Aspartoacylase is a dimer of two identical monomers of 313 amino acids and uses a zinc cofactor in each.[5][11] 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.[12] 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.[5]
The zinc cofactor is found at the active site and is held by Glu-24, His-21, and His 116.[13] The substrate is held in place by Arg-63, Asn-70, Arg-71, Tyr-164, Arg-168, and Tyr-288.[5] 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.[13]
A monomer of aspartoacylase with the N-domain in green, C-domain in yellow, and zinc cofactor in red. Generated from 2I3C.[5]Active site of aspartoacylase with a bound N-phosphonamidate-L-aspartate. This is a tetrahedral intermediate analogue with phosphorus replacing the attacked carbon. In the structure, zinc, Arg-63, and Glu-178 are stabilizing the tetrahedral intermediate. Generated from 2O4H.[13]
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.[14] Aspartoacylase follows the deprotonated water mechanism.[13] 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.[5] This leads to a tetrahedral intermediate that is stabilized by the zinc, Arg-63, and Glu-178.[13] 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.[14]
Aspartoacylase mechanism.[13] All the coordinating residues are not shown for clarity.
Aspartoacylase is used to metabolize N-acetyl-L-aspartate by catalyzing its deacylation. Aspartoacylase prevents the buildup of N-acetyl-L-aspartate in the brain. It is believed that controlling N-acetyl-L-aspartate levels is essential for developing and maintaining white matter.[5] It is not known why so much N-acetyl-L-aspartate is produced in the brain nor what its primary function is.[15] However, one hypothesis is that it is potentially used as a chemical reservoir that can be tapped into for acetate for acetyl-CoA synthesis or aspartate for glutamate synthesis.[15][16] This way, N-acetyl-L-aspartate can be used to transport these precursor molecules and aspartoacylase is used to release them. For example, N-acetyl-L-aspartate produced in neurons can be transported into oligodendrocytes and the acetate released can be used for myelin synthesis.[12][17] Another hypothesis is that N-acetyl-L-aspartate is essential osmolyte that acts as a molecular water pump that helps maintain a proper fluid balance in the brain.[18]
Mutations that lead to loss of aspartoacylase activity have been identified as the cause of Canavan disease.[19] Canavan disease is a rare autosomal recessive disorder that causes spongy degeneration of the white matter in the brain and severe psychomotor retardation, usually leading to death at a young age.[12][20] The loss of aspartoacylase activity leads to the buildup of N-acetyl-L-aspartate in the brain and an increase in urine concentration by up to 60 times normal levels.[19] Though the exact mechanism of how loss of aspartoacylase activity leads to Canavan disease is not fully understood, there are two primary competing explanations. The first is that it leads to defective myelin synthesis due to a deficiency of acetyl-CoA derived from the acetate product.[20] Another explanation is that the elevated levels of N-acetyl-l-aspartate interfere with its normal brain osmoregulatory mechanism leading to osmotic disequilibrium.[21]
There are over 70 reported mutations of this enzyme, but the most common ones are the amino acid substitutions E285A and A305E.[12] E285A reduces activity of aspartoacylase down to as low as 0.3% of its normal function and is found in 98% of cases with Ashkenazi Jewish ancestry.[22] The mutation A305E is found in about 40% of non-Jewish patients and reduces activity to about 10%.[22] Of these two mutations, a crystal structure of the E285A mutant has been taken, showing that the loss of the hydrogen bonding from glutamate leads to a conformational change that distorts the active site and alters the substrate binding, leading to the much lower catalytic activity.[12]
Distortion of the active site caused by the E285A mutation. Wild type ASPA is on the left (2O4H[5]) and E285A on the right (4NFR[12]).
^ abZhang C, Liu X, Xue Y (January 2012). "A general acid–general base reaction mechanism for human brain aspartoacylase: A QM/MM study". Computational and Theoretical Chemistry. 980: 85–91. doi:10.1016/j.comptc.2011.11.023.
^Baslow MH (April 2002). "Evidence supporting a role for N-acetyl-L-aspartate as a molecular water pump in myelinated neurons in the central nervous system. An analytical review". Neurochemistry International. 40 (4): 295–300. doi:10.1016/s0197-0186(01)00095-x. PMID11792458. S2CID34902757.
^ abNamboodiri AM, Peethambaran A, Mathew R, Sambhu PA, Hershfield J, Moffett JR, Madhavarao CN (June 2006). "Canavan disease and the role of N-acetylaspartate in myelin synthesis". Molecular and Cellular Endocrinology. 252 (1–2): 216–23. doi:10.1016/j.mce.2006.03.016. PMID16647192. S2CID12255670.
^Baslow MH, Guilfoyle DN (April 2013). "Canavan disease, a rare early-onset human spongiform leukodystrophy: insights into its genesis and possible clinical interventions". Biochimie. 95 (4): 946–56. doi:10.1016/j.biochi.2012.10.023. PMID23151389.
^ abZano S, Wijayasinghe YS, Malik R, Smith J, Viola RE (January 2013). "Relationship between enzyme properties and disease progression in Canavan disease". Journal of Inherited Metabolic Disease. 36 (1): 1–6. doi:10.1007/s10545-012-9520-z. PMID22850825. S2CID34211521.
