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Sandbox Reserved 346

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This Sandbox is Reserved from January 10, 2010, through April 10, 2011 for use in BCMB 307-Proteins course taught by Andrea Gorrell at the University of Northern British Columbia, Prince George, BC, Canada.
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PDB ID 1b4x

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1b4x, resolution 2.45Å ()
Ligands: ,
Activity: Aspartate transaminase, with EC number 2.6.1.1
Resources: FirstGlance, OCA, PDBsum, RCSB
Coordinates: save as pdb, mmCIF, xml



Aspartate AminotransferaseAspartate Aminotransferase

Aspartate Aminotransferase (AST), also known as Glutamic aspartic transaminase, glutamic oxaloacetic transaminase, and transaminase A., is an enzyme that is a member of the class-I pyridoxal-phosphate-dependent aminotransferase family [1]. It is coded by the gene GOT1[2]. It is a homodimer that is 413 amino acids long and serves a critical role in amino acid and carbohydrate metabolism, ureogenesis, and transfer of reducing equivalents into the mitochondria and chloroplast[3]. Within prokaryote cells it is exclusively found in the cytosol, but in eukaryotic cells there are cytosol, mitochondrial, and chloroplast isozymes[1][4].

In the human body it is produced in the brain, skeletal muscles, liver, pancreas, red blood cells, and kidneys [5][6]. The wide range of tissues in which it is made, separates it from the similar enzyme alanine transaminase (ALT) which is found primarily in the liver[6]. The level of AST in the body can be used as a marker for tissue disease or damage[6]. As well, AST and ALT levels can be compared to pinpoint whether tissue damage is primarily found within the liver[6].

StructureStructure

Ligand

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is a homodimer that contains 16 alpha helices and a Beta-sheet formed from 7 parallel and antiparallel strands[4]. Each subunit contains an equivalent active site[4]. The subunits connect at two sites: between their large domains and between the N-terminal residues and the large domain on the other subunit[4]. This structure of AST varies minutely among organisms ranging from E. coli to humans[4][3]. As well, the structure of the active site is highly conserved with a sequence homology of 25%[4].

Each subunit of the homodimer is further divided into a small and large domain[4]. The is comprised of the amino acids from the N-terminus to Pro 48 residue and from Met 326 to the C-terminus[4]. The remaining amino acids make up the , and the are connected by a long alpha helicx consisting of 32 amino acids[4].

The large domain is where the active site of AST is found and to accommodate this, the core contains many alpha/beta supersecondary structures[4]. This is contrasted with the core of the small subunit which is formed from two alpha helices and two beta strands[4]. In multicellular organisms there is a kink at the 325th residue which acts as a hinge for the small domain, which allows for the resulting conformational changes that take place upon the binding of inhibitors to the enzyme[4].

As was stated above, the active site of AST is situated on the large domain of the subunit[4]. Within the active site is the amino residue Lys 258, also known as the internal aldimine, which binds with the cofactor Pyridoxal 5'-phosphate () forming what is called a Schiff base[4][3]. Upon the addition of an amino acid substrate, a new Schiiff base forms between PLP and the amino acid[4].




FunctionFunction

 

AST catalyzes the reversible transamination of the alpha-amino group from L-aspartate to alpha-ketoglutarate forming oxaloacetate and alpha-ketoglutamate[3]. This reactivity is lower in E.coli than in higher eukaryotes, and has broader substrate specificity[4]. However, the reaction takes place in the same way[4]. Upon introduction of an amino acid substrate, a new Schiff base will form between it and the PLP cofactor[4][7]. This causes the amino acid to lose a hydrogen and form a quinoid intermediate, and reprotanation takes place resulting in a ketimine[4][7]. Next, the structure is hydrolyzed forming an alpha-keto acid and pyridoxamine phosphate[7]. 2-methyl aspartate acts as an inhibitor of AST when it forms a Schiif base with the PLP cofactor, rather than aspartate[7][4]. This results in the process stopping at the step prior to the alpha protein elimination[7][4].

This reaction is essential to maintaining homeostasis in organisms. The four different molecules that can form as a result of this transanimation (oxaloacetate, alpha-ketoglutarate, aspartate, L-glutamate) our critical to a number of metabolic processes[8][9][10][3]. Oxaloacetate and alpha-ketoglutarate play a critical role in the Krebs cycle and the varying forms of aspartate are important molecules in the urea cycle and participate in gluconeogenesis[8][9][10][3].

Clinical ApplicationsClinical Applications

The levels of AST in the body are indicative of tissue damage and disease[11]. Normally AST is found in minimal amounts within the blood, however when the organs mentioned above are damaged, AST is released into the blood[11]. The amount released is proportional to the level of damage sustained[11]. AST levels have been shown to rise substantially within 6 hours of the initial tissue degradation and can stay elevated for up to 4 days[11]. AST levels when compared with the levels of other enzymes can be used by physicians to determine where in the body the damage has taken place[6]. Comparisons with ALT have proven particularly useful in identifying liver damage such as cirrhosis and hepatitis[6]. Under normal condition, AST levels within men are 6-34 IU/L and for women it is 8 - 40 IU/L[11].

Additional ResourcesAdditional Resources

ReferencesReferences

  1. ↑ 1.0 1.1 Han Q, Robinson H, Cai T, Tagle DA, Li J. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV. Biosci Rep. 2010 Oct 26. PMID:20977429 doi:10.1042/BSR20100117
  2. ↑ DeLorenzo RJ, Ruddle FH. Glutamate oxalate transaminase (GOT) genetics in Mus musculus: linkage, polymorphism, and phenotypes of the Got-2 and Got-1 loci. Biochem Genet. 1970 Apr;4(2):259-73. PMID:4193185
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 Jeffery CJ, Gloss LM, Petsko GA, Ringe D. The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase. Protein Eng. 2000 Feb;13(2):105-12. PMID:10708649
  4. ↑ 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20 Kamitori S, Okamoto A, Hirotsu K, Higuchi T, Kuramitsu S, Kagamiyama H, Matsuura Y, Katsube Y. Three-dimensional structures of aspartate aminotransferase from Escherichia coli and its mutant enzyme at 2.5 A resolution. J Biochem. 1990 Aug;108(2):175-84. PMID:2121725
  5. ↑ Palaiologos G, Hertz L, Schousboe A. Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons. Neurochem Res. 1989 Apr;14(4):359-66. PMID:2569674
  6. ↑ 6.0 6.1 6.2 6.3 6.4 6.5 Tran A, Longo F, Ouzan D, Bianchi D, Pradier C, Saint-Paul MC, Sattonnet C, Laffont C, Dantin S, Piche T, Benzaken S, Rampal P. Effects of 1-year interferon-alpha 2a treatment in patients with chronic hepatitis C and persistently normal transaminase activity. Scand J Gastroenterol. 2000 Apr;35(4):433-7. PMID:10831269 Cite error: Invalid <ref> tag; name "Liver damage" defined multiple times with different content
  7. ↑ 7.0 7.1 7.2 7.3 7.4 Martinez-Carrion M, Tiemeier DC, Peterson DL. Conformational properties of the isoenzymes of aspartate transaminase and the enzyme-substrate complexes. Biochemistry. 1970 Jun 23;9(13):2574-82. PMID:5450225
  8. ↑ 8.0 8.1 Tretter L, Adam-Vizi V. Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress. J Neurosci. 2000 Dec 15;20(24):8972-9. PMID:11124972
  9. ↑ 9.0 9.1 Tretter L, Adam-Vizi V. Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress. J Neurosci. 2000 Dec 15;20(24):8972-9. PMID:11124972
  10. ↑ 10.0 10.1 Jungas RL, Halperin ML, Brosnan JT. Quantitative analysis of amino acid oxidation and related gluconeogenesis in humans. Physiol Rev. 1992 Apr;72(2):419-48. PMID:1557428
  11. ↑ 11.0 11.1 11.2 11.3 11.4 Gonzalez-Flecha B, Cutrin JC, Boveris A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J Clin Invest. 1993 Feb;91(2):456-64. PMID:8432855 doi:http://dx.doi.org/10.1172/JCI116223

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OCA, Luke Spooner, Andrea Gorrell
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