A general acid–general base reaction mechanism for human brain aspartoacylase: A QM/MM study

Aspartoacylase is a crucial enzyme for the Canavan disease, which is a fatal autosomal recessive disorder that affects the central nervous system. In this work, we employed a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to investigate the reaction mechanism of human brain aspartoacylase. Two possible pathways were considered: the promoted-water and the anhydride mechanisms. The calculated results indicate that the enzymatic reaction proceeds favorably through the promoted-water process with Glu178 serving as the general base and general acid. The free-energy calculations suggest that the first nucleophilic attack step is rate-determining with a barrier of 15.9 kcal/mol, which is consistent with experimental kcat value. During the formation of the tetrahedral intermediate, the Zn2+ ion activates the zinc-bound water nucleophile and stabilizes the transition state by interacting with the substrate carbonyl oxygen atom. In addition, the Arg63 plays a key role in the transition state stabilization.