The simultaneous application of chemical modification of primary amines and the insertion of a lgF probe have been used to detect the nature and physical properties of the amino group influencing a selected region of the active center of the enzyme aspartate aminotransferase. The enzyme, carbamylated at the NH,-terminal alanine (transaminase’, produced from cyanate-treated holoenzyme) or at the active site Lys-258 (transaminase*, inactivated by carbamylation of Lys-258 with KNCO), can accept phosphopyridoxyl trifluoroethylamine instead of the constitutive coenzyme pyridoxal phosphate. The spin-lattice relaxation time, T,, of the fluorine atoms in the enzyme-bound fluorinate compound is 0.12 s and the spin-spin relaxation time, TO, is 6.4 ms. Both T, and T, values remain constant after carbamylation of Lys-258. Assuming a simple treatment of the dipolar relaxation mechanisms as being due to IH-lYF interactions, a molecular rotation correlation time TV value of -80 ns is calculated for native and carbamylated enzyme. The complexes of phosphopyridoxyl trifluoroethylamine and transaminase’ or transaminase*, although catalytically inactive, retain the same affinity as those with native transaminase for the competitive inhibitors succinic acid and acetate anion. Increasing ionic strength results in increasing values for the observed pK of Lys-258 determined from a pH dependence of the rate of enzymatic inactivation by KNCO treatment. The ionic strength effects are identical with those previously observed in the pK value of the protein group