Characterization of two multispecific aspartate aminotransferase isozymes purified from bushbean shoots capable of transaminating aromatic amino acids and several DL-chloro-phenylalanines

Two electrophoretically distinct isozymes of L-phenylalanine aminotransferase (Enz I, Enz II) purified from a total soluble shoot extract of bushbean have been characterized. The M rs of Enz I and Enz II were 100 000 and 110 000, respectively. Both isozymes showed pH optima of 8.5. Enz I was able to use either 2-oxoglutarate (2-OG) or oxaloacetate (OAA) equally as a keto acid substrate when L-phenylalanine was the amino donor, while Enz II preferred 2-OG. Neither isozyme was able to use glyoxylate or pyruvate in the presence of L-phenylalanine. When tested with a range of protein amino acids, both Enz I and Enz II showed the highest rate of transamination with L-aspartate, indicating that both isozymes were L-aspartate aminotransferases capable of also showing L-aromatic aminotransferase activity. L-Phenylalanine aminotransferase activity relative to L-aspartate aminotransferase activity was found to be 0.6 % for Enz I and 3.3% for Enz II. Lineweaver-Burk plots of kinetic data gave apparent K m values (mM) for Enz I of 2.3 ( L-Asp), 55.0 ( L-Phe) and 9.0 (2-OG) and for Enz II, 2.8 ( L-Asp), 320.0 ( L-Phe) and 8.2 (2-OG). The values were confirmed by treatment of the data by Hill plots. When tested with a series of 12 ring-substituted DL-chlorophenylalanines, Enz I was active only with the 3-chloro- and 4-chloro-compounds, while Enz II was active with all three monochloro-compounds as well as with the 2,4-, 2,6- and 3,4-dichlorophenylalanines. The activity of Enz II with 4-chlorophenylalanine was very high, 222 % higher than that observed with DL-phenylalanine. Enz I was completely inhibited by 1.0 mM Ca 2+ while Enz II was unaffected by this cation, which suggested different subcellular locations for each isozyme. Cell fractionation studies indicated, however, that both Enz I and Enz II were cytoplasmic. Different isozymes of this multispecific aspartate—aromatic aminotransferase were found in the chloroplasts and mitochondria of bushbean shoots.