Aspartoacylase is a hydrolytic enzyme (, also called aminoacylase II, ASPA and other names) that in humans is encoded by the ASPA gene. ASPA catalyzes the deacylation of N-acetyl-l-aspartate (N-acetylaspartate) into aspartate and acetate. It is a zinc-dependent hydrolase that promotes the deprotonation of water to use as a nucleophile in a mechanism analogous to many other zinc-dependent hydrolases. It is most commonly found in the brain, where it controls the levels of N-acetyl-l-aspartate. Mutations that result in loss of aspartoacylase activity are associated with Canavan disease, a rare autosomal recessive neurodegenerative disease. Structure Aspartoacylase is a dimer of two identical monomers of 313 amino acids and uses a zinc cofactor in each. There are two distinct domains in each monomer: the N-terminal domain from residues 1-212 and the C-terminal domain from residues 213–313. The N-terminal domain of aspartoacylase is similar to that of zinc-dependent hydrolases such as carboxypeptidaseA. However, carboxypeptidases do not have something similar to the C-domain. In carboxypeptidase A, the active site is accessible to large substrates like the bulky C-terminal residue of polypeptides, whereas the C-domain sterically hinders access to the active site in aspartoacylase. Instead, the N-domain and C-domain of aspartoacylase form a deep narrow channel that leads to the active site. The zinc cofactor is found at the active site and is held by Glu-24, His-21, and His 116. The substrate is held in place by Arg-63, Asn-70, Arg-71, Tyr-164, Arg-168, and Tyr-288. The zinc cofactor is used to lower the pKa of a ligated water molecule so that an attack on N-acetyl-L-aspartate may occur and to stabilize the resulting tetrahedral intermediate along with Arg-63, and Glu-178. Mechanism There are two types of possible mechanisms for zinc-dependent hydrolases depending on what is the nucleophile. The first uses deprotonated water and the second attacks with an aspartate or glutamate first forming an anhydride. Aspartoacylase follows the deprotonated water mechanism. Zinc lowers the pKa of a ligated water molecule and the reaction proceeds via an attack on N-acetyl-l-aspartate when the water molecule is deprotonated by Glu-178. This leads to a tetrahedral intermediate that is stabilized by the zinc, Arg-63, and Glu-178. Finally, the carbonyl is then reformed, the bond with nitrogen is broken, and the nitrogen is protonated by the proton taken by Glu-178 all in one concerted step. Biological function Aspartoacylase is used to metabolize N-acetyl-L-aspartate by catalyzing its deacylation. Aspartoacylase prevents the buildup of