Enzymes of nitrogen metabolism in legume nodules: Partial purification and properties of the aspartate aminotransferases from lupine nodules

The two aspartate aminotransferase isoenzymes, AAT-P 1 and AAT-P 2, found in the plant cytosol fraction of lupine nodules have been separated and partially purified. Both isoenzymes showed a broad pH optimum between 7.0 and 9.5 and demonstrated high substrate specificity. Molecular weights determined by gel filtration chromatography were 105,000 and 96,000 for AAT-P 1 and AAT-P 2, respectively. AAT-P 1 demonstrated a subunit molecular weight of 47,000 and AAT-P 2, 45,000. Both isoenzymes showed oxaloacetate substrate inhibition and gave similar K m values for aspartate (2.2 and 2.6 m m) and 2-oxoglutarate (0.26 and 0.2 m m) for AAT-P 1 and AAT-P 2, respectively. However, AAT-P 2 showed a fivefold lower K m for oxaloacetate (0.02 m m) and a 1.8-fold lower K m for glutamate (12 m m) than did AAT-P 1 for these substrates. The parallel nature of the 1 V vs 1 [ S] plots together with the product inhibition kinetics were consistent with a ping-pong-bi-bi mechanism of action. The results are discussed in terms of the possible physiological significance of these plant aspartate aminotransferases in ammonia assimilation in lupine nodules.