Characterization of human brain kynurenine aminotransferases using [ 3H]kynurenine as a substrate

Abstract

The brain metabolite kynurenic acid is an established broad-spectrum antagonist at ionotropic excitatory amino acid receptors. In the human brain, two distinct enzymes are capable of synthesizing kynurenic acid from its bioprecursor l-kynurenine. Using [ 3H]kynurenine as the substrate, the two kynurenine aminotransferases (kynurenine aminotransferase I and kynurenine aminotransferase II) are now characterized using partially purified enzyme preparations. When assayed at its pH optimum of 10.0, kynurenine aminotransferase I showed pronounced oxo acid specificity (pyruvate ≫2-oxoglutarate). This co-substrate selectivity was lost when assays were performed at pH 7.4. Kynurenine aminotransferase I activity was potently inhibited by 2mM glutamine, tryptophan or phenylalanine, but not by 2mM α-aminoadipate or glutamate. In contrast to kynurenine aminotransferase I, kynurenine aminotransferase II showed a shallow pH curve with an optimum of about 7.4, displayed virtually equal activity with all of the nine 2-oxo acids tested and was not susceptible to inhibition by any of 10 amino acids (2 mM) which are known to serve as substrates for enzymatic transamination. Kinetic analyses, performed at pH 7.4 (kynurenine aminotransferases I and II) and 10.0 (kynurenine aminotransferase I), and using various concentrations of kynurenine, pyruvate or 2-oxoglutarate, respectively, substantiated the differences between the two enzymes and further elucidated the pH dependence of kynurenine aminotransferase I activity [apparent K m values for kynurenine with 1 mM 2-oxoglutarate: 515 μM (pH 7.4) and 22 μM (pH 10.0)]. Taken together, these data suggest that under physiological conditions, human brain kynurenic acid may derive preferentially from kynurenine aminotransferase II. However, kynurenine aminotransferase I activity could become a significant factor in kynurenic acid biosynthesis under conditions which decrease the cerebral concentration of amino acids such as glutamine, tryptophan and phenylalanine, and in pathological situations which result in an alkalotic cellular milieu.