CHARACTERIZATION OF HUMAN BRAIN KYNURENINE AMINOTRANSFERASES USING [H-3] KYNURENINE AS A SUBSTRATE

The brain metabolite kynurenic acid is an established broad-spectrum a ntagonist 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 [H-3]kynurenine as the subs trate, the two kynurenine aminotransferases (kynurenine aminotransfera se I and kynurenine aminotransferase II) are now characterized using p artially purified enzyme preparations. When assayed at its pH optimum of 10.0, kynurenine aminotransferase I showed pronounced oxo acid spec ificity (pyruvate much greater than 2-oxoglutarate). This co-substrate selectivity was lost when assays were performed at pH 7.4. Kynurenine aminotransferase I activity was potently inhibited by 2 mM glutamine, tryptophan or phenylalanine, but not by 2 mM alpha-aminoadipate or gl utamate. In contrast to kynurenine aminotransferase I, kynurenine amin otransferase 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 transamina tion. Kinetic analyses, performed at pH 7.4 (kynurenine aminotransfera ses I and II) and 10.0 (kynurenine aminotransferase I), and using vari ous concentrations of kynurenine, pyruvate or 2-oxoglutarate, respecti vely, substantiated the differences between the two enzymes and furthe r elucidated the pH dependence of kynurenine aminotransferase I activi ty [apparent K(m) values for kynurenine with 1 mM 2-oxoglutarate: 515 muM (pH 7.4) and 22 muM (pH 10.0)]. Taken together, these data suggest that under physiological conditions, human brain kynurenic acid may d erive preferentially from kynurenine aminotransferase II. However, kyn urenine aminotransferase I activity could become a significant factor in kynurenic acid biosynthesis under conditions which decrease the cer ebral concentration of amino acids such as glutamine, tryptophan and p henylalanine, and in pathological situations which result in an alkalo tic cellular milieu.