stepwise mechanism where a proton from a conserved His159 is transferred first to the Ni atom of the peptide and, after a transient intermediate is located, the Cys25 attacks the carbonyl carbon atom. The stabilization of the activated Cys25 is achieved by an effect of the local environment through interactions with residues Trp26, Gly160, and His159, rather than by a less complex Cys25S(-)/His159H(+) ion pair. In contrast, the deacylation stage, which was proposed to occur via a general base-catalyzed reaction whereby His159 activates a water molecule that attacks the peptide, would take place through a concerted mechanism. In this stage, the role of some residues of the active site, such as Gln19, Asn175, and Trp181, appears to also be crucial. Interestingly, the local environment of His159 would be modulating its pK(a) value to act as an acid in the acylation stage and as a base in the following deacylation stage.