Purification, characterization and identification of rat liver mitochondrial kynurenine aminotransferase with alpha-aminoadipate aminotransferase.
Kynurenine aminotransferase (L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7) was purified 378-fold from rat liver mitochondria by digitonin solubilization, heat treatment, DEAE-Sepharose CL-6B chromatography, Sephadex G-100 gel filtration, hydroxyapatite chromatography and chromatofocusing. Elution patterns of alpha-aminoadipate aminotransferase (EC 2.6.1.39) activity were identical with those of kynurenine aminotransferase activity on all column chromatographies. The ratios of the two specific activities were constant throughout the purification. On polyacrylamide gel electrophoresis both activities were detected at the same position. Both enzymatic activities showed the same inactivation curves upon heat inactivation at various temperatures. alpha-Aminoadipate showed competitive inhibiton against kynurenine or 3-hydroxykynurenine. alpha-Ketoadipate was utilized in the kynurenine aminotransferase reaction as an amino acceptor in place of alpha-ketoglutarate. The Km value for alpha-ketoadipate was 10 microM, lower than for alpha-ketoglutarate. These observations indicate that kynurenine aminotransferase is identical with alpha-aminoadipate aminotransferase. The Km values of purified kynurenine aminotransferase were determined at pH 6.5 as: kynurenine, 4.3 mM; pyridoxal 5'-phosphate, 4.2 microM; alpha-ketoglutarate, 20 microM (kynurenine substrate), and 3-hydroxykynurenine, 5.7 mM; pyridoxal 5'-phosphate, 1.7 microM; alpha-ketoglutarate, 13 microM (3-hydroxy-kynurenine substrate). The enzyme was strongly inhibited by Hg2+ and p-chloromercuribenzoate.
D051307:2-Aminoadipate Transaminase; D000818:Animals; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008190:Lyases; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D011773:Pyruvates / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D011773:Pyruvates; D000637:Transaminases; D051307:2-Aminoadipate Transaminase; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases