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Aspartoacylase
Structure of aspartoacylase dimer. Generated from 2I3C.[1]
Identifiers
EC no.3.5.1.15
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Aspartoacylase (EC 3.5.1.15, aminoacylase II, N-acetylaspartate amidohydrolase, acetyl-aspartic deaminase, acylase II) is a hydrolase enzyme with system name N-acyl-L-aspartate amidohydrolase.,[2][3] which breaks down N-acetylaspartate. A deficiency is associated with Canavan disease.This enzyme catalyses the following chemical reaction



Structure

Aspartoacylase is a dimer of two identical monomers of 313 amino acids and use a zinc cofactor in each.[1][4] 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.[5] The N-terminal domain of aspartoacylase is similar to that of zinc-dependent hydrolases such as carboxypeptidases A. 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 polypetides, 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. [1]

The zinc cofactor is found at the active site and is held by Glu-24, His-21, and His 116.[6] The substrate is held in place by Arg-63, Asn-70, Arg-71, Tyr-164, Arg-168, and Tyr-288. [1] The zinc cofactor is used to lower the pKa of a ligated water 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. [6]

A monomer of aspartoacylase with the N-domain in green, C-domain in yellow, and zinc cofactor in red. Generated from 2I3C.[1]
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.[6]

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 asparate or glutamate first forming an anhydride. [7] Aspartoacylase follows the deprotonated water mechanism.[6] 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.[1] This leads to a tetrahedral intermediate that is stabilized by the zinc, Arg-63, and Glu-178. [6]. 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. [7].

Aspartoacylase mechanism.[6] All the coordinating residues are not shown for clarity.


Biological Function

Aspartoacylase is used to metabolize N-acetyl-L-aspartate by catalyzing its deacylation. Aspartoacylase prevents the build up of N-acetyl-L-aspartate in the brain. N-acetyl-L-aspartate is one of the most abundant amino acids found in the brain with concentrations of up to 10mM and makes up about 1% of the brain's dry weight. [8] It is believed that controlling N-acetyl-L-aspartate levels is essential for developing and maintaining white matter.[1] It is not known why so much N-acetyl-L-aspartate is produced in the brain nor what its primary function is.[9] 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. [8] [9] [10] 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.[5] [11]

Disease Relevance

Mutations that lead to loss of aspartoacylase activity have been linked to Canavan disease.[12] Canavan disease is a rare autosomal recessive disorder that causes spongy degeneration of the white matter in the brain and severe psychomotor retardation. [5] [13]The loss of aspartoacylase activity leads to the buildup of N-acetyl-L-aspartate in the brain and patients . Though the primary mechanism of how loss of aspartoacylase activity leads to Canavan disease is not fully understood, one explanation is that it leads to defective myelin synthesis due to a deficiency of acetyl CoA derived from the acetate product. [13].

References

  1. ^ a b c d e f g Bitto, E; Bingman, CA; Wesenberg, GE; McCoy, JG; Phillips GN, Jr (9 January 2007). "Structure of aspartoacylase, the brain enzyme impaired in Canavan disease". Proceedings of the National Academy of Sciences of the United States of America. 104 (2): 456–61. PMID 17194761.
  2. ^ Birnbaum, S.M. (1955). "Aminoacylase. Amino acid aminoacylases I and II from hog kidney". Methods Enzymol. 2: 115–119. doi:10.1016/S0076-6879(55)02176-9.
  3. ^ Birnbaum, S.M., Levintow, L., Kingsley, R.B. and Greenstein, J.P. (1952). "Specificity of amino acid acylases". J. Biol. Chem. 194: 455–470. PMID 14927637.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  4. ^ Herga, S; Berrin, JG; Perrier, J; Puigserver, A; Giardina, T (30 October 2006). "Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease". FEBS letters. 580 (25): 5899–904. PMID 17027983.
  5. ^ a b c Wijayasinghe, YS; Pavlovsky, AG; Viola, RE (5 August 2014). "Aspartoacylase catalytic deficiency as the cause of Canavan disease: a structural perspective". Biochemistry. 53 (30): 4970–8. PMID 25003821.
  6. ^ a b c d e f Le Coq, J; Pavlovsky, A; Malik, R; Sanishvili, R; Xu, C; Viola, RE (18 March 2008). "Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue". Biochemistry. 47 (11): 3484–92. PMID 18293939.
  7. ^ a b Zhang, Chenghua; Liu, Xiaoqiang; Xue, Ying (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:doi:10.1016/j.comptc.2011.11.023. {{cite journal}}: Check |doi= value (help)
  8. ^ a b Clark, JF; Doepke, A; Filosa, JA; Wardle, RL; Lu, A; Meeker, TJ; Pyne-Geithman, GJ (2006). "N-acetylaspartate as a reservoir for glutamate". Medical hypotheses. 67 (3): 506–12. PMID 16730130.
  9. ^ a b Moffett, JR; Arun, P; Ariyannur, PS; Namboodiri, AM (26 December 2013). "N-Acetylaspartate reductions in brain injury: impact on post-injury neuroenergetics, lipid synthesis, and protein acetylation". Frontiers in neuroenergetics. 5: 11. PMID 24421768.
  10. ^ Hershfield, JR; Madhavarao, CN; Moffett, JR; Benjamins, JA; Garbern, JY; Namboodiri, A (October 2006). "Aspartoacylase is a regulated nuclear-cytoplasmic enzyme". FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 20 (12): 2139–41. PMID 16935940.
  11. ^ Chakraborty, G; Mekala, P; Yahya, D; Wu, G; Ledeen, RW (August 2001). "Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase". Journal of neurochemistry. 78 (4): 736–45. PMID 11520894.
  12. ^ Moore, RA; Le Coq, J; Faehnle, CR; Viola, RE (1 May 2003). "Purification and preliminary characterization of brain aspartoacylase". Archives of biochemistry and biophysics. 413 (1): 1–8. PMID 12706335.
  13. ^ a b Namboodiri, AM; Peethambaran, A; Mathew, R; Sambhu, PA; Hershfield, J; Moffett, JR; Madhavarao, CN (27 June 2006). "Canavan disease and the role of N-acetylaspartate in myelin synthesis". Molecular and cellular endocrinology. 252 (1–2): 216–23. PMID 16647192.
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