Specific labeling of cytosolic and mitochondrial aspartate aminotransferases

The apoisozymes of cytosolic and mitochondrial aspartate aminotransferase are both irreversibly inhibited by α‐ N ‐fluorodinitrophenyl‐ β ‐ N ‐phosphopyr‐idoxyldiaminopropionate, an affinity‐labeling reagent analog of the coenzyme. Analysis of the modified peptides shows that the active‐site Lys‐258, which in the holoenzyme binds the coenzyme pyridoxal 5′‐phosphate, is labeled in both isozymes. Comparison with the results obtained using the parent compound 4′‐ N ‐fluorodinitrophenylpyridoxamine 5′‐phosphate, which labels only the cytosolic enzyme, provides information about differences in active‐site reactivity and geometry. Labeling external to the active site occurs in both isozymes. In the cytosolic enzyme the very reactive Cys‐45 is modified, in the mitochondrial enzyme the surface residue Lys‐342 reveals a peculiar reactivity.