Kinetics of the Inhibition of Aspartate Aminotransferase Isozymes by dl‐Glyceraldehyde 3‐Phosphate

The inhibitor dissociation constants and types of inhibition of the isozymes of aspartate aminotransferase from rat liver by the time‐dependent inhibitor dl‐glyceraldehyde 3‐phosphate were determined from residual enzyme activity after equilibration of each isozyme with the inhibitor and one substrate in the preliminary incubation mixtures. The results showed that the inhibition of the cationic isozyme was completely competitive with respect to α‐ketoglutarate and completely noncompetitive with respect to l‐aspartate. The pyridoxamine form of the cationic isozyme was approximately 10 times more sensitive to inhibition by dl‐glyceraldehyde 3‐phosphate (K_i= 0.084 mM) than the pyridoxal form (K_i= 0.98 mM). Inhibition of the anionic isozyme was mixed partially competitive‐partially noncompetitive with respect to α‐ketoglutarate and partially noncompetitive with respect to l‐aspartate. The pyridoxamine form of the anionic isozyme was approximately 5 times more sensitive to inhibition by dlDL‐glyceraldehyde 3‐phosphate (K_i= 0.39 mM) than the pyridoxal form (K_i= 1.9 mM). Both forms of the cationic isozyme were more sensitive to inhibition than the corresponding forms of the anionic isozyme. The degree to which inhibition of the anionic isozyme was partial was assessed by determining the interaction constants α and β. These were α= 6.9 and β= 0.32, and indicated that the anionic isozyme, when fully combined with dl‐glyceraldehyde 3‐phosphate, retained 32% of its catalytic capacity. The cationic isozyme was completely inhibited with α=∞ and β= 0. Comparison of the K_i values for the pyridoxamine and pyridoxal forms of the isozymes of aspartate aminotransferase with the concentrations of d‐glyceraldehyde 3‐phosphate reported for perfused liver and for incubated erythrocytes suggests that inhibition may occur in vivo.