Evidence of phenylalanine transaminase activity in the isoenzymes of aspartate transaminase.

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. Ada, 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 mu, and tyrosine = 3.9 mM for the supernatant enzyme; and phenylalanine = 18.4 mM, tryptophan = 12 mM, and tyrosine = 3.3 mu for the mitochondrial enzyme. It can be shown that phenylalanine reacts with the pyridoxal form of both isozymes (absorbance, 350 to 360 mn) to generate the pyridoxamine form of the enzyme (absorbance, 330 nm). This form reconverts into the pyridoxal form upon addition of either phenylpyruvate or cr-ketoglutarate, completing a full transamination cycle between an amino acid and its analogous keto acid or with acceptor keto acid. Other nonaromatic 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 a-ketoglutarate as acceptor are 12 and 200 nmoles of glutamate formed per hour per pg. 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 mu for the mitochondrial enzyme and phenylalanine = 25 XnM and phenylpyruvate = 0.6 InM for supernatant enzyme. These values are comparable to those of purified brain phenylalanine transaminase. Immunological and electrophoretic studies reveal identity