Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases.
BACKGROUND
Kynurenine aminotransferase (KAT) catalyzes the transamination of kynunrenine to kynurenic acid (KYNA). KYNA is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of N-methyl-D-aspartate receptors. Four KAT enzymes, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. Because of the substrate overlap of the four KAT enzymes, it is difficult to assay the specific activity of each KAT in animal brains.


RESULTS
This study concerns the functional expression and comparative characterization of KAT I, II, III, and IV from mice. At the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse KAT I and IV, equimolar methionine inhibited only mouse KAT III and equimolar aspartate inhibited only mouse KAT IV. The activity of mouse KAT II was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. pH optima, temperature preferences of four KATs were also tested in this study. Midpoint temperatures of the protein melting, half life values at 65 degrees C, and pKa values of mouse KAT I, II, III, and IV were 69.8, 65.9, 64.8 and 66.5 degrees C; 69.7, 27.4, 3.9 and 6.5 min; pH 7.6, 5.7, 8.7 and 6.9, respectively.


CONCLUSIONS
The characteristics reported here could be used to develop specific assay methods for each of the four murine KATs. These specific assays could be used to identify which KAT is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of KAT-involved human diseases.
Han
Qian
Q
Department of Biochemistry, Virginia Tech, Blacksburg, 24061, USA.
Cai
Tao
T
Tagle
Danilo A
DA
Li
Jianyong
J
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008297:Male; D008715:Methionine / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014364:Tryptophan / Q000494:pharmacology
D000596:Amino Acids; D020033:Protein Isoforms; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014364:Tryptophan; D008715:Methionine; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase