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-phosphopyridoxyldiaminopropionate, an affinity-labeling reagent analog of the coenzyme. Analysis of the modified peptides shows that the activity-site Lys-258, which in the holoenzyme binds the coenzyme pyridoxal 5'-phosphate, is labeled in both isoenzymes. 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 isoenzymes. In the cytosolic enzyme the very active Cys-45 is modified, in the mitochondrial enzyme the surface residue Lys-342 reveals a particular reactivity.