|Bonner, C.; Jensen, R.
|Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris
|Arch Biochem Biophys
|A prephenate aminotransferase enzyme that produces L-arogenate was demonstrated in extracts from cultured-cell populations of Nicotiana silvestris. The enzyme was very active with low concentrations of prephenate, but required high concentrations of phenylpyruvate or 4-hydroxyphenylpyruvate to produce activity levels that were detectable. It is the most specific prephenate aminotransferase described to date from any source. Only L-glutamate and L-aspartate were effective amino-donor substrates. Prephenate concentrations greater than 1 mM produced substrate inhibition, an effect antagonized by increasing concentrations of L-glutamate cosubstrate. The enzyme was stable to storage for at least a month in the presence of pyridoxal 5'-phosphate, EDTA, and glycerol, and exhibited an unusually high temperature optimum of 70 degrees C. The identity of L-arogenate formed during catalysis was verified by high-performance liquid chromatography. DEAE-cellulose chromatography revealed two aromatic aminotransferase activities that were distinct from prephenate aminotransferase and which did not require the three protectants for stability. The aromatic aminotransferases were active with phenylpyruvate or 4-hydroxyphenylpyruvate as substrates, but not with prephenate. Both of the latter enzymes were similar in substrate specificity, and each exhibited a temperature optimum of 50 degrees C for catalysis. The primary in vivo function of the two aromatic aminotransferases is probably to transaminate between the aspartate/2-ketoglutarate and glutamate/oxaloacetate couples, since activities with the latter substrate combinations were an order of magnitude greater than with aromatic substrates. The demonstrated existence of a specific prephenate aminotransferase in N. silvestris meshes with other evidence supporting an important role for L-arogenate in tyrosine and phenylalanine biosynthesis in higher plants.