Specific labeling of cytosolic and mitochondrial aspartate aminotransferases.

The apoisozymes of cytosolic and mitochondrial aspartate aminotransferase are both irreversibly inhibited by alpha-N-fluorodinitrophenyl-beta-N-phosphopyridoxyldiaminopropi onate, 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.