Evidence of Phenylalanine Transaminase Activity in the Isoenzymes of Aspartate Transaminase

Abstract

Abstract The transamination by pig mitochondrial and supernatant aspartate transaminase of aromatic amino acids was studied in order to determine if an analogy exists between these enzymes and a previously reported brain phenylalanine transaminase (George, H., and Gabay, S. (1968) Biochim. Biophys. Acta, 167, 555). Supernatant and mitochondrial aspartate transaminase from pig heart can utilize aromatic amino acids as substrates. Phenylalanine and other aromatic amino acids are competitive inhibitors of the transamination between glutamate and aspartate and their respective keto acids. The inhibition constants (Ki) are: phenylalanine = 6.8 mm, tryptophan = 4.1 mm, and tyrosine = 3.9 mm for the supernatant enzyme; and phenylalanine = 18.4 mm, tryptophan = 12 mm, and tyrosine = 3.3 mm for the mitochondrial enzyme. It can be shown that phenylalanine reacts with the pyridoxal form of both isozymes (absorbance, 350 to 360 nm) to generate the pyridoxamine form of the enzyme (absorbance, 330 nm). This form reconverts into the pyridoxal form upon addition of either phenylpyruvate or α-ketoglutarate, completing a full transamination cycle between an amino acid and its analogous keto acid or with acceptor keto acid. Other non-aromatic amino acids, with the exception of methionine, are considerably poorer substrates. At pH 8.3 the transamination rates of isolated pig heart supernatant and mitochondrial aspartate transaminase with phenylalanine as amino donor and α-ketoglutarate as acceptor are 12 and 200 nmoles of glutamate formed per hour per µg. The substrate dissociation constants, as determined by direct spectrophotometric titrations of the isoenzyme's active site, are: phenylalanine = 18.3 mm and phenylpyruvate = 0.4 mm for the mitochondrial enzyme and phenylalanine = 25 mm and phenylpyruvate = 0.6 mm for supernatant enzyme. These values are comparable to those of purified brain phenylalanine transaminase. Immunological and electrophoretic studies reveal identity between the isoenzymes of heart aspartate transaminase and brain phenylalanine transaminase. Antibodies against the heart enzyme inhibit the brain enzyme, with a concomitant decrease in both the aspartate transaminase and phenylalanine transaminases activities. After inhibition by heart's supernatant and mitochondrial aspartate transaminase antibodies, the ratio of aspartate transaminase activity to phenylalanine transaminase remains constant throughout the brain's enzyme purification procedure. It appears that because of substrate specificity, substrate affinity, transamination rates, inhibition by phenylalanine, inhibition by antibodies and electrophoretic mobility, pig brain phenylalanine transaminase is aspartate transaminase. Brain contains both the cytoplasmic and mitochondrial isoenzymes. Because of a higher rate of utilization of phenylalanine by the mitochondrial enzyme, it is suggested that the production of phenylpyruvate in brain might preferentially occur, in vivo, through the participation of mitochondrial glutamate aspartate transaminase.