Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l ‐kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000‐fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2‐oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K m of 2.0 m M and 10.0 m M for l ‐kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l ‐glutamine, l ‐phenylalanine, and l ‐tryptophan, using either pyruvate (1 m M ) or 2‐oxoisocaproate (1 m M ) as a cosubstrate. l ‐Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K i = 480 µ M ) and competitively with regard to l ‐kynurenine ( K i = 200 µ M ). Anti‐KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine‐pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.